Handbook Of Analysis And Quality Control For Fruit And Vegetable Products [2 ed.] 9780074518519, 0074518518

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VEGETABLE LRODUGRES

HANDBOOK OF

ANALYSIS AND QUALITY CONTROL FOR FRUIT AND VEGETABLE PRODUCTS Second Edition

HANDBOOK OF ANALYSIS AND QUALITY CONTROL FOR FRUIT AND VEGETABLE ~ PRODUCTS Second Edition

S. RANGANNA Area Coordinator Manpower Development Central Food Technological

Research Institute Mysore

Education

McGraw Hill Education (India) Private Limited NEW DELHI New Delhi

NewYork

Kuala Lumpur

StLouis’

Lisbon. San Juan

San Francisco

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London Madrid Mexico City Milan Montreal Santiago Singapore Sydney Tokyo Toronto

Hill Education

McGraw Hill Education (India) Private Limited

Copyright © 1977, 1986 by The McGraw-Hill Education (India) Private Limited 23rd reprint 2016 RDDZLCRLQLZLX No part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publishers. The program listings (if any) may be entered, stored and executed in a computer system, but they may not be reproduced for publication.

This edition can be exported from India by the publishers, McGraw Hill Education (India) Private Limited ISBN (13): 978-0-07-451851-9 ISBN (10): 0-07-451851-8

Published by McGraw Hill Education (India) Private Limited, P-24, Green Park Extension, New Delhi 110 016, and printed at Sai Printo Pack Pvt. Ltd, New Delhi 110 020 Visit us at: www.mheaucation.co.in

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Foreworc

The purpose of conservation and processing of food in a developin; country like India is to stimulate socio-economic progress towards a bette quality of life. The development of this industty stimulates agriculture and othe sectors of the economy, generates employment, reduces imports, increase foreign exchange earnings, and brings about transformation essential for acce erating the pace of progress. To build a sound agro-food industry, it is essenti: to develop food regulations and to ensure their systematic and meaningft compliance. Prior to 1947, some food regulations had been introduced in India. Partici lar mention may be made of the Agriculture Produce (Grading and Marketing Act (AGMARK) in 1937. Subsequently, the Fruit Products Order (FPO) we promulgated under the Essential Supplies Act in 1943 which has since bee revised and modified to meet the changing requirements of agro-industri: development. With the socio-technological changes which began to take place rapidl after independence it became necessary to move towards building a compre hensive legal framework for this purpose. The first step, therefore, was t compile food laws of various Provinces, subsequently known as States, anc bring them together in the form ofa book. This was done by the CFTRI in 1954 within only six years of its establishment. — The publication on the above laws and a detailed study of the social an economic requirements for the development of agro-industries resulted in th: first comprehensive law, known as the Prevention of Food Adulteration (PFA’ which was passed by the Parliament in 1954. These laws have since continue to evolve, and specifications have been formulated for a number of foods unde them. Simultaneously, the Indian Standards Institution was established in 194 to assist the industries to raise their standard of quality. Its Agricultural and Foo: Product Development Council (AFDC) has helped to evolve a number

voluntary food standards, and these have proved most valuable to the industi and the consumers. The benefits of food laws to the consumers and the processing industr depend upon the effectiveness with which the laws are implemented. Th: requires not only a well organised national infrastructure for inspection an quality control, but availability of reliable methods of analysis which could b used to check on the quality standards and safety so that the industries can b advised to make improvements in their products and legal action taken whe necessary to protect the consumer interest.

viii

Foreword

Considering the above requirements, Dr S.Ranganna prepared a compre-

hensive volume known as Manual of Analysis of Fruit and Vegetable Products in 1977. The value of this publication and its success can be seen from the fact that it had to be reprinted in 1978 and again in 1979. Since then, a considerable amount of knowledge has accumulated in the field of food analysis, which includes inter alia, the use of sophisticated instruments. This necessitated revision, addition of much new information and rearrangement, and the result

is this more valuable Handbook of Analysis and Quality Control for Fruit and Vegetable Products. Thirty-five years of Dr Ranganna’s rich experience of dealing with the problems of food industries, and active involvement in research and training has gone into it. The present volume will certainly meet the requirements of not only scientists and technologists involved in analysis and quality control of fruit and vegetable products, but will also be useful toa much wider range of users in India and abroad.

H.A.B. PARPIA

Preface to the Second Edition

The first edition of the book well-served the purpose of bringing together methods for analysis and quality control of fruit and vegetable products. The overwhelming response to the first edition was a great source of inspiration to revise the book. To increase its utility, a number of new chapters have been added and some of the old chapters have been revised in this edition. Sampling techniques, methods for the determination of some B-group vitamins, maturity of fruits and vegetables, and quality control of raw-materials like sugar, salt, oils and fats and flavouring materials used in the preparation of fruit and vegetable products, and packaging materials like glass containers and flexible packaging materials in addition to tin containers have been added. The chapter on measurement of consistency has been enlarged to include measurement of consistency constants of non-Newtonian fluids and texture of fruits and vegetables. New chapters on the analysis of specific processed products are frozen products, preserves and confections, pickles and je le and fruit juices, concentrates and beverages. FAO/WHO Codex Alimentarius Commission has issued international standards for fruit and vegetable products, and elaborated methods of analysis in some cases. Like many other countries, in India too, specifications for fruit and vegetable products are likely to be revised on similar lines. Hence, typical examples of Codex Alimentarius standards are given under each class of products for illustration. Statistics play a great role in the acceptance sampling of raw materials and finished products, control of processes, and interpretation of results of analysis

or of sensory evaluation. To meet these needs, chapters on Statistical Methods and Statistical Quality Control have been added, and statistical analysis of results of sensory evaluation illustrated. These additions necessitated changes in the order of presentation and the title ofthe book. I hope that the readers will find this edition more useful. S. RANGANNA

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Preface to the First Edition

The analysis, and quality control of fruit and vegetable products involve physical, chemical and microbiological methods. In addition to making routine analyses, laboratory personnel are required to examine the technical problems which arise in the course of production and handling, and in the examination of

products by public analysts. No single book contains all the information required by the specialist. The methods involved are varied, and since standard reference works are not readily available, this manual is intended to be a compendium for these needs. I have included whatever has been found essential! and, in my experience, not readily ' available, in the text. The topics included thus reflect my personal experience and choice to a great extent, since most of the. methods included are those which have been found satisfactory by me and my colleagues. The matter under each head has been chosen and carefully arranged to avoid repetition. A Guide to Analytical Methods provides extensive cross-references to the methods suitable for a particular product or raw material. Specifications for each fruit or vegetable product as laid down under the Fruit Products Order are also included in the Guide. Methods of determining common constituents of fruit and vegetable products are given in chapters 1 to 6. Those involving methods for specific products are included in chapters 7 to 10. Colour, texture, and sensory quality are the three criteria by which the consumer judges the quality of the product. Books have recently appeared on each of these topics. In chapters 12 to 14, these quality aspects are discussed briefly, but adequately for practical application in evaluating quality. Since objective measurement of texture has not been very well applied in practice, the scope of chapter 13 has been restricted to consistency. Availability of appropriate-quality water in adequate quantities is a prerequisite for any plant. Chapter 16 is, therefore, devoted to quality specifications and analysis of water. Tin containers are mostly used for packing fruit and vegetable products. To enable canners to understand trade practices in the manufacture and sale of tin plate, and to test the quality oftin plate and lacquers, chapter 17 has been added on this subject. Chapter 18 is devoted to double-seaming, which is as important as adequate processing. The next six chapters, 19 to 24, relate to microbiological examination. Adequate processing is the heart ofthe canning process. The National Canners

Association have evolved, over yeats, process time and temperature for canned

xii

Preface to the First Edition

foods, but for many tropical fruits and vegetables, processing schedules have yet to be developed. Although there are several books on this subject, one has to study a number of original papers to get at the actual methodology. Chapter 23 is devoted to this subject. S. RANGANNA

Acknowledgements Additions to the revised edition was possible by the permission given by the associations and publishers to include the published material for whom I am grateful. Among them are the Association of Official Analytical Chemists, U.S., American Society for Testing and Materials, U.S., American Oil Chemists’ Society, U.S., Flavour Manufacturers Association, U.S., Institute of Food Technologists, U.S., Biometrika Trustees, London, The AVI Publishing Company Inc., Food and Nutrition Press Inc., John-Wiley & Sons, Inc., McGraw-Hill Interna-

tional Book Company, Hutchinson Publishing Group Ltd., Blackie and Son Ltd., The Academic Press Inc. (London) Ltd., and D. Reidel Publishing Company. Iam grateful to the Literary Executor of the late Sir Ronald A Fisher, F.R.S., to

Dr. Frank Yates, F.R.S. and to Longman Group Ltd., London for permission to reprint part of Tables II] and VII from their book Statistical Tables for Biological, Agricultural and Medical Research (6th Edn., 1974).

I record my sincere gratitude to Mr. K.R. Kumar for reviewing the chapter on flexible packaging materials; to Dr. M.C. Misra for help in preparing the manuscript on measurement of consistency, and to Mr. S. Dhanraj for reviewing the chapter on statistics as well as for checking the statistical analysis of results of sensory evaluation. Special thanks are also due to Mr. D. Sampath for typing the manuscript, and to Mr. K. Nanjundaiah for drawing figures and illustrations of this edition. I wish to record my deep debt of gratitude to Mr. P.G.K. Menon for his invaluable help in reading the proofs. - The efforts of Tata McGraw-Hill Publishing Company Limited, New Delhi in bringing out this edition is gratefully acknowledged. S. RANGANNA

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Contents Foreword Preface to the Second Edition

Preface to the First Edition Acknowledgements . Proximate Constituents . FPeCuin

. Polyphenols . Plant Pigments . Vitamins

. Minerals . Maturity Indices and Quality Criteria . Sugar Salt ODNANRWNH

. . . .

Edible Oils and Fats Flavouring Materials Chemical Additives Water-Standards and Analysis

. Tin Containers

. . . . . . . . . . . . .

Glass Containers Flexible Packaging Materials Colour Measurement Measurement of Viscosity, Consistency and Texture Sensory Evaluation General Instructions for Microbiological Examination Bacteriological Examination of Water Assessment of Surface Sanitation Microbiological Examination of Spoilage Micro-Analytical Examination for Extraneous Matter Determination of Thermal Process Time Examination of Canned Products Frozen Fruits and Vegetables 28. Fruit Juices, Concentrates and Beverages 29. Jam, Jelly, Marmalade and Preserve

30. Tomato Products

xvi

Contents

51. Dehydrated Fruits and Vegetables

32. a0. 34. opt 36.

Pickles, Chutneys and Vinegar Statistical Methods Statistical Quality Control Standard Solutions Tables

Guide to Analytical Methods and FPO Specifications Index

CHAPTER

1

Proximate Constituents

THE quantitative analysis of fruits and vegetables and their products may broadly be divided into proximate analysis and ultimate analysis. The former gives useful information, particularly from the nutritional and biochemical point of view, while the cre tefets to the determination of a particular el¢e- -

ment or a compound present in the material. The proximate analysis consists in determining the percentages of the moisture, ash, acidity, crude fat or ether extractives, protein, sugars and the crude

fibre. Their sum total subtracted from 100 represents primarily the amount of carbohydrates other than sugars, but includes starch, pectin, gums, etc. In addition,

all the errors in the determinations of all the other constituents

ate also reflected in the calculation of the carbohydrates other than sugars. SAMPLING

Foods and food products are variable in composition. Plant foods are more variable in composition than flesh foods. Processing methods cause additional changes in composition. In addition to differences in composition between individual fruits and vegetables of the same variety and maturity, there is a difference in composition between the various parts of the same fruit or vegetable. Some constituents vary more than others. Carbohydrate, fat and protein content of foods of vegetable origin are more constant than the content ot the mineral elements. In sampling foods and food products, sufficient material must be taken to compensate for the variability involved. It is also advisable to analyse replicate samples considering the variability and the requirements of certain analytical techniques. The amount of material to be selected for analysis can be estimated statistically when the extent of variability of the individuals of a sample are available. When repeated chemical analyses are done, it is advisable to make a preliminary determination of the variability of the sample. The number of individual samples to be selected may be Pas from the following expression:

n=Cr/N where » = number of individuals to be selected; C = a factor which represents the degree of accuracy desired in the sample; and, N = lot size. Where the extent of variability is not known, it is advisable to select at least ten times the amount to be taken as a sample for analysis.

2.

Analysis of Fruit and Vegetable Products _

The sample selected should be representative, and reflect all the homogeneous parts of the heterogeneous population. Generally, the errors in sampling are the following: a. Failure to select the individuals composing the sample at random. b.Changes in the composition of the product during sampling such as loss or absorption of moisture, loss of volatile constituents, or deterioration in fresh

fruits and vegetables due to respiration, enzyme activity or mechanical injury. c. Difficulties in obtaining a uniform sample.

Preparation of Sample Fresh fruits and vegetables: Remove the adhering soil or sand by washing or wiping the surfaces with a damp cloth. Avoid excessive washing to prevent leaching of soluble solids. Separate the fresh tissue into core, outer, and inner tissue depending on the objectives of analysis. Separate the shells from nut kernels. Remove the pits from the flesh of stone fruits. Comminute the prepared material in a blender. Enzymes naturally present may cause undesirable changes. They may be inactivated with steam or boiling alcohol. Sufficient alcohol should be used to give a strength of 80% in the mix. Cut the material into small pieces and drop directly into boiling alcohol. When

intended

for estimation of sugars, add sufficient

calcium carbonate to neutralize the acidity, Heat the material preferably under . reflux for 30 min.

- Plant:tissues may be prepared for analysis by drying in a vacuum oven at 60° C. Drying at atmospheric pressure even at a lower temperature causes undesirable changes. The extent of drying should be sufficient enough to render the dried matter stable during storage, but should not be carried too far. When the moisture content is 6% or above, nonenzymatic browning will occur at room temperature, but at lower moisture contents at which browning is inhibited, lipids may become rancid. Powder the dried matter to pass through a 60 mesh sieve. Canned fruits and vegetables: When analyses are to be made on the composite sample, mix and comminute the entire contents. When the analyses are to be made on solid and liquid portions separately, drain the contents on a sieve as described in Chapter 26, and comminute the solid matter. Dried fruits: Pass the sample through a food chopper three times, and thoroughly mix after each grinding. If need be, grind initially with a coarse cutting blade, and do final grinding with a nut butter blade. Pureed products: Shake thoroughly pureed products such as tomato puree, ketchup, fruit pulps and strained fruits and vegetables before sampling. . Frozen products: Comminute by grinding while frozen’ in a cold room. Fruit juice beverages: Render fruit juice beverages containing insoluble matter, thoroughly uniform, by blending in a high speed blender. Powdery or granular materials: Sample the material by the technique of ‘quartering as follows: Spread the material from the container ona large sheet, and mix witha spatula.

Proximate Constituents

3

Draw a cross over the heaped material. Remove two diagonally Opposite segments, and return them to the original container. Remix the-remaining segments, and draw a cross over the heap. Remove the two opposite segments, and transfer to the original container. Repeat the process until about 250 g remains. If need be, grind the granular material at this stage. Transfer the material to a sample jar, and tightly stopper.

Storage and Preservation of Samples’ Prepared samples may undergo changes in composition through evaporation » or absorption of moisture, or by the action of enzymes or microorganisms. The components which are likely to change (e.g. ascorbic acid) should be analysed immediately after preparation. Store the prepared samples in hermetically sealed. inert containers like glass jars or wide-mouthed bottles with screw caps or friction top tin containers. Store dried fruits and fruit pees containing 20-30% moisture in glass or plastic containers. Hydrolytic changes caused by the enzymes may be prevented by dropping the sample into boiling alcohol. Store the alcoholic extracts below 0° C to prevent changes in composition. Products which are likely to undergo microbial spoilage may be preserved by using acetic. acid or sodium benzoate as preservative, by freezing, or by drying. Fruits and fruit products for sugar analysis may be preserved for several-days by adding excess of neutral lead acetate to a weighed sample. Toluene or thymol used as a preservative in liquid products aré not efficient, and care should be taken to ensure the distribution of toluene throughout the mass. In the estimation of enzyme activity, toluene is generally used, but mezcthiolate (0.1%) is superior. . Freezing of samples in air and moisture proof containers by rapid freezing, and storage at less than -6.7° .C (20° F) prevents microbial activity, but not enzyme

activity, which continues to occur although at a slower rate at temperatures down

to -40° C (-40° F). Storage of dried products at 0° to 10° C minimizes deterioration. While taking samples stored at low temperature foranalysis, either the.entire sealed container should be warmed to room temperature,or a portion transferred quickly to aclean, ‘dry stoppered container to avoid changes in moisture content. | MOISTURE

Water, particularly in plant foods, may occur in any of the followiiig three different forms: (i) as a dispersing’ medium for the colloids and as a solvent for the crystalloids present, i.e. as ‘free water’; (ji) it may be adsorbed on ‘the surfaces of the colloidal particles in the protoplasm, the cell walls, and cell constituents such as proteins, starches, cellulose which hold water very tenaciously; and (iii) as water of hydration in chemical combination with various substances like carbohydrates and hydrates of various salts. The “botnd water’ found in biological materials and the water in colloidal systems may. exist as (a)

|

4

Analysis of Fruit and Vegetable Products

occluded water, (b) capillary water, (c) osmotic water, (d) colloidal bound by physical forces, or (e) chemically ‘bound water’. Whatever be the true nature of ‘bound water’, a certain proportion of the total present in the biocolloids will not separate readily when frozen or when dried at high temperatures; this proportion is retained for longer than the remainder. Moisture content of foods may be determined (i) by drying in an (ii) by distillation with an immiscible solvent, and (iii) by chemical

water may water even times oven, and

physical methods. The first two methods are described below in detail and a

teference is made to other methods. Oven Drying This method consists in medstiring- the weight lost by foods due to the evaporation of water. Drying methods are generally used as they give accurate results. However, this loss of weight may not be a true measure of the water content of the sample, as some volatile constituents present may be lost at the drying temperature employed. Further, with some foods high in protein content, only a proportion-of the ‘free water’ present may be evaporated at the drying temperature, and the remainder, generally referred to as the ‘bound water’, may still remain associated with the proteins present. The proportion of ‘free water’ lost increases as the temperature of drying increases. So it is important to compare the results obtained using the same conditions of drying. Further, if decomposition is likely to occur, as in fruit products, which contain an appreciable proportion of sugars, it is advisable to dry at lower drying temperatures, such as 60-70° C and preferably in vacuum. Use an oven whose temperature can be centrolled accurately. Since the temperature tends to be different on different shelves, a drying oven fitted

with an internal fan for circulation of air is to be preferred. The sample, after determination of moisture, the-determination of ether extractives or ash.

could

be used

either for

PROCEDURE

Inio a flat-bottom metallic dish, spread a thin layer of finely divided asbestos

(Gooch grade). Dry at 110° C for 1 hr; cover the dish, cool and weigh. Spread 20 g of sample uniformly over the asbéstos layer. Weigh as quickly as possible to avoid loss of moisture. Remove the cover and dry in a hot air oven at atmospheric pressure. Maintain a temperature of 70°C in the case of fruits

or their products, ‘or 100° C in the case of vegetables or their products. The duration of heating will vary with the type of tissues; 16-18 hr are sufficient for most tissues. After drying replace the lid, cool the sample in a desiccator, and reweigh. Reheat the sample, if necessary, until the consecutive: weighings do not.. vary by more than 3-5 mg. Tissues or products which contain volatile organic tonstituents or high percentages of sugars cannot be brought

'

Proximate Constituents

5

to a constant weight. In such cases, a compromise procedure must be adopted. A standard technique should be employed. Drying at 55° C for 4 days is generally suitable. The sample after determination of moisturg content could be used for estimation of ether extractives. If the dried sampl¢ is to be used for ashing and estimation of mincrals, dry in a silica dish-without any filter (asbestos) aid. Drying in Vacuum Oven

Use a dish preferably with a flat-bottom and having a_tight desing cover. @ Prepare the dish and spread the sample as described in the oven method. Predry the liquid products such as juices, beverages, syrups and purees by evaporation on a boiling water bath or in an oven at 55-60°C till samples reach apparent dryness. ‘Tomato products, vegetable purees and comminuted products may be dried to an apparent dtyness in a vacuum oven at 70°C at ‘a pressure not to exceed 450 mm Hg. Place partially dried samples in a vacuum oven maintained at 70°C. The bottom of the dish should be in direct contact with the shelf. The temperature variation from onc part of shelf to another should not be more than +2°C. Admit dry air (by bubbling through H;SO,)

into the oven at the rate of 2-4 bubbles per sec. .Dry the samples at 70°C (oven temperature may become as low as 65° C at the start of drying, but must reach 70° C within 1 hr) at a pressure not more than 100 mm Hg. As the dried sample will absorb moisture on standing, cover quickly, cool in a desiccator and

weigh as soon as possible after the sample reaches the room temperature. If necessary, redry the sample until consecutive weighings made at intervals do not vary by more than 3 mg: Drying for 6 to 7 hr is generally found sufficient. Hine fruit juice powders should, however, be dried for 16 hr. Immiscible

Solvent Distillation

Method

A rapid and fairly accurate method for determining relatively small quantities of moisture is the immiscible solvent distillation method. This method is of special importance, when it is desired to distinguish between water and volatile matter present, as for example, iin spices which contain volatile oils. The method involves the reflux distillation of the food with an immiscible solvent having a higher boiling point and a lower specific gravity than water, e.g. toluene, heptane or xylene. The refluxed water settles as the solvent floats in a graduated tube, in which it can be measured by volume. Though at times low values may be obtained with the distillation method, it has its advantages. It needs little attention once the apparatus has been set up, and secondly, volatile oils which distil over and mix with the solvent are not measured. _ PROCEDURE

Set up the apparatus as shown in Fig. 1.1. Weigh into a 300 ml flask, a sample

6

Analysis of Fruit and. Vegetable Products

which will give 2-5 ml of water.

If bumping is likely to occur, place dry sand

at the bottom of the flask before

adding the sample.

Add sufficient toluene

to cover the sample completely (75 ml or more). Connect the flask to the side arm of the Bidwell-Sterling tube. Pour toluene threugh the condenser, until

the tollecting tube is filléd. Heat the flask to boiling and distil slowly (about

2 drops/sec) until most of the water has distilled over. Then increase the distillation raté to 4 drops/sec until no more is distilled. Wash down the condenset with toluene. If water droplets adhere to the condenser, brush down witha burette brush saturated with toluene. Continue the distillation until ‘no more water distils over and repeat the washing process. Discontinue heating and allow the collecting tube to attain room temperature. Read the volume of water distilled and, assuming its specific gravity to be 1.0, calculate the percentage of water present in the sample.

Fig. 1.1: Apparatus for determination of moisture by immiscible solvent distillation method.

Physical and Chemical

Methods

Infrared Heating Infrared moisture balance is an instrument for measuring the moisture y : * var . content of matetials that do not change their chemical structure while losing

‘water under exposure to infrared radiation. A graduated scale gives a continuous percentage reading of the loss of weight of the sample due to the loss of moisture. Since drying and weighing are simultaneous, this instrument 1s

Proximate Constituents

7.

capecitity useful in measuring the moisture content of substances that quickly teabsorb moisture and for control purposes.

Rapid Physical Methods Methods based on the measurement of nuclear magnetic resonance abedtption of hydrogen nuclei in water are suggested for the determination of water content of potatoes! and apple ‘tissue?. Methods based on the use of electronic devices like Brown-Duvel and the Steinlite instruments? and photoelectric colorimetric methods! are also of interest. Rapid Chemical Methods

Several chemical methods for the determination of moisture contents have been reported. The Karl Fischer method has been _tecommended by the British Standards Institute.6 The methed is based on ‘the nonstoichiometric reaction of water with iodine and sulphur dioxide in pyridine-methanol solution. It is applied for the determination of moisture in dehydrated vegetables.®? ; Another quick method is based on the reaction of water with calcium carbide to produce acetylene and calculating the moisture content, either from loss in weight or from an increase in the pressure so prcduced. The procedure has been applied for the quick determination ,of moisture content in fresh and frozen sweet corn.® A. method based on oxidation with dichromate and by electrometric titration with ferrous sulphate has Been ‘reported for determination of moisture in fresh and frozen fruits and vegetables.10 References

. Shaw, T.M. & R.H. Risken, J. Agr. Food Chen., 4, 162 (1956). . Palmer, K.J.~&

R.H. Eisken, J. Agr. Food Chem., 4, 165 (1956).

. Ceise, E.E., P.G. Homeyer & R.G. Fischer, Food Technol. (Chicago), 5, 250 Rs : Sididappa, G.S. & D.P. Das, Curr. Sci., 23, 157 (1954). . Brit. Standard No. 2511, British Standards Institute (1954). . McComb, E.A. & H.M. Wright, Food Technol. (Chicago), 8, 73 (1954). pw N —AvAY rl . Peters E.D. & J.L. Jungnickel, Anal. Chem., 27, 450 (1955).

. Potter, E.F., Food Ind., 23 (1), 85 (1951). . Tomimatsu, Y. & H.F. Launer, Food Technol. (Chicago), 6, 281 (1952). (Chicago), 9, 453 (1955). ~ ° : Sterling, C., Food Technol. ~! \o_c

(

ASH AND MINERAL MATTER Ash content of a foodstuff represents inorganic residue remaining after destruction of organic matter. It may not necessatily be exactly equivalent to the mineral matter as some changes may occur due to volatilization or some

interaction’ between ‘constituents. High ash content and/pr a low alkalinity ai)

8 — Analysis of Fruit’ and Vegetable Products

of the ash may in some cases be suggestive of the presence of adulterants. The atid-insoluble ash is a measure of sand and other silicious matter present. Difficulty of effecting complete combustion in some samples, and the possible loss by volatilization on ignition may be overcome by moistening the substance to be ignited or the carbonaceous residue therefrom with concentrated sulphuric acid. Total Ash

- Note the tare weight of three silica dishes (7-8 cm dia). Weigh 5-10 g (or more if minerals ate to be estimated, see under Minerals) of: the sample into each. If moist, dry on a water bath. (After determination of moisture con-

tent, the same dishes may be used for ashing (see under Moisture, page 3.) Igriite the dish. and the contents on a Bunsen burner. Ash the material at not more than 525° C for 4 to 6 hr; if need be, ash overnight, in a muffle furnace. Cool

the dishes and weigh. The difference in weights gives the total ash content and is expressed as percentage. PRECAUTIONS

1. If ashing is not complete, break up the ash with a platinum wire, and reignite. 2. Spattering of on occurs when the ash is fluffy. Ones such ash with a petri dish after placing the silica dishes in the desiccator prior to weighing. Water-Soluble

and -Insoluble

Ash

Boil the ash in one of the silica dishes with 25 ml water, filter through an ashless filter paper and thoroughly wash with hot water. The combined filtrates and washing should not exceed 60 ml. Transfer the filter paper to the original dish, ignite, cobl and note the weight. The tare weight deducted from this weight gives water-insoluble ash content. Water-soluble

ash = Total ash — Water-insoluble Acid-Insoluble

To the ash in the second wt/wt), cover with a watch Filter using an ashless filter water. Retutn the paper to soluble ash.

ash

Ash

dish, add 25 ml of dilute hydrochloric acid (10% glass and boil gently over a low flame for 5 min. paper. Wash the filter papet thoroughly with hot the original dish, ignite,.cool and weigh the in-

Alkalinity of the Ash

To the ash in the third dish, add 10 ml of 0.1 N HCI or H,SO,. Dissolve by warming on a water bath, cool, and titrate the excess acid ‘with 0.1 N NaOH using methyl orange indicator. Let the alkali consumed be B ml. Carry out a blank titration using 10 ml of 0.1 N HCl. Let the blank value be A ml. Cal-

Proximate Constituents

9

culate the alkalinity of ash as potassium carbonate (K,CO,). One ml of 0.1 N acid is equal to 0.00691 g of potassium carbonate.

g of Potassium carbonate per gram of ash

(A — B) x 0.00691 Oty hata

Bema

Alkalinity may also be expressed as number of millilitres of 0.1 N acid required to neutralize the ash from 100 g of the sample.

Alkalinity of Water-Soluble Ash Titrate the water-soluble ash with 0.1 N H,SO, ot HCl using methyl orange indicator and calculate the alkalinity. Alkalinity

of Insoluble Ash

Add 10:.ml of 0.1 N HCI to the insoluble ash in the dish, cover with watch

glass. Heat gently to boiling over a low flame, cool and titrate with 0.1 N NaOH using methyl orange indicator. Carry out a blank and calculate alkalinity. Sulphated

Ash

Moisten the ash with a few drops of concentrated sulphuric ignite gently to constant weight to get the sulphated ash.

acid and

TITRABLE ACIDITY

To obtain the samples in suitable liquid forms, the following procedures are employed. Juices: Mix thoroughly by shaking and filter through previously washed and dried muslin cloth. Jellies and syrups: Mixthoroughly. Dissolve a known weight of the sample in water. Heat on steam bath to dissolve, if necessary. Cool and make up to a known volume. Take aliquots for determination. If insoluble material. is present, filter before taking aliquots. Fresh fruits, dried fruits, preserves, jams and marmalades: Pulp the sample in a blender or in mortar, and mix thoroughly. Weigh the pulped material, add water and boil for 1 hr replacing the water lost by evaporation. Cool, transfer to a volumetric flask and make up to volume, Filter, if necessary. PROCEDURE

i

Colourless or slightly

coloured

solutions:

Dilute

an aliquot of the sam-

ple prepared as above with recently boiled distilled water. Titrate with 0.1 N NaOH using a few drops of 1% phenolphthalein solution as indicator. Note the titre value. Calculate the results as per cent anhydrous citric acid or

other acids.

10

Analysis of Fruit and Vegetable Products

Highly coloured solutions: Dilute the sample with distilled water and titrate just below end point with 0.1 N NaOH, using phenolphthalein° indicator. ‘Transfer a measured quantity (2 or 3 ml) of this solution into approximately 20 ml of neutral water in a small beaker. (In this extra solution, colour of fruit juice becomes so pale that the phenolphthalein colour is easily seen). If the test shows that the end point has not been reached, pour extra diluted portions back into the original solution, add more alkali and continue the titration to the end point. By comparing dilutions in a small beaker, diffe-. rences produced. by a few drops of 0.1 N alkali can be easily observed. The coloured solutions can also be titrated by diluting a small volume of sample (e. g. 5 ml of purple grape juice) with a large volume of distilled ‘water (300—400 ml). The colour becomes so pale that the indicator colour change during titration can easily be observed. - CALCULATION

Litre > % Total

acid =

Normality _ Volume of alkali “* made up*”>

Equivalent wt of acid

iia Volume of sample taken | Wt or volume of x 1000 for estimation* sample taken

*This does not apply if the sample is directly taken for estimation as given in juices.

CRUDE FAT OR ETHER EXTRACTIVES’ Ether soluble material in a food is extracted from an oven-dried sample using a Soxhlet extraction apparatus. The ether is evaporated and the residue weighed. The ether extract or crude fat of a food represents, besides the true fat (triglycerides), other materials such as phospholipids, sterols, essential oils, fat-soluble pigments, etc.,

extractable with ether.

Water-soluble materials are

not extracted since the sample has been thoroughly dried prior to extraction with anhydrous ether or petroleum ether. PROCEDURE

Transfer the dried sample remaining. after moisture determination to a thimble and plug the top of the thimble with a wad of fat-free cotton. Drop the thimble into the fat extraction tube of a Soxhlet apparatus. Attach the bottom of the extraction tube to a Soxhlet flask. Pour approximately 75 ml or more of anhydrous ether through the sample in the tube into the flask. Attach

the top of fat extraction tube to the condenser.

Extract the. sample

for

16 hr or longer on a water bath. The water bath should be regulated so that the ether which volatilizes condenses and drops continuously upon the sample without any appreciable loss.

Proximate

Constituents

11

At the.end of the extraction period, remove the thimble from the apparatus and distil off most of the ether by allowing it to collect in the Soxhlet tube. Pour off the ether when the tube is nearly full. Fig. 1.2 shows a sample apparatus for extraction of fat and removal of the solvent. (Save the thimble containing the extracted sample for the crude fibre estimation). When the ether has reached a small: volume, pour it into a small, dry, (previously weighed) beaker through a small funnel containing a plug of cotton. Rinse the flask and filter thoroughly, using several small portions of ether. Evaporate the ether on a steam bath at low heat, preferably under a current of air. Dry at 100° C for 1 hr, cool and weigh. The differende in the ven gives the ether-soluble material present in the sainple.

Fig. 1.2: Soxhlet apparatus (A) for extraction of fat and (B) for removal of solvent. Both units must have ground glass joints of the same size to enable interchangeability. CALCULATION

fe de SBee ftir CHoIcE OF SOLVENT

Wt of ether-soluble material X 100 "Wt

of ‘sample

Extraction of fat from food materials is ordinarily done with anhydrous ethyl ether or petroleum ether (b.p. 35-45° C). Of these two, the. petroleum ether is cheaper and requires no special preparation beyond a possible fractionation to secure material boiling within the desired limits. It has also the advantage that it is not affected by traces of moisture present in the

12.

Analysis of Fruit and Vegetable Products

material to be extracted and does not take up moisture during the ex-. traction. Ethyl ether, though a better solvent for the fat than the petroleum ether, must be specially freed from water and kept so during the determination since moist ether will dissolve sugar and other materials that should not be included in the true ether extract. Reference

,

1. Cohen, E.H.,J. Assoc. Offic. Anal. Chem., 54, 212 (1971)

SUGARS 1. Lane

and Eynon

Method}?

Invert sugar reduces the copper in Fehling’s solution to red, insoluble cuptous oxide. The sugar content in a food sample is estimated by determining the volume of the unknown sugar solution required to completely reduce a measured volume of Fehling’s solution. REAGENTS

1. Fehling’s solution (A): Dissolve 69.28 g of copper sulphate (CuSQ,. 5H,O) in water, dilute to 1,000 ml and, if necessary, filter through No. 4 Whatman paper.

2. Fehling’s solution (B) : Dissolve 346 g of Rochelle salt (potassium sodium tartrate, KNaC,H,O,.4H,O) and 100 g NaQH-in water and make up to 1,000 ml.

3. Methylene blue indicator: Dissolve 1 g of methylene blue in 100 ml of water. 4. 45°% Neutral lead acetate solution: Dissolve 225 g of neutral lead acetate in water and dilute to 500 ml. 5. 22°% Potassium oxalate solution: Dissolve 110 g potassium oxalate (K,C,O,.H,O) in water and dilute to 500 ml. An excess of lead acetate in the sugar solution will result in an error in the titration. Determine the exact amount of potassium oxalate solution necessary to precipitate the lead from the lead acetate solution. To obtain this value, pipette 2-ml aliquots of the lead acetate solution into each of six 50-ml beakers containing 25 ml water. To the beakers, add 1.6, 1.7, 1.8, 1.9, 2.0\and 2.1 ml potassium oxalate solution respectively. Filter each through a 41H Whatman paper and collect the filtrate in a 50-ml conical flask.

To each of the filtrates, add

a few drops of potassium oxalate solution. The correct amount of potassium oxalate required is the smallest amount which, when added to 2 ml of lead acetate solution, gives a negative test for lead in the filtrate. In the presence of lead, the filtrate gives white precipitate with HCl or yellow precipitate -with potassium chromate solution. The equivalent volume should be marked on the bottle and employed when the solution is used in sugar determinations.

Proximate

Constituents

13

6. Standard invert sugar solution :Weigh accurately 9.5 g of AR sucrose into a 1-litre volumetric flask. Add 100 ml water and 5 ml conc HCl. Allow to stand for 3 days at 20-25° C or 7 days at 15°C for inversion to take place, and then make up to mark with water. This solution is stable for several months. Pipette 25 ml of the standard invert solution into a 100-ml volumetric flask and add about 50 ml water. Add a few drops of phenolphthalein indicator and neutralize with 20°, NaOH until the solution turns pink. Acidify with 1. N HCl adding it dropwise until one drop causes the pink colour to disappear. Make up to mark with water (1 ml = 2.5 mg of invert sugar).

STANDARDIZATION

OF THE FEHLING’S SOLUTION

Mix equal quantities of Fehling’s solutions (50 ml of .4 and 50 ml of B). Accurately pipette out 10 ml of the mixed solution into a 250-ml. conical flask. Add 25 to 50 ml of water. Take the standard invert sugar solution prepared by inversion of sucrose in a 50-ml burette. Add to the mixed Fehling’s solution almost the whole of the standard invert sugar solution (18 to 19 ml) required to effect the reduction of all the copper, so that not more than 1 ml will be required later to complete the titration. Heat the flask containing the cold mixture over a hot plate or burner covered with asbestos filled wire gauze. When the liquid begins to boil, keep it in moderate ebullition for 2 min. Without removing from the flame, add 3 drops of methylene blue indicator solution and complete the titration in a further one minute, so that the reaction mix-

ture boils altogether for 3 min without interruption. The end point is indicated by the decolourization of the indicator. Note the volume of the sugar solution required for completely reducing 10 ml of Fehling’s solution. The equivalent volume should be 20.37 + 0.05 ml. Small deviations. from the tabulated factors may arise from variations in the individual procedures or composition of the reagents. If the variation is too wide, adjust the concentration of the Fehling’s solution such that the equivalent volume of neutralized

sugar solution for 10 ml of Fehling’s solution is 20.37 + 0.05 ml. Factor for Fehling’s solution (g of invert

PREPARATION

sugar)

‘Titre x 2.5 1000

OF SAMPLE

a. Fruit juices: Weigh 25 g of filteced (Whatman No. 4) juice and transfer to 250-ml volumetric flask. Add about 100 ml of water and neutralize with 1 N NaOH. Add 2 ml of lead acetate solution. Shake and let it stand for 10 min. Add the necessary amount of potassium oxalate solution to remove the excess of lead, make up to volume with water, and filter.

14

Analysis of Fruit and Vegetable Products

b. Jams, jellies and marmalades: Place 50 g of the blended jam in a 500-ml beaker and add 400 ml of water. Neutralize the solution with 1N NaOH using phenolphthalein indicator. Boil gently for 1 hr with occasional stirring. Add boiling water to maintain the original level. Cool and transfer to a 500-ml volumetric flask. Make up to volume and filter through No. 4 Whatman paper. Pipette a 100-ml aliquot into a 500-ml volumetric flask. Add 2 ml of neutral lead acetate solution and about 200 ml of water. Let it stand for 10 min, then precipitate the excess of lead with potassium oxalate solution. Make

up to mark and filter. c. Preserves and candied peel: Grind the sample in a blender or using a pestle _ and mortar. Proceed as described under (b).

PROCEDURE : REDUCING

SUGARS

Method of titration: The sugar solution should be neutral. The concentration of the sugar solution should be such that the titre value ranges between 15 and 50 ml. For this purpose, adjust the sugar concentration in the solution taken for titration so as to contain 0.1 to 0.3 g of sugar per 100 ml, when 10 ml of mixed Fehling’s solution is used. Initially, titrate by the incremental method. When the correct dilutions are established, perform subsequent titrations by the standard method. The incremental method of titration: Pipette 10 ml of the mixed Fehling’s solution into a 250-ml flask. Add 50 ml water. Fill the burette with the clarified sugar

solution.

Add from

the burette,

sugar solution sufficient

to reduce

almost completely the Fehling’s solution used. Mix and heat to boiling on hot plate or burner covered with a clean asbestos-filled wire gauze. Boil for’ 15 sec. If the colour remains blue (indicating that the Fehling’s solution is not completely reduced), add further 2-3 ml of the sugar solution. Boil the solution for a few seconds after each addition until only a faintest perceptible blue colour remains. Add 3 drops of methylene blue solution and complete the titration by adding the sugar solution dropwise until the indicator is completely decolourized. Record the volume of solution required. The accuracy of the incremental method is increased by attaining the end point as rapidly as possible and by maintaining a total boiling period of 3 min.

Standard method of titration: Pipette 10 ml of mixed Fehling’s solution into each of two 250-ml conical flasks. Fill the 50-ml burette with the solution to be titrated. Run into the flask almost the whole volume of sugar solution required to reduce the Fehling’s solution, so that 0.5 ml to 1.0 ml is required later-to complete the titration. Mix the contents of the flask, heat to boiling and boil moderately for 2 min. Then add 3 drops of the methylene blue solution, taking care not to allow it to touch the side of the flask. Complete the titration within 1 min by adding 2 to 3 drops of sugar solution at 5 to 10 sec

Proximate

Constituents

15

intervals, until the indicator is completely decolourized. At the end point, the boiling liquid assumes the brick-red colour of precipitated cuprous oxide, which it had before the indicator was added. Note the volume of the solution required. Nore: The indicator is so sensitive that the end point can be determined within one

drop of the sugar solution. The complete decolourization of the methylene blue is usually indicated by the whole reaction liquid becoming bright red or orange in colour. In case of doubt, remove frora the flame and hold the flask against a sheet of white paper on the bertch. The liquid will appear bluish if the indicator is not completely decolourized. Do not interrupt the boiling for more than a few seconds as the indicator undergoes back oxidation rapidly when air has free access into the flask.

TOTAL

SUGARS

Pipette 50 ml of the clarified solution into a 250-ml conical flask.

of citric acid and 50 ml of water. inversion of sucrose, then cool.

Add 5 g

Boil gently for 10 min to complete the

Transfer to a 250-ml

volumetric flask and

neutralize with 1 N NaOH using phenolphthalein as indicator. Make up to volume. For inversion at room temperature, transfer 50 ml aliquot of clarified and-

deleaded

solution

to a 250-ml

flask. Add 10 ml of HCl (1 +1) and allow to

stand at room temperature (20°C or above) N,OH solution and make up to volume.

for 24 hr. Neutralize with conc

Talse an aliquot and determine the total sugars as invert sugars. CALCULATION

. “ mg of Invert sugar < Dilution x 100 a. % Reducing sugars = =—-?——___> Titre pH meter. Transfer the contents toa 100-ml volumetric flask and dilute to volume. Pipette 1 ml aliquot of this solution into a 100-ml volumetric flask, and follow the procedure described for the determination, of reducing sugars. Calculate the: amount of sucrose frorn the difference between the reducing sugars content before and after inversion, and multiply by the factor of 0.95. Calibration: To construct the standard curves, take known glucose solution containing 0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14 g of sugar per 100 ml. Take fructose solution similarly. Draw standard curves for glucose and fructose at both 100° and 55° C. At 100° C glucose and fructose have similar rates of oxidation, and the curves are identical. At 55° C, the values for fructose are about eight times

higher than those for glucose of same concentrations. All curves obey the Beer's law, and pass through the origin. CALCULATION

Total reducing sugars: From the standard curve find the & value for the total reducing sugars, and calculate using the formula k= c/a (1) where & = factor for unit absorbence, or slope of curve. ¢ = concentration in gram reducing sugars per 100 ml a= ‘absorbence of solution at that concentration The & values at different sugar concentrations are averaged and designated as K. The total reducing sugar content, 5, of the sample is calculated from the formula: S=

Kx AxD

where S = total reducing sugar concentration of sample (mg/100 ml) K = average slope of curve A = absorbence of sample

D = dilution factor

(2)

Proximate Constituents

21

Fructose

The K¢ and Kg values for fructose and glucoseirespectively, « ents at 55°C, are obtained from the standard curves of these two sugars in the manner described for K. The ratio Kg to Kr or Q is used in the calculation of true glucose and fructose of the sample ‘with the following simultaneous equations. } GtF =S (3) (4) G/Q+F=L where G = % glucose in sample F = % fructose in sample S = % total reducing sugars (sum of G. aind aie L =.% apparent fructose Q = ratio between Ky and K; (i.e. K,/Kp

In a mixture of glucose and fructose, both sugars-present are assumed to be fructose. The apparent fructose L, is calculated from Formula 2, substituting L for S,and K; for K. Solving equations 3 and 4 simultaneously, one obtains Formula 5 for percentage of glucose in the sample. iD

G=(S—L)x (@/(Q— 1)]

(5).

The quotient, Q/(Q-1),is used as a constant in calculations of true glucose value. True fructose value can be obtained by difference. Reference

1. Ting, S.V., J. Agr. Food Chem., 4, 263 (1956).

NITROGEN

Micto-Kjeldahl Method Nitrogen content is estimated by the Kjeldahl method which is based on the determination of the amount of reduced nitrogen (NH, and NH) present

in'the sample. The various nitrogenous compounds are converted into ammonium sulphate by boiling with conc H,SO,. The ammonium sulphate formed is decomposed

with an alkali (NaOH), and the ammonia liberated is absorbed

in excess of neutral boric acid solution and then titrated with standard acid.

APPARATUS

1. Kjeldahl digestion flasks: 250-ml capacity. 2. Distillation apparatus (Fig. 1.3): This is a modification of the ParnasWagner apparatus with the rubber connections eliminated. It should be made of Pyrex glass and built in two compact units joined glass-to-glass with a short tubber tubing B. This will render the apparatus less rigid, and reduce the danger of breakage due to bumping when. water boils in the steam generator A. The whole apparatus may be conveniently clamped on to an iron stand. For the steam generator, use a 1-litre round-bottom flask with a, side arm for.

22 ~—sAnalysis of Fruit and Vegetable Products

Fig. 1.3: Modified Parnas--Wagner distillation apparatus fodmicro-Kjebdahi\ determination of nitrogen. A Steam generator, B rubber tubing.C steam mp

D pinch clamp, E funnel, F condenser, G distilling flask.

refilling. When

it is initially filled two;thirds

with ‘Uistilled water , and

heated, \it will give enough steam for 8 to 12 determination’, REAGENTS

1. Mixed indicator: Prepare 0.1% bromocresol green and 0.1% methyl red indicators in 95% alcohol separately.- Mix 10 ml of the bromocresol. green with 2 ml of the methyl red solution in a bottle pravided-with a dropper which e deliver about 0.05 ml per 4.drops. , 2. 2% Boric acid: Dissolve 10 goof.boric acid (crystals) in i 500 ml of boiling

distitled water. After cooling, transfer the solution

into.a

glass-stoppered

bottle. It keeps indefinitely. ‘3. 0.01 N Hydrochloric acid: Check the. concentration of the final solution against pure sodium carbonate. , 4. 30% Sodium hydroxide: Dissolve 150 g of\sodium hydroxide pellets in 350 ml of distilled water. Store the solution in a.bottle closed with a rubber stopper. 5. Catalysts for digestion: Mix 2.5 g of powdered selenium dioxide (SeO,),. 100 g of potassium sulphate (K,SO,) and 20 8 of ote sulphate (CuSO,.

5H,0).

PROCEDURE

Weigh 4 to 6-g of fruit or 2 to 4 g of vegetable, and transfer to a 250-mi

;

Proxishate Constiguents

23

Kjeldahl flask taking care to sce that no portion of the sample clings to the

neck of the flask. Add 1 to 2 g of catalyst mixture and; (25 nd! of conc H,SO,. Place the flask in an. inclined positton on the: $tand in the digestion chamber and digest. Heat the flask’ gently over a low: flame until the initial frothing ceases and the mixture boils briskly at a moderate rate. During heating, rotate the flask several times. Continue heating for about an hour‘or more until the . colout of the digest is pale blue. Cool the digest and 4dd slowly, 30 to 40 ml: ‘water in 5 ml portions with mixing. Cool and transfer the digest to a 100-ml ° volumetric flask. Rinse the digestion flask 2 or 3 times wih re transfer to the volumetric flask, cool and make to volume with water. Carry outa blank .

digestion without the sample and make the digest to 100 m}. Distillation and titration: Set up the, distillation apparatus ((ooinas ails , available) as shown in Fig. 1.3. Place a flask under’ tHe condenser. Boil the distilled water in the steam generator A using Bunsen burner. Close’ ‘stop~ m cock E and pinch clamp D. Run cold water through the condenser, from_ which about 5 ml of distillate should collect per minute.

whereupon. the condensate

Remove the burner,

in the distilling flask Gis sucked back into the

.steam trap C. Fill funnel E with distilled water, and

open the stopcock mo-

mentarily to drain the water into G, Replace the burner under the steam’ generator for about 20 sec and reméve it again. Pipette’5-ml of 2% boric acid _ and add 4 drops of the mixed indicator into a clegh conical flask. Fill the

micro burette with 0.01 N HCI to the zero mark. By this time, the distilling flask G would’ have become empty. Replace burnet under the steam generator, and open pinch clamp D to remove liquid fron the steam trap C. Leave the

pinch clamp on the glass tubing through which the steam escapes. Replace the beaker under the condenser with the c@nical flask’ containing boric acid, and support the flask in an oblique position, so that the tip of the comic ser is completely immersed in the liquid. Open the stopcock E with one hand and with the other hand, pipette’5.0 ml of the digest into G. Rinse the funnel twice with about 2 to 3 ml portions of distilled water. Thea introduce 10 ml of 30°4 NaOH and close stopcock E. Replace the pinch cock Don the rubber tubing,

whereupon

steain enters G,

stirs up the digestion mixture

and sodium hydroxide, and/liberates ammonia which escapes with steam through the condenser into the boric acid ‘solution. The boric acid changes from bluish purple to bluish green as soon as it comes in contact with ammonia. The change, which is very sharp, takes placé between 20°to 30 sec after the pinch clamp is closed. Three to five minutes after the boric acid has changed colour, lower the conical flask so. that the condenser tip is 1 cm above the liquid. Wash the end of the condenser with a little distilled water. Continue distillation for another minute ‘and then remove the burner. Titrate with standard hydrochloric acid until the blue colour disappears. (If preferred, continue the titration until a faint pink tinge appears and subtract from the burette reading 0.02 ml. There_is no danger of missing the end point, because after appearance of pink tinge the

intensity of pink colour increases considerably

with a trace

more of 0.01

©

24 ~~ Analysis of Fruit and Vegetable

N HCl). The titration may be done in day light or artificial light. By this time, the distilling flask in the apparatus would have become again empty. Wash E with distilled water as described above, and ).

Weigh 20 mg of purified crystalline 0-dianisidine dihydrochloride into a tube, and

dissolve in 2 ml of water and 1 ml of ethanol. Into another tube, weigh 2 mg of horseradish peroxidase, and dissolve in 5 ml of pH 5.5 buffer, then add .0.3 ml of glucose oxidase prepared from Aspergillus niger, Type V, containing 1000 unjts/ml. Transfer the solution in the two tubes to a 100-ml volumetric flask,

make up to volume with pH 5.5 buffer, and store in an amber coloured glassstoppered bottle. Prepare the reagent just before use. 6. Glucose standards: Weigh 100 mg of glucose, dissolve in water, and make up to 1000 ml. Pipette 10, 20, 40 and 60 ml aliquots into 100-ml volumetric flask, and make up to volume to get standard solutions having 10, 20, 40 and 60 yg of glucose per ml respectively. PROCEDURE Render the sample free of sugars by the procedure given in the previous

method, or proceed as follows: A. Take known weight of the sample containing 0.1-0.2 g of starch into a 250-ml centrifuge bottle, add 150 ml of 80% ethanol, cover with a watch glass, and heat on

a boiling water bath for 1 hr. Centrifuge for 10 min and decant the supernatant. Add 50 ml of 80% ethanol, centrifuge again for 10 min, and decant the | supernatant. Note; The alcohol from the supernatant solutions may be evaporated and the residue used for sugar

estimation.

B. If the sample contains much fat and protein, add 150 ml of hot ethanolic solution to the residue, cover with a watch glass, and heat on a boiling water bath

with occasional

stirring for 1 hr. Centrifuge for 10 min, and discard the

supernatant. To the residue, add 50 ml hot 80% ethanol, centrifuge and discard the

supernatant. To the residue, either in A or B, add 30 ml of water, and heat on a boiling water

bath for 1 hr to remove traces of alcohol. Cover the mouth of the centrifuge bottle with an aluminium foil, and continue heating for 2 hr to gelatinize the starch. Remove the*ottle and cool to 40° C. Add 20 ml of 0.2 N sodium acetate and

then 30 ml of 0.2 N acetic acid (pH 4.5), and mix. Add 2.0 ml of freshly prepared amyloglucosidase enzyme reagent, and mix. Add 2 drops of toluene, cover the bottle with aluminium foil, and incubate overnight (16 hr) at 55-58° C. Cool the contents, make up to 100 ml with water in a volumetric flask, mix and allow to

settle. \Dilute 2.0 ml of the clear supernatant to 100 mf with water.

30

Analysis of Fruit and Vegetable. Products

Take glassestoppered test tubes and proceed as follows:

Standard solution, of glucose

Samp:

(ug/ml)

Blank-10 (20

digest

40

60

oe

Water

mM

10—-

—

—

—

—

Glucose standards

ml

—

10

10

10

10

-_-

—

Sample digest non Glucose oxidaseperoxidase-chromogen

—-

—-

—

—

—

10

1.0 ;

2.0

2.0

2.0

20

2.0

20

20

reagent .

8 N.H2SO,

ml

—

Stopper, mix and incubate at 37° C for 1 hr.

ml 5.0 5.0 5.0 5.0 5.0

5.0

5.0

Mix, cool to room temperature, and measure the colour at 526 nm using distilled water to set the instrument to 100% transmission. oF \

\

CALCULATION Correct the absorbence of the standards for the blank value. Draw a calibration curve of concentration of glucose (ug/ml) versus corrected absorbence. ~ Correct the absorbence of the sample for the blank value, find the glucése concentration from the calibration curve, and calculate as follows:

% Starch =

wg ot glucose/mlx 100 x 100 x 100 x 0.9 1.x 2 X wt of sample x 1,0 00,000 cos | Oe ug of glu REEe/mlTEAL weight of sample (g)

SF

References i} McRae, J.C,, Planta (Berlin), 96, 101 (1971).

2; te a D.R. & P. Voogt, Thb Analysis of Nutrients in Foods, Acddemit Press, London, 1978, p.139.

’

CHAPTER 2.

Pectin

Pecrins from different sources have different compositions resulting in their varying characteristics. The beet pectin contains acetyl group which inhibits jelly formation. The pectins of fruits vary in their methoxyl content and in jellying power. Methoxyl content of commercial pectins generally

varies from 8 to 11.0% and can form high sugar (65.0%, and more) gels. Low methoxyl pectins (methoxyl content less than 7.0 of, which cannot form high sugar gels, can form gels with. lower concentrations of sugar, and even Without ‘sugar in the presence of polyvalent cations. While this property has" been made use of with advantage to prepare low calorie jellies, degradation (Of high methoxyl -pectins to low methoxyl pectins by enzymes causes loss of cloud stability and/or gelation of citrus concentrates. The methods necessary to analyse and characterize the pectin have been standardized by Owens ef al. at the Western Regional

Research

Laboratory,

U.S.A.

The

methods described for analyses and characterization of pectin are mainly based on these methods and have been included here with their kind petmission. Reference 1. Owens, H.S., R.M. McCready, A.D. Shepherd, S.H. Schultz, E.L. Pippen, H.A! Swenson, J.C. Miers, R.F. Erlandsen & W.D. Maclay, Methods used at Western Regional Research Laboratory for Extraction and Analysis of Pectic Materials, AYC-340, Western, Regional

Research Laboratory, Albany, California (1952).

EXTRACTION

AND

PURIFICATION

OF

PECTIN |

' Macerate fresh sample in a blender or grind the dried tissue. Weigh 100 g of fresh material after maceration or 10 g of ground dried tissue and transfer to

the tared 1000-ml beaker containing 400 ml water.. Add 1.2 g of freshly ground sodium hexametaphosphate. Adjust the pH to 4.5 and heat with stirring at 90-95° C for 1 hr. Check the pH at intervals of 15 min and ensure that it is 4.5. Adjust pH, if necessary, with citric acid or NaOH. Replace iwater Igst by-evaporation at intervals. Do not add water during the last 20 ‘min of the extraction period. Add-4 g of filter aid and 4 g of ground paper pulp. Check the weight. Filter rapidly through a fast filter paper coated with 3 g of moistened, fast filter aid. Collect not less than.200 ml of the filtrate.

-€ool the filtrate as ‘rapidly as possible and note its weight i in the tared container.

32. ~— Analysis of Fruit and Vegetable Products

If the concentration of pectin is below 0.2% in the filtrate, concentrate to that Jevel under vacuum before precipitation with alcohol. Pour the cooled, weighed, filtrate into 3 volumes of ethanol, iso-propanol, ‘or acetone containing 0.5 M HCl (the pH of the slurry should be between 0.7 and 1.0). Stir for 30 min. Centrifuge, filter, or separate the precipitate on coarse mesh nylon. Wash again at the same pH to remove all but traces of ash. Wash repeatedly in 400-ml portions of 70%, -alcohol or acetone until the precipitate is essentially chloride ion-free or the pH is above 4.0. Dehydrate the precipitate further in 400 ml of acetone. Dry overnight iz vacuo (5 mm of

Hg pressure) with a slow stream of dry air passing through the oven. Weigh the precipitate. Use this pectin for further analysis. Qualitative test for ammonia: The dried pectin should be free from ammonia which otherwise will interfere with many of the proposed analytical methods. Therefore, test the pectin for ammonia as follows: Add 1 ml of 0.1. N NaOH to a small amount of dried sample. On heating, the presence of ammonia can be detected by its odour or, better, by moistened litmus paper. Wash out ammonium ion, if present,. with acidified 60% alcohol, followed by neutral alcohol to remove the acid, and dry.

CHARACTERIZATION OF PECTIN Store the dry pectin samples prepared by the foregoing procedure under dry, cool conditions. Expose the samples to the laboratory atmosphere for 1 or 2 days until they reach an equilibrium moisture level. Then determine the moisture content and apply correction for it in all other analyses and physical measurements. Results of analyses and physical measurements should be expressed on a moisture and ash-free basis.

Commercial pectins are likely to contain reducing sugars which should be removed by washing with 60% alcohol and finally with acetone. Moisture

Weigh 1 g of sample, ground to pass 80-mesh, into a tared metal dish (5 cm in diameter with cover). Dry i# vacuo (5 to 20 mm of Hg) for 4 hr at 100°C. Cool in a desiccator over phosphorus pentoxide. Do not use this sample for subsequent measurement as pectin would have been degraded. If the sample is to be used for other measurements, drying should be done at 70° C for 16 hr. Add 1% to the per cent moisture observed to obtain agreement with the Fischer method. Reference

te

Johnson, C.M., Ind. Eng. Chem., Anal. Edn., 17, 312 (1945).

_

Pectin

«© 33

Ash

Weigh 1 to 2 ¢ of pectic substance ground to pass 80-mesh cible. Ignite slowly, then heat for 3-4 hr at 600° C. Cool the temperature in a desiccator and weigh. To determine the ash, dissolve the ash in 25 ml of 0.1 N HCl. Heat gently to

into a tared crucrucible to rogm — alkalinity of the boiling and cool.

Titrate with 0.1. N NaOH using phenolphthalein as indicator. (The normality of HCl and NaOH used should be the same or else carry out a blank titration using 25 ml of the HCI used.)

CALCULATION sg

Wt of ash X 100 “Wt of pectin

as

;

Titre x Normality of NaOH

Alkalinity We as carbonate —"

Carbonate free ash 94

x 60 X 100

bige aiitcoblaslh bakiO00 VLzoLe,

=

Ash % — Carbonate %

Reference 1.

Owens, H.S. ef al., ibid.

Equivalent Weight Equivalent weight is used for calculating the anhydrouronic acid content and the degree of esterification. It is determined by titration with sodium hydroxide to pH 7.5 using either phénol red or Hinton’s indicator. REAGENTS

1, Ethanol. 2. 0.1 N Standard sodium hydroxide. 3a. Phenol red indicator: Grind 0.1 g of the dry powder in a mortar with 28.2 ml of 0.01 M NaOH. Dilute to 250 ml with distilled water ; or

b. Hinton’s indicator: Mix together 20 ml of 0.4% bromothymol blue, 60 ml of 0.4% phenol red, 20 ml of 0.4% cresol red and 20-ml of distilled water: Use sodium salts of the indicators for preparing the solutions. 4. Carbon dioxide-free distilled water: Boil distilled water for 15 min. Cool to room temperature. Protect from atmospheric carbon dioxide.

PROCEDURE

y-

Weigh 0.5g of pectic substance (ammonia- and ash-free) into a250-ml conical flask. Moisten with 5 ml ethanol. Add 1 g of sodium chloride to sharpen the end point. Add 100 ml of carbon dioxide-free distilled water and 6 drops of

phenol red or Hinton’s indicator. Make sure that all the pectic substance has dissolved and that no lumps are retained on the sides of the flask. Titrate slowlv

Axe) shiv possible deesterification) with 0.1 N’NaOH until the colour of the wae Changes (pH7.5); the colour change should persist for at least 30 sec. iituioa’s indicator gives a magenta end point.

34.

Analysis of Fruit and Vegetable Products

_-The neutralized solution can be used for methoxyl determination.

CALCULATION ivalent Equ valen

- Wt of sample x 1000 weight = —— ———_ __ __ ml of alkal . i x< Norm i __ alit of alkal y __

weigh

Methoxyl Content The methoxyl content or degree of esterification is an important factor in controlling the setting time of pectins, the sensitivity to polyvalent cations, and their usefulness in the preparation of low solid gels, films. and fibres. It is determined by saponification of the pectin and titration of the liberated carboxyl group. REAGENTS

1. 0.25 N and 0.1 N Standard sodium hydroxide. 2. 0.25 N Standard hydrochloric acid. PROCEDURE

To the neutral solution titrated tor equivalent weight, containing 0.5 g of pectic substance, add 25 ml of 0.25 N sodium hydroxide, shake thoroughly; and aliow to stand for 30 min at room temperature in a stoppered flask. Add 25 ml of 0.25 N HCl (or an amount equivalent to the base added) and titrate with 0.1 N’ NaOH to the same end point as before. CALCULATION

Meo

ement

;

oe

ml of Alkali x Normality of alkali x 3.1. Wt of sample

References

1.

Owens, H.S. ¢f al., ibid.

2.

Gee, M., E.A. McComb

& R.M. McCready, Food Res., 23, 72 (1958).

Anhydrouronic Acid Pectin, which is a partly esterified polygalacturonide, contains 10% or more of organic material composed of arabinose, galactose and perhaps sugars. Estimation of anhydrouronic acid content is essential to determine the purity and degree of esterification, and to evaluate the physical properties. ProcepurE

Making use of the equivalent weight, methoxy! content and the alkalinity

of the ash data, calculate the anhydrouronic acid from the expression below.

Anhydrauronic ac! a where

given

in Alkali for’ m.e. Alkali for |m.e. Titrafree acid saponification table ash ) kine Fe Wt of sample (mg) HAM

m.¢. == milli equivalents

(Pectin

35

Reference

1.

Owens, H.S. e¢ al., ibid..

Acetyl Value

Sugar-beet pectin contains acetyl group. Perhaps other pectin may ales contain this group. If acetyl group’ is present in pectin, it inhibits jelly formation. Analysis for this functional group by simple alkalirie saponification procedure followed by back titration does not yield satisfactory results. Modified Clark’s method as applicable to pectic substances isgiven here.’ A colorimetric method based on hydroxamic acid reaction has been described. by McComb and McCready.’ REAGENTS

1. 0.05

N and 0.1 N Standard sodium hydroxide solutions.

' 2, Magnesium sulphate—sulphuric acid solution:

Mix 100 g of magnesium

‘sulphate crystals and 1.5 g of H,SO, and dilute to 180 ml. 3. Phenol red eeleaoe

PROCEDURE

Weigh 0.5 g of pectin into a 250-ml conical flask and add 25 salof60.1 N NaOH. Stopper the flask and stir the contents until the pectin ‘is dissolved. Set aside for at least 1 hr or preferably overnight. Dilute the contents to 50 ml with water. Pipette 20 ml into the distillation apparatus. Add 20 ml of magnesium sulphate-sulphuric acid solution. Steam distil and collect about 100 ml of distillate, keeping the volume in the distillation flask low. Titrate the ace-

tic acid with 0.05 N NaOH to a phenol red end point. Catry out a blank dis- tillation using 20 ml water and 20 ml of the magnesium sulphate—sulphuric acid solution and titrate the distillate as described in the case of the sample.. The titre should be less than 0.1 ml of the standard alkali.

CALCULATION

i 7 Normality of NaOH Xx ml of NaOH x 4.3 Woes = Wt of samiple in aliquot (g) References 1.

Pippen, E.L., R.M. McCready & H.S. Owens, Anal. Chem., 22, 1457 (1950).

2. McComb, E.A.:&

R.M. McCready, Anal. Chem., 29, 819 (1957):

Viscosity’ The contribution that pectin makes to the viscosity of the food products, to the firmness of texture and to jelly formation is due in part to its molecular weight.

The simplest measureof ‘viscosity average molecular weight is the intrinsic

viscosity. This is defined'as the limiting value for the ratio 7,—1/C es C, the.

36 — Analysis of Fruit and Vegetable Products

“concentration, approaches zero; 7, is the viscosity of the

solution

relative

to the solvent. The intrinsic viscosity is indicated as 7 or 7). Pectin contains carboxyl groups, which, when ionized, contribute to the

flow behaviour of pectin solutions,

because

electrostatic repulsion between

them causes lengthening of the chain. This effect can be reduced by addition of either salt or acid. PROCEDURE

Weigh exactly 0.1 g of pectic substance (ash- and moisture-free basis). Dis-

‘solve in 50 ml of water. Stir for 2 hr. Add 0.8 g of sodium chloride and 0.2 g of sodium hexametaphosphate or neutral versene in 15 ml of distilled water and stir for another hour. Adjust the pH, if necessary, with dilute acid or alkali

to6+ 0.2. Rinse the electrodes of the pH meter into the solution, transfer to a 100-ml volumetric flask and make to volume. Stir rapidly and thoroughly for 1 min and stopper the flask. If the solution is cloudy or contains dust or fibres, centrifuge in covered tubes or filter using a coarse sintered-glass funnel. Determine the viscosity of the solution within an hour after the pH adjustment, using an Ostwald-Cannon-Fenske No. 50 pipette with 10 ml of solution at 25 + 0.03° C. Determine the efflux time in the same instrument for the solvent (0.894 sodium chloride and 0.2% sodium hexametaphosphate svlution). Since the density difference between the solution and the solvent is only 4 parts in 10,000, the relative viscosity is essentially equal to the ratio of the time of efflux for solution to that for solvent. Read the intrinsic - viscosity directly from the plot in Fig. 2.1. The plot is based on Martin’s exponential equation,” in which the constant, k’, is assumed to be 0.40. Maximum errors in intrinsic viscosity resulting from deviations in k’ (K.) with citrus or apple pectic substances are not more than 0.06 at 7; = 3.5 and 0.20 at 7; = 7.0. If greater accuracy is required, determine the relative viscosity at three concentrations, such as 0.15, 0.10 and 0.05 g per 100 ml, plot

the ratio (n, — 1)/C against C on a semi-log paper, and extrapolate to zero concentration to get the intrinsic viscosity. Care should be exercised in cleaning the viscometer. When in doubt, use cleaning solution. When determinations are made in series on the same day, rinse thoroughly five times with distilled water followed by a pure 95% ethanol or acetone rinse and dry by suction. - The degree of esterification influences the intrinsic viscosity value which decreases with increasing esterification. The viscosity value can be a useful index of jelly grade or molecular weight. References 1.

Owens,>H. S. ef a/.,

2...

Owens, H.S., H. Lotzkar, T.H. Schultz & (1946).

ibid.

W.D. Maclay, J. Amer. Chem. Soe. 68, 1628

Pectin.

37

pazaGaNabaca Meo EShECg Sale NGS) aSRE

eT Tee a

NUS

Kar ted |

VISCOSITY, INTRINSIC DL.PER G.

| sle Nat

de del UStea oee 8 mata cebLe

PN

Fig. 2.1: Relation of relative viscosity to intrinsic viscosity of pectin solution. (Courtésy: Western Regional Research Laboratory, U.S.A.—R. M. McCready) —

Number Average Molecular Weight

The number average molecular weight is an important factor controlling jellying with pectin. Many investigators recommend converting the pectin into the nitrate, then measuring intrinsic viscosity of the nitrate in acetone solution, and relating that to molecular weight measured by osmotic pressure.!»2 To reduce the chance of degradation and of fractionation, direct measurement ©

is to be preferred. Owens et a/.3 recommend the Bull osmometer*® slightly modified as shown in Fig. 2.2. Instructions for its use are given below. Prepare solutions of pectic substances (ash- and moisture-free basis) at three concentrations preferably below 0.6% in 0.2 M sodium chloride at pH 4.5. Preserve the solutions with 0.001% phenylmercuric nitrate. Introduce 0.5 ml of toluene into the capillary of the osmometer. Fill the chamber of.

section A with the salt solution. Tie a 5-in. section of water-swollen uncoated cellulose sausage casing (wall thickness 20 microns; diameter 12/32, 18/32 or 8/32 in). Trim to proper length and tightly fasten to the lower end of section B with a rubber band or linen thread. Test the casing by immersion in water, and application of air pressure through the top of! section B. Remove excess water, rinse the inside of the casing with pectin-solution, and fill to the desired height, removing all air bubbles. The difference in the levels

38

Analysis of Fruit’ and Vegetable Products

in’ sections’ A and‘B should be about the estimated osmotic pressure of the | peetin solution. It is helpful to mark these two levels on the tubes with - marking pencil in advance. Grease the joints with stopcock grease. Join sections A and B with the stopcock open. Restore the shape of the casing and

—

12 mm. 0.D.

‘E

a; aewm Sa

2

Y

©

o

Stop-cock

7

2 mm. bore

ae =

6S

+

hd

.

8 mm. 0.0.

22 mm.0.D.

a 9.5 mm.-0.D. 4mm. [.D.

8 mm o0.\

21 mmo.

| ;Section A

Section B

Fig. 2.2; Modified Bull osmometer.

(Courtesy: Western Regional Research Laboratory, U.S.A.—R. M. McCready)

remove bubbles, if any, from the region of stopcock by blowing on section 'B. Add salt solution to section A up to the desired level. Remove any bubbles from

toluene column

by

suction,

Retard

evaporation

from

tubes

by

-coveting them with small vials or slotted corks or stoppers. Immerse osmo-meter in water bath at 25° + 0.01° C. After 30 min, read liquid levels in all the three arms by means of a cathetometer or travelling telescope. Close stopcock. Read toluene and pectin solution levels every day until difference is constant. At least 5 days are required with commercially available tubing. (Before reading the toluene level, it should be temporarily raised about 1 mm by gentle suction. If the capillary is clean, the level will return to its undisturbed _ position in a few seconds. Calculate the osmotic pressure from the equation:

;

P = 1.01 (S, — B,) + 0.866 (T, — T,) — (S, — S,) where P =

osmotic pressute in cm of water at 4° C; S, = initial level of

Pectin

| 39

pectin solution; S, = final level of pectin solution; B, = initial level of salt solution; T, = initial level of toluene; T, = final level

f toluene; and 1.01

= density ofthe pectin in sodium chloride solution as Nescribed above. Divide osmotic pressure by pectin concentration expressed as percentage

(g per 100 ml of solution) and plot P/C vs C. The intercept at C = 0 is the limiting value for the ratio P/C and is used in the calculation of molecular weight. If temperature during measurement is maintained at 25° C, the molecular weight is calculated from the equation: Number average molecular weight =

25.3 x 104 (P/ C)e_.o

Significant diffusion or leaking of pectin through the membratie can be detected by testing the salt solution by means of' carbazole (see page 42). The-osmometer must be clean so that capillarity differences will not introduce errors. If aqueous solution menisci do not have the same shape orif the toluene does not return to the same level after being raised repeatedly about 0.2 cm, clean the osmometer with “cleaning solution” before the next determination. Measure in duplicates on each concentration. If (S;—B,) is set a little above the expected osmotic pressure in one determination and below it in another, .a plot of apparent osmotic pressure vs time will increase the, accuracy and. enable determination of thé equilibrium value in a shorter period of time. .

References

2. 3.

1.

Hanglien, F.A., Makromol. Chem., 1, 70 (1947). Speiser, R. & C.R. Eddy, J. Aw. Chem. Soc., 68, 287 (1946). Owens, H.S. ef al., sbid.

4.

Bull, H.B., J. Biol. Chem., 137, 143 (1941).

5.

Bull, H.B. & B.T. Currie, J. Am. Chem. Soc., 68, 742 (1946).

Molecular Weight Weigh pectin sample equivalent to 0.1 gon ash-and moisture-free basis in a 50ml beaker. Wet the sample with a few drops of alcohol, and dissolve in about 50 ml

of 1% sodium hexametaphosphate solution with warming, if necessary. Cool, transfer to a 100-ml volumetric flask, and make up to volume using 1% sodium hexametaphosphate solution. Filter the solution through Whatman 41H filter paper. Take 7 ml of the pectin solution in a No. 100 Ostwald-Cannon-Fenske

viscometer, and place in a water bath maintained at 20°C for equilibration. Determine the viscosity by noting the time required for the solution to flow from

the mark at the neck of the bulb to the bottom mark. Determine the viscosity of the solvent (i.e., 1% sodium hexametaphosphate solution only). Calculate the _ molecular weight using the following formula. —

:

Molecular weight =

(ne!?=1) Xx p CK

40

Analysis of Fruit and Vegetable Products

where

7, = relative viscosity C = concentration in terms of grams of galacturonic acid/100 ml P = any number when the deviation of the intrinsic viscosity is minimum, which has been found to be equal to 6

K = 47 X 10° (constant) EXAMPLE A sample of low methoxyl pectin (LMP) prepared by ammonia-deesterification procedure from lime peel had’ 7.22% moisture and 0.71% ash. In the determination of methoxyl content, using 0.25 g of LMP, the initial titre value was 7.5 mland saponification titre value was 3.1 ml of 0.1 N NaOH, the total being (7.5 + 3.1). = 10.6 ml: Hence, the volume of 0.1 N NaOH required for 1.0 g of LMP is

(1.0/0.25) X 10.6 = 42.4 ml.

1.0 ml of 0.1 N NaOH = 2.3 mg of Na 42.4 ml of 0.1 N NaOH = 97.52 mg of Na

23 mg of Na

= 176 mg of galacturonic acid

97.52 mg of Na = a.

wee 746.24 mg of total galacturonic acid

= 810.4 mg on ash and moisture free basis: To determine relative viscosity, 0.1 g of LMP is to be taken on ash and moisturefree basis. Hence 0.1086 g of sample is equal to 0.1 g of LMP onash and moisturefree basis. This contains 0.08104 g of total galacturonic acid. The flow time for the 0.1% solution of pectin in 1% sodium hexametaphosphate solution was 75 sec, and for the hexametaphosphate solution was 58 see in a No. 100 Ostwald-Cannon-Fenske viscometer. Hence, n; = 75/58 = 1.293. Substituting the values in the formula Molecular weight =

(1.29381) X 6

0.08104 X 4.7 X 105 = 68930

Reference

Smit, CJ.B. & Bryant, E.F., J. Food Sci., 32, 197 (1967).

ESTIMATION 1.

OF PECTIC SUBSTANCES

Pectin as Calcium

Pectate

Pectin extracted from plant material is saponified with alkali and precipitated as calcium pectate from an acid solution by the addition of calcium chloride. The calcium pectate precipitate is washed until free from chloride, dried and weighed. REAGENTS

torn Acetic acid (approxirnate): Dilute 30 ml of glacial acetic acid to 500 | ml with water. 2.1 N Calcium chloride (approximate): Digsolve 27.5 g of anhydrous CaCl, in water and dilute to 500 ml.

Pectin

41

3. 1% Silver nitrate: Dissolve 1 g of AgNO, in 100 ml of water. 4.0.05 N HCl. PROCEDURE

Fresh material: Weigh 50 g of blended sample into a 1000-ml beaker. Extract with 400 ml of 0.05 N HCI for 2 hr at 80-90° C. Replace water lost by evaporation. Cool, transfer the conténts to a 500-ml volumetric flask and ‘make up to mark with water. Shake and filter through No. 4 Whatman paper into a 500-ml conical flask. Repeated extraction of pulped vegetable or fruit in cold water followed by boiling the mixed extract prior to filtration, or boiling. the pulped material with water without any addition of acid has been recommended by some workers. To solubilize the insoluble pectin, acid extraction is considered essential. Alternate procedure of acid extraction : Boil initially with 0.01 N HCl for 30 min. Filter under suction and wash the residue with hot water. To the residue, add 0.05 N HCl, boil for 20 min and filter as before. To the residue,

add 0.3 N HCl, boil for 10 min and filter. Combine the filtrates, cool and make up to volume. Dried pectin extracted and purified as before (see page a1may also be used. Weigh 200 mg of dried pectin into a 1000-ml beaker. Wet with 2 or 3 m! of alcohol. Add 400 ml of water with stirring. Heat to boiling and cool. Transfer to a 500-ml volumetric flask and make up-to volume. Jam, jelly or marmalade. Weigh 50 g into a 1000-ml beaker and prepare a solution with 400 ml of water and by heating on a water bath. While heating, disintegrate the tissue with a glass rod. Coo] and transfer the contents to a 500-ml! volumetric flask. Make up to volume and filter through No. 4 Whatman paper. Pipette 100-200 ml aliquots each into two 1000-ml beakers. Add 250 ml

water. Neutralize the acid with 1 N NaOH using phenolphthalein as indicator. Pipette 10 ml of 1 N NaOH in excess with constant stirring. Allow to stand overnight. Then add 50 ml of 1 WN acetic acid and after 5 min, add 25 ml of 1 N calcium chloxide solution with stirring. After allowing it to stand for 1 hr, boil for 1 or 2 min. Filter through a previously prepared filter paper (wet the filter paper in hot water, dry in oven at 102° C for 2 hr, cool in a desiccator and weigh in a covered dish). Wash the precipitate with watet which is almost boiling, until free from chlorides. Test using silver ni_ trate. Transfer the filter paper containing the calcium pectate to the original ‘weighing dish, dry overnight at 100° C, cool in a desiccator and weigh. CALCULATION

of.Chietasd Becta=

Wt

Ici tate xx 500500 x of calcium pectate x 100 100° ml of Filtrate taken for estimation x Wt of sample

Carre and Haynes! ascribed to the calcium pectate, the empirical formula

42

Analysis of Fruit and Vegetable Products

C,7HygO,¢Ca, while King? gave the formula C,,H,,O,,Ca. The calcium pectate yield of highly purified pectinic acids is usually about 110% of the ‘pectinic acid. According to Deuel ef a/.,3 the amount of calcium in calcium pectate: ‘is equi-| valent to the amount of carboxyl groups. Therefore, the theoretical yield of calcium pectate precipitate from pure galacturonic anhydride is 110.6% of weight taken. References 1.

Carre, M.H. & S. Haynes, Biochem. J.,16, 60 (1922).

2. King, J., Analyst, $0, 371 (1925). 3.

Deuel, H., G. Huber & L. Anyas-Weisz, Helv. Chim. Acta, 33, 563 (1950).

2. Colorimetric Method?”

The colorimetric method is based on the reaction of galacturonic acid, the basic structural

unit of pectin molecule,

with carbazole in the presence

of

H,SO, and measurement of the colour at 525 nm. Depending upon the standard used, the results may be expressed as anhydrogalacturonic acid (AuA), pectic acid, or in terms of a stated pectin or calcium pectate. However, it is desirable to present the results as AuA, since it is the basic structural unit of

pectin. %

REAGENTS

*

1. 959% Ethyl alcohol. 2. 60% Ethyl alcohol: Dilute 500 ml of 95% alcohol with 300 ml of water. _ 3. Ethyl alcohol purified: Reflux one litre of 95% ethyl alcohol with 4 g of zinc dust and 2 ml of conc H,SO, for 15 hr and distil in an all-glass distillation apparatus. Redistil using 4 g of zinc dust and 4 g of potassium hydroxide. 4. 1.0 N and 0.05 N Sodium hydroxide. 5 H,SO,, AR. 6. 0.1% Carbazole reagent: Recrystallize reagent grade carbazole from toluene. Weigh 100 mg of recrystallized carbazole, dissolve and dilute to 100 ml with purified alcohol. PROCEDURE

Weigh 100 mg of pectin, make it into solution and dilute to 100 ml with 0.05 N NaOH solution. Allow to stand for 30 min to deesterify the pectin. Dilute 2 ml of this solution to 100 ml with distilled water. Develop the colour as given below. Sample: Pipette 2.0 ml pectin solution and 1.0 ml of carbazole reagent. A white flocculent precipitate will form. Add 12.0 ml of conc H,SO, with constant agitation. Close the tubes with rubber stopper and allow them to stand for 10 min for the colour to develop.

Pectin

43

Blank: Proceed as in the sample except that in place of 1 ml of carbazole reagent, add 1 ml of purified ethyl alcohol. Exactly 15 min after adding acid,

measure the colour of the sample at 525 nm setting the instrument to 100% transmittance with the blank. Standard curve: Accurately weigh 120.5 mg of galacturonic acid monohydrate (mol wt 212) (vacuum dried for 5 hr at 30° C), and transfer to a 1000-ml volumetric flask. Add 10 ml of 0.05 N NaOH and dilute to volume. Mix thoroughly and allow the solution to stand overnight. One ml of this standard solution will contain 100 yg of AuA. Dilute in separate volumetric flasks, 10, 20, 40,

50, 60 and 80 ml of the standard solution to 100 ml with water. Pipette 2 ml of each of these solutions for colour measurement. Run the working standards in triplicates. Develop and measure the colour as in the case of the sample solution. Plot the absorbence against 1the concentration of anhydro‘galacturonic acid (mol wt 176). CALCULATION

From the standard curve, read ‘the concentration of the anhydrogalacturonic _acid (AuA) corresponding to the reading of the sample and calculate as shown below: e

pg of AuA found in the aliquot x Dilution

x 100

ADA oe ml taken for estimation X Wt of pectin of sample X 1,000,000 In the procedure developed by McComb and McCready,’ 12 ml of conc H,SO, is taken in a test tube and cooled to about 3° C in an ice bath. Two ml of the deesterified pectin solution is added and again cooled to 5° C. ‘The tube is then heated for 10 min in a boiling-water bath, cooled to about 20° C,

1 ml of 0.15% carbazole reagent in ethyl alcohol is added, allowed to stand at room temperature for 25 +5 min and the colour measured at 520 nm. Standards are developed similarly. The anhydrouronic acid content in Carre-Haynes calcium pectate may be ‘calculated by determining the calcium content and assuming all of the calcium to be associated with carboxyl group of the pectate.

AuA% =

% Calcium found x Wt of calcium pectate x 90.5 — 10.25 x. Wt of the original sample

where 90.5 and 10.25 represent AuA% and calcium % respectively calcium pectate, References

1.

Dische,.Z.J.,

2.

Dietz, J.H: and A.H. Rouse, Food Res., 18, 169 (1953).

Biol. Chem.,

167, 189 (1947).

3.

McComb, E.A. and R.M. McCready, Anal. Chem., 23, 1630 (1952).

in pure

44

Analysis of Fruit and Vegetable Products

3. Optical Rotation Method light by pectin solutions is a characteristic property polarized The rotation of pectin when the specific rotation is known." determine to used which can be PROCEDURE

Dissolve 0.5 g of pectin in 75 ml of water, adjust the pH between 4.5 and 7 and make up to 100 ml. Pour the solution into a 2-dm tube and measure the optical rotation in a polarimeter or saccharimeter at 25° C. With a saccharimeter which has a Bates-Jackson scale, the specific rotation is obtained by the equation: Observed rotation x 100 x 0.346

pened

eae

‘Pectin from oranges has a specific rotation of £230 when the methoxyl content is about 10%. Dete~mination of pectin in extracts of orange peel by optical rotation procedure is given below. Filter 100 ml of the solution containing about 0.5% pectin with filter aid, discarding the first 25 ml. Measure the optical rotation of the filtrate in a 1-dm tube. To 25 ml of this solution, add 25 ml of copper sulphate solution. (The copper sulphate solution should contain 9.4 g of the pentahydrate, 27.2 g of sodium acetate trihydrate and 12 ml of glacial acetic acid made to one litre.) Filter the precipitated copper pectinate and measuré the rotation of the filtrate in a 2-dm tube. The difference between this and the rotation of the pectin extract is the net rotation due to the pectin alone and is used in the calculation: Pectin %, =

Net rotation x 0.346 x 100 230

Yields by this method are higher than those by uronide determination. Reference 1.

McCready, R.M., A.D. Shepherd, H1.A. Swenson, R.F. Erlandsen & W.D. Maclay, Chem, 23, 975 (1951).

GRADING

Anal,

OF PECTIN

“Grade” of pectin means the weight of sugar with which one part by weight of pectin will, under suitable conditions, form a satisfactory jelly. This jelly, subjected to the usual finger testing, should have the proper texture, resilience and consistency. The method given bclow has been made use of by

the U.S. Department of Agriculture in pectin purchases.’

Standard 109-grade Pectin and Standard Jelly For determining the grade and setting time, make test jellies with the test

Pectin

45

sample and compare with a jelly made under similar conditions with a standard 100-grade pectin sample. Requirements for making comparison are: 1. 65% Sugar jelly 2. pH 3 + 0.05 3, Time lapse after making jelly should be at least 18 hr for both unknown and standard. Marerrats Usep

1, Sugar (sucrose). 2. Distilled water. 3. Citric acid solution: Dissolve 50 g of citric acid (monohydrate, mol wt 210) in water and dilute to 100 ml. 4, Sodium citrate solution: Dissolve 25 g of sodium citrate in distilled water and dilute to 100 ml. —

5. Vessel for making jelly: Should be made of aluminium, stainless steel or glass and of capacity 850 ml. 6. Ladle or spoon.

7. Laboratory balance, capacity 200 g + 1 g sensitivity. 8. Jelly glasses with covers — 240 ml capacity. 9. Laboratory balance, sensitivity + 1 mg. PROCEDURE

Note the tare weight of empty Legirs vessel and jelly ladlé,or spoon. Add 320 ml of cold distilled water. Weigh 500 g of sugar. If the assumed grade of pectin is 150, weigh 3.33 g of pectin (Table 2-1) and mix the pectin with TABLE 2-1:

Weights of Pectin to be Used in Testing for Different Grades (U.S. Department of Agriculture, 1942)

Grade

fog

Weight, g

# OF WE

50.00

20

25.00

30

16.66

_

{jie 3b

Grade

gages ;

Weight, g

*

ep)

130

3.85

140

3.57

40

15.50

150

3-33

50

10,00

160

Bale

60

8.33

“170

2.94

7o

7.14

180

2.78

80

6.25

190

2.63

90

5-55

200

2.50

100

5-00

210

2.38

110

4-55

220

2.2%

Binal

46

Analysis of Fruit and Vegetable Products

about 5 times its weight of sugar. To the water in the kettle, add 0.5 ml of the citric acid solution and 1 ml of the sodium citrate solution. Then add the pectin-sugar mixture into the water. Stir to ensure dispersion. Place the mixture over a hot fire. Heat rapidly to boiling while stirring constantly to prevent lumping or sticking to the sides of the kettle. Boil for 30 sec, remave from flame, stir until pectin has gone into solution completely and then add the remaining sugar. Heat the solution again to boiling, stir continuously and boil down to a net weight of 770 g. Remove from the fire occasionally to check the weight, as otherwise, too much water may be evaporated. When the correct weight has been reached, remove from the fire and allow the jelly to cool for 30 sec. Skim off the foam. While the jelly is boiling, add 2 ml of citric acid solution and 0.5 ml of so-

dium citrate solution to each of the three jelly glasses. Pour the hot jelly into these jelly glasses and stir for 2 or 3 sec with a glass rod to ensure thorough mixing of the acid, sodium citrate and hot jelly. Note the time itnmediately after pouring hot jelly into the glasses. Allow the jellies to stand’ at 26° C for 18 hr and note the time required for the product to set into a cohe-

rent mass initially, At the end of the 18-ht period, transfer the jellies from the glasses on a flat surface. Compare overall firmrtess and degree of resilience of the test jelly with a standard jelly prepared under similar conditions. Cut a piece of jelly and squeeze between the thumb and forefinger until the jelly breaks. Compare with the standard jelly. Differences up to 5% can be detected in this way by an experienced operator. Jelly ‘strength can also be. measured by the use of instruments described later.

If the jelly does not have_

the strength of a standard one, prepare a jelly by increasing the amount of pectin and compare with the standard jelly. Pectin at canst Based on « Jelly Grade Sunkist Growers, U.S.A., make use of the following expression for calculating the amount of a certain grade of pectin equivalent to a given weight _Of pectin of another grade.

Wtof pectin required =

Wt

Rate

x Grade of pectin being used Grade of pectin to be used of Setting

If the jelly sets in 10 to 25 min, the pectin is considered ‘rapid setting’ and if the time required for the setting of jelly is more than 25 min, it is ‘slow setting. Reference sg

1.

Se

Kertesz, Z.1.,The Pectic Substances,

P» 485.

Interscience Heeianee

CES

Inc., New

York, 1951,

.

|

Pectin.

47

Determination of Jelly Grade by Measuring Relative Viscosity Although sugars, proteins and starches influence the viscosity of fruit juices and extracts to a slight extent, the main influence on the viscosity is due to pectin. The viscosity 9f extract is an indication of the quality and the quantity of pectin present ardxcan be used as an index for the quantity of sugar to be added. Although this method is in practice, it is only useful for approximate and preliminary evaluation. The relative viscosity of pectin extracts or solutions is the ratio of the time of flow of the pectin solution through a given orifice compared to the time of flow of an equal quantity of water at the same temperature. A simple capillary pipette called the jelmeter has been viscosity determinations.

developed by Baker! for

such

APPARATUS

The apparatus consists of an Ostwald pipette cut off at the bottom of the capillary (Fig. 2.3). The pipette consists of a capillary tube about 10 cm long

eS

we ow wm ee owe we meen ee

Fig. 2.3 : Baker’s

jelmeter

(viscosity

pipette).

sealed to a bulb of about 2 ml capacity. The bulb is blown on the end of a glass tube of about 4mm inside diameter. The tube is slightly constricted as it enters the bulb chamber and extends about 5 cm from the bulb. At A, the place of constriction, and at B, about 2 cm below: the bulb on the capillary, calibration marks are etched. The. capillary should be so small that it takes water at room temperature (22-26° C) about 40 sec to’

run between the marks A and. B,

.

48

Analysis of Fruit and Vegetable Products

PROCEDURE

Bring the pectin extract or extracted juice to room temperature. Draw up

through the capillary into the bulb above the constriction marked A. Note the time in sec for the liquid to flow back of its own free will, from mark A to mark B, when the apparatus is held upright. Wash the pipette thoroughly and note the time required for water of same temperature to flow between the same marks.

CALCULATION Relative viscosity

_

at temperature T

Time

in seconds

for the extract to flow through

Time in seconds for the water to flow through

Using this device, Baker and Woodmansee* made viscosity measurements of apple and citrus pectin solutions (0.5 and 1.0%) av pH 2.5 and 26° C. The approximate grade of pectin can be read from the curves given in Fig. 2.4. When the relative viscosity exceeds 20, the solution should be diluted with water for measurement.

SO

75

100

+125 # 150

175

Fig. 2.4.

Approximate grades of apple and citrus pectin on the basis of A 0.5% and B 1.0% solution, determined by measuting relative viscosity (RV).

Jelly grade can also be determined from the intrinsic viscosity by means of the curve shown in Fig. 2.5. Further work will be necessary to establish its reliability, because it represents data from only a few citrus pectins and molecular weight heterogeneity probably has a marked influence on the curve. . sit

Pectin

49

© Slow-set pectins © Rapid-set pectins

RIDGELIMETER JELLY BY: GRADE 1

2

3 & § 6 INTRINSIC VISCOSITY,

7 8 dil perg

9

Fig. 2.5 : Measurement of jelly grade from intrinsic viscosity.

(Courtesy: Western Regional Research Laboratory, U.S.A.—R.M. McCready) References 1.

Baker, G.L., Food Ind., 6, 305

2, 3.

Baker, G.L. & C.W. Woodmansee, Owens, H.S. et al., ibid.

(1934).

MEASUREMENT

Delaware Agr. Expt. St. Bull., 272 (1948).

OF JELLY STRENGTH

The various methods based on different equipment used for measuring jelly: strength can be divided into two broad groups.! The methods under the first group measure the breaking strength of jellies when they rupture after exceeding their elastic limits, while those of the second group measure jelly strength by taking into account the deformation of jellies within their elastic limits. The methods included in group I are: (i) Sucharipa’s jelly disc method,? (ii) Penetrometet method,’ (iii) Pektinometer method,’ and (iv) Delaware jelly strength tester.5 The following methods come under group

II: (i) Bloom’s gelometer,® (ii) Sag method,” (iii) Wageningen sag method,® and (iv) B.A.R. jelly tester® or F.I.R.A, tester. Of these methods, the Pektinometer‘ has been ‘used extensively in Germany,

the B.A.R. or F.I.R.A. tester® in Great Britain, the Wageningen sag method? in the Netherlands, and the Delaware tester and the Sag Method?’ in the U.S.A. and many other-countries. ‘The committee set up by the Institute of Food

50

Analysis of Fruit and Vegetable Products

Technologists, U.S.A., after a study of the available methods, has described

modifications!® to the Sag method.’ It may, therefore, be expected that this method will be increasingly used for grading. high methoxyl pectins and hence is described in detail. The Sucharipa’s jelly disc method? and the _ Pektinometer method*-are also described strength based on elastic limits.

to. demonstrate

measurement of gel

References al e

Christensen,

P.E., Food Res., 19, 163 (1954).

Sucharipa, R., J. Assoc. Offic. Agric. Chem., 7, 57 (1923).

Fellers, C.R. & J.A. Clague, Ind. Eng. Chem., Anal. Edn, 4, 106 (1932). Liiers, H. & K. Lochmiille:, Kolloid-Zschr, 42, 154 (1927). Baker, G.L., Ind. Eng. Chem., 18, 89 (1926).

Bloom, O.T., U.S. Pat., 1,540979 (1925). Cox, R.E. & R.H.. Higby, Food Ind., 16, 441 (1944). Doesburg,

J.J., Voeding, II, 138 (1950).

ica SS ed rt se I Campbell, L.E., J. Soc. Chem. -_

©

Ind., §7, 413 (1938).

IFT Committee on Pectin Standardization, Food Technol. (Chicago), 13, 496 (1959).

Sucharipa’s Jelly Disc Method APPARATUS

The apparatus is shown in Fig. 2.6 and the inset.

Fig. 2.6 : Sucharipa’s jelly tester for measuring

the strength of jellies.

Take a glass tube (4) open at both ends. To one end, cement a metal disc

(B) having a round hole of 1 cm diameter.

Fit the glass tube with a rubber

Pectin

51

lid (‘C” in inset) to close the hole. Smear vaseline on the rubber lid to prevent jelly from’ adhering to it. Pipette 3 to 5 ml of cooked jelly into it and allow to

set. After the jelly has set, insert the rubber lid into the apparatus and bring to the desired temperature. Gradually increase the air pressure through (E) in the chamber (D). When the jelly breaks, note the préssure on the mano-

meter (F). Since the test jellies may differ not only-in the pressure required to break them, but also in their elasticity, the jelly strength may be evaluated by taking a simultaneous reading on manometer (G), just before jelly breaks. The pressure in the lower Spanier indicates the extent of compression ‘caused by ‘pouching’ of the jelly.~ ‘The method is more suitable for testing firin jellies.. A thin jelly slab may be damaged while removing the lid. This: may be overcome by: pouring the jelly " i a container into which a set of parallel circular metal plates, supported by side rods has been inserted: /This gives a number of jelly discs from the same lot. Instead of-air pressure, oil may be poured directly into the chamber and the height of the oil measured. Reference

1.

Sucharipa, R., sbid.

Liiers-Lochmiiller Pektinometer . The

jelly is allowed

to set in a.special container with corrugated sides.

The force required. to pull the disc.embedded in the jelly upward is: measured. The apparatus’ is shown in Fig..2.7,. The moving load is put on one arm of a balance while the disc in the jelly is attached to the other arm. The jeliy container is held in a fixed position on the base of the balance. (B). /

Fig. ‘2.7 : Liiers and Lochmiiller said aietees

$2.

Analysis of Fruit and Vegetable Products

Place disc (A) held by an arm and suspended on a balance in the corrugated

container (C). Pour hot jelly on it. Allow to cool for at least one hour in

running cold water. The disc becomes embedded in the jelly. Apply weights on pan (W) and measure the force needed to break the jelly by the pressure of the disc which is submerged in it. The corrugated side of the vessel ptevents slipping of the jelly in the container. The indicator (I) indicates the extent of compression of the jelly caused by weight (VV). Henglein? suggested a breaking value of 200 g, while Gudjons* tecommended 300 ++ 15 g for’a standard jelly strength. A modification described by Hamer‘, which enables simultaneous observation of the resistance of the

jelly to deformation in addition to measuring the force required to pull the disc upward, is given below.

. Suspend a brass disc, 0.5 to 0.75 inch in diameter, in a beaker containing (the jelly by means of a wite attached to one beam of an analytical balance.

Keep the beaker containing the jelly on an adjustable platform. Connect a calibrated capillary tip to a burette containing mercury. Add mercury to a beaker on the other pan at a constant rate of 16 g per min. As the mercury is added, go on adjusting the height of the platform manually so that the pointer is always at zero. Note the height of the adjustable platform at frequent

intervals until the jelly is broken. _, The gel strength in grams per square:cm is equal to the weight of mercury divided by the area of the disc. Resistance of the jelly to deformation is indicated by the rate of change in the height of the platform. The drawbacks

of the. method are that the jelly may slip in the beaker and the manual operation of the platform may introduce an element of arbitrariness. References

1, Léers, H. & K.Lochmiller, ibid. 2. Henglein, F.A., Z. Lebensm-Untersuch.

3. Gudjons, H., Z. Lebensm-Untersuch.

U. Forsch., 90, 417 (1950).

U. Forsch., 90, 426 (1950).

4. Hamer, W.J., J. Research, Nat. Bur. Standards, 39, 29 (1947).

Cox-Higby Sag Method i The apparatus described by Cox and Higby! and tater by Joseph and Baier? is based on the ridgelimeter principle developed by Lockwood ‘and Hayes, for determining the strength of gelatine and agar jellies. PRINCIPLE

_ The method is based on measurement of. petcentage sag or slump occurring whena test jelly is removed from its supporting container and inverted

upon a glass plate,

Pectin

-53

APPARATUS

Figure 2.8 shows the tester with a jelly in méasuring position and: jelly glasses with and. without sideboards. The glasses have a standard depth of

Fig. 2.8: Exchange Ridgelimeter. Ridgelimeter glasses are shown with

and without the sideboards.

79.4 mm (3.125 in.).The micrometer screw has 32 threads to an inch so that | one revolution moves the point 0.03125 in. or 1% of the supported height *”

of the jelly (and is equal to 1% sag). t an Jelly glasses: The ridgelimeter glasses are Hazel-Atlas No. 85 tumblers which have been ground so that the inside height is exactly 3.125 in. The sidewalls are extended about 0.5 in. (12 mm) higher with removable gummed paper. Scotch drafting tape 0.75 in. wide and 9.5 in. long may also be used to make the sideboards. The strip should cover 0.25 in. of the glass and extend 0.5 in.

above the glass.

4

PREPARATION OF JELLY

The IFT- Committee gn standardization of pectin? has observed that pH of-the test jelly is a critical item and it is not appropriate to test pectins at jelly pH values above 3. In such jellies (use range of pH 3 to 3.4), the jelly strength increased upon standing. ‘Acid in glass’ procedure at pH below 3 would give mote reliable results in one day than at higher pH: values. Test jellies as described earlier may be used for

measuring the gel strength. ‘The

procedure adopted by IFT Committee is given below.

54

—Analysis of Fruit and Vegetable Products

Dissolve 48.8 g of tartaric acid in distilled water and make to 100 ml in a volumetric flask. Calculate the weight of pectin to be used by dividing 650 by the value of an assumed grade for the pectin. Jellies must contain 65% TSS (650 g of TSS in 1000 g of jelly). The ratio of the actual weight of pectin to that

of sugar in the jelly is defined as ‘assumed grade’ for the pectin in the jelly.

Hence weight of pectin to be used is 650/ assumed grade. Example: If the assumed grade is 150, 4.33 g (650/150) of pectin is required. Weigh 646 g of sugar and 4.33 g of pectin. Mix the pectin thoroughly with

about 20-30 g of weighed sugar in a dry beaker. Note the tare weight of a 3quart stainless steel sauce pan and a stirter. Pour 410 ml of distilled water in-

to the pan followéd dy the sugar-pectin mixture.

Stir gently for about 2 min.

Place the sauce pati on a-stove or a gas burner. If an electric heater is used, preheat the heater and heat with stirring till the contents come to a full rolling

boil. Add the remaining sugar and continue heating and stirring until the sugar has dissolved. Continue boiling until the net weight of jellyis 1015 g. If the net weight is less,-add distilled water in slight excess and boil down to the correct weight. The entire heating time should not exceed 5-8 min. After, removing’ from the scale, allow the jelly to stand for 1 min and then slightly tip the-pan on side so that the contents are nearly ready to overflow. Skim off any foam or scum. Remove-the stirrer, place a thermometer in the pan

and stir gently with it, until the temperature is exactly 95°C.

Then pour the

jelly quickly-into three Ridgelimeter glasses, each containing 2 ml of tartaric acid solution. While pouring the jelly into the Ridgelimeter glasses, stir vigorously with a glass rod, Pour the jelly rapidly until the glasses are filled part way up the side board and then pour more slowly so that the glass can be filled completely full to the point of overflowing. The acid solution gets mixed with the jelly when rapid pouring is done. Fifteen min after filling, cover the glasses with regular metal lids which fit snugly over the side boards. Store jellies for 20-24 hr at 25° 3°C MEASUREMENT

At the end of storage period, tear off the side board. Slice off the exposed jelly with a tightly stretched wire which is clean and wet (cheese cutter supplied along with the instrument). While slicing, hold the glass upright and ' turn slowly part way around so that a smooth cut is made. Remove the detached layer and discard.

To detach the jelly, hold the glass slightly tilted at an angle of about 45°, insert the tip of a spatula between the jeltysaméthe glass wall to start!sepatation and slightly rotate the spatula, if necessary. Invert the glass. catéfully just above the square glass plate provided along with the instrument, sd that the jelly slides down. Do not drop the jelly. Start a stop watch as soon as the jelly is put on the glass. Place the jelly and the plate carefully'on the base of the Ridgelimeter so that the jelly is

Pectin

55

centered under the micrometer screw. After exactly 2 min, bring the point of the micrometer screw just into contact with the jelly surface. The lowest line on the vertical scale beyond which the lower edge of the circular micrometer head has passed is the per cent and the number on the micrometer head nearest the vertical scale denotes the tenth of a per cent sag. Read the

sag to the nearest 0.1%. PRECAUTIONS

- 1. When

Ridgelimeter readings on different glasses from same jelly batch

differs more

than 0.6, remake

the

batch.

2. Note the TSS with the help of a refractometer and apply temperature correction. A variation of + 1% from 65% at 20°C can mean an error 3 to 4%. CALIBRATION

CURVE

No definition of standard firmness for test jellies thave been reported. Each manufacturer has his own conception of standard firmness. Cox and Higby! prepared test jellies from five different types of pectin above and

1.204 EXCHANGE RIOGELIMETER CALIBRATION CURVE. for standardization of Pectin

1.15

FACTOR X ASSUMED GRADE = TRUE GRADE

1.10 FIRM JELLIES

1.05 1.00 FACTOR 0.95

TG

2et

0.90

weAK

“i

JELLIES

085

19 Fig. 2.9:

20

2 22 23 2) RIOGELIMETER READING

25 26 27 (% sag of jelly)

28

Relation between ratio ‘true grades/assumcd grades’ (factor) and per cent of sag. (Courtesy: Sunkist Growers)

56

Analysis of Fruit and Vegetable Products

below the specified grade. After the percentage was determined on each glass of jelly, a curve (Fig. 2.9) showing the percentage sag and the ratio true grade/

assumed grade (factor) was drawn. A jelly sag of 23.5% was assumed. as “standard firmness.” From the sag measured, the true grade of the test ‘pectin may be ascertained from Fig. 2.9 or by referring to Table 2-2. . Test jellies which are 20% above or below standard firmness may be graded with accuracy. Assuming that a jelly was made at 200 grade and that it showed 26.0% sag, it will be seen from the Fig. 2.9 that the true grade is 0.9 of the assumed gtade or (200 x 0.9) = 180.

References ‘Cox, R.E.

& R.H. Higby, sbid.

Joseph, G.H. & W.E. Baier, Food Technol. (Chicago), 3, 18 (1949). Lockwood, H.C. & R.S. Hayes, J. Soc. Chem.

Ind., 50, 145T, April 24 (1931).

ars ea LF.T. Committee Report on Pectin Standardization, Food Technol., 13, 496 (1959).

GRADING

OF LOW-METHOXYL

PECTIN

Jelly strength of low methoxyl pectin is influenced not only by their pectin content and pH, but also by the calcium to pectin ratio. The latter is dependent on the manner of preparation of low methoxyl pectin and on their methoxyl content. Hills e¢ a/.1 have carried out grading tests in gels witha sugar content of 35%, and pH values ranging between 2.9 and 3.6 by addition of a constant amount of calcium biphosphate Ca(H,PO,),, sodium citrate and citric acid. Lange ef a/.2 have modified this method. A method described by Joseph for grading low methoxyl pectin is given below. REAGENTS

1. Sodium citrate (Na;C,H;O,.2H,O) solution: 6% in water. 2. Citric acid (CgH,O,.H,O) solution: Dissolve 60 g of citric acid in water and make to 100 ml. 3. Calcium chloride (CaCl,.2H,O) solution: Dissolve 22.05 g of calcium chloride dihydrate in 1 litre of water. PROCEDURE

Measure 425 ml of water into a previously tared 3-quart sauce pan. Add 10 ml of sodium citrate solution and 5 ml of citric acid solution. Mix 6: g of standardized low methoxyl exchange pectin with 30 g of sugar and pour into sauce pan. Heat the mixture to boiling with constant stirring, add 150 g\of sugar, and again bring to boiling. When the mixture is boiling, add 25 ml of

calcium chloride solution and boil the mixture to a final weight of 600 g. Pour the jelly into jelly glasses previously prepared for making jellies for

Pectin

' TABLE 2-2: Exchange Ridgelimeter

57

Factors for the Standardization of Pectin

Multiply ‘Assumed Grade’* by Factor to get True Jelly Grade ———

MWY

_—__

Ridgelimeter geading

20.1

rrr

——— ——

ESESSeSESSeSSSeSFSse

Ridgelimeter

Factor

fading

Factor

1.150 1.146

23.1

T.Of7

20.2

23,2

1.013

20.3

1.142

23.3

1.008

20.4

1.137

23.4

1.004

20.5

1.133

23.5

1.000

20.6

1.128

23.

0.995

20.7

1.124

23teT,

0.991

20.8

1.119

23.8

0.987

20.9:

1.115

23.9

0.982

21.0

I.III

24-0

a

t.106

24.1

0.977 0-973

2162

1.102

24-2

0.969

2163 21.4

1.097 1.093

24.3 24.4

0-964 0.960

21-5

1,088

24.5

0.956

21.6

1,084

24.6

0.952

21.7

1.080

24.7

0.948

21-8

1.075

24.8

0.944

21.9

1,070

24-9

9.940

22-0

1.066

25.0

03936

25et Tag.2

0.932 0.928 0.924 0.920

22.1

TCO

22.2

‘1.058

22.3 2204

1.053 1.048

25 +3 25-4

2205

1.044

25155

0.917

22.6

1,040

25.6

0.913

2237

1.039

25-7

0.969

22.8

1.031

25.8

0-905 |

22.9

1.926

25-9

0.902

23,0

¥.022

26.0

0.898

Oe

‘

Values calctlated byH. H. Holton and G. H. Joseph in January 1951 from calibration curve illustrated in Fig. 2.9.

Ridgelimeter

** Assumed gtade’’ Actual weight of total soluble solids in a particular jelly divided by the weight of pectin used in that jelly.

Ridgelimeter factors must be applied only to the

‘assumed grade’ of the pectin in the particular jelly being examined. (Courtesy :Sunkist Research Department, U.S.A.)

58

Analysis of Fruit and Vegetable Products

sag. measurement (see page 52). Store at 24-26°C for 18-24 hr. and then test. If the pH of the finished jelly is not 30.05, make changes in the quantity of citric acid added to get the required value. The percentage of sag for normal firmness of this type of gel is 20-21% as against 23.5% for high methoxyl pectin jellies. Owens ef a/.4 reported that a pectin prepared by the acid precipitation method, with a uniform

methoxyl

content of 3.2 + 0.3%

and an intrinsic

viscosity of 3.4 + 0.5, will be satisfactory for gel purposes. For films and fibres, the lower limit of viscosity is about 3.5. Higher methoxyl content requires additional calcium ion for gelation, while higher viscosity requires less calcium. By varying calcium ion concentration, pectinic acids outside the mentioned range can be applied for formation of gels, etc. References

1.

Hills, C.H., J.W. White & G.L. Baker, Proc. Inst. Food Technol., 47 (1942).

z. 3.

Lange, D., W. Bock & K. Taufel, Ernabrungsforsch, 6, 65 (1961). Joseph, G.H., Low Methoxyl Pectin, California Fruit Growers’ Exchange, fornia (1947).

4.

Owens,

H.S.

Ontario,

Cali-

ef al., ibid.

SETTING

TIME AND TEMPERATURE METHOXYL PECTIN

OF HIGH

Setting time is the interval of time which elapses between the instant at which all the constituents of a jelly batch (sugar, pectin and acid) are filled into cans or bottles after cooking and the instant at which jellification or gelation of the whole into a coherent mass occurs. Doesburg and Grevers! considered

- the setting time as the time elapsed between the moment of addition of the acid after cooking and the moment when jellying to a coherent mass was observed.

The temperature at the moment of jellying is designated as the set-

ting temperature. The setting temperature of a pectin jelly is a determinate physical property dependent on pH; increase of pH causing a rapid fall of the former. The concentration of soluble solids and pectin, nature of

ing have subsidiary effects.»? The

buffer cations, and heat-

setting temperature does not appear to be

related to jelly strength in any direct way.? The setting time of jellies, containing pectins with a low ash content, increases with a decrease of the degree of esterification of pectins to about 50%, while jellies from pectins with lower degrees of esterification exhibit shorter setting times. Addition of calcium increases the setting temperature. In the manufacture of jams and marmalades, a rapid set pectin is required to hold the fruit, pulp or slices uniformly distributed in the container, thus preventing floating. In jellies, use of rapid set pectins may cause entrapping

of air and hence slow set pectins are preferred. The rapid set pectins avail-

Pectin

59

able commercially begin to form a jelly at about 88°C while the slow set ones start jellying at about 54°C. If the jelly sets in 10 to 25 min, the pectin is considered rapid setting; when the time taken is more than 25 min, it is slow setting. Usually, a high molecular weight pectin with 10.5% or more of methoxyl groups [degree of esterification (DE) = 70%] will be a rapid set pectin; those with 7 to 10% methoxyl content (DE 50 to 70%) will be a slow set one. When the methoxyl content is lower than 7%, the low methoxyl pectin tends to be rapid set and the presence of polyvalent cations drastically decreases its setting time.4 Methods to determine setting time and temperature have-been reported by Olsen ef a/.,5 Joseph and Baier,® Hinton,?

Doesburg

and Grevers,! and

Pilnik.? Determination of Setting Temperature? Place an aluminium pan containing 3 litres of water on a burner and maintain the temperature at 95° C. Place a wire cage containing 6 boiling tubes with stoppers in the water bath. As soon as the jelly is ready, pour immediately into . each boiling tube about 40 ml of jelly, put on the stoppers and replace the — tubes into the cage. Into one of the tubes, insert a thermometer. When all the six tubes have been charged, start cooling the water bath by removing the soutce of heat. Continuously stir while cooling. After a reasonable interval of time, examine the content of a tube. If no jellying has occurred, replace the stopper, allow to cool in air and examine at short intervals for signs of

setting. As soon as this is observed, set it aside for further observation. Repeat the procedure until a tube on removal from bath shows signs of a weak jelly when the tube is tipped up. In deciding when the setting -has begun, do not consider the formation of a slight surface skin, as this might be due to a little evaporation and the resulting concentration at the surface. Proper setting usually starts from the bottom of the tube. Note the time elapsed since pouring, and the temperature shown by the thermometer inserted in one of the tubes. Determination

of Setting Time®

Use a water bath having glass windows on opposite sides and a support for jelly glass placed between the windows so that when the tank is filled to the over-flow outlet with distilled water at 30° C, the jelly glass is surrounded with water almost to the top and is held in place by a clip provided for the purpose. Place a light behind the rear window to obsetve the jelly through the front

window. Pour the jelly into the jelly glass. Use the jelly glasses and the’ procedure described in the Sag method of determining jelly strength (see page 52). Note the time with a stop watch as soon as the first glass is filled. Fill other glasses in the same way. Pay close attention to the first glass which is in the water

bath.

60

Analysis of Fruit and Vegetable Products

Give a slow and easy twist at intervals to the glass in the water bath. Observe the setting through the front of the tank. The setting progresses from the bottom to the top of. the jelly. At first, the particles of jellywill be seen to rotate only in the direction in which the glassis turned. As the jelly begins to set, the jelly particles in the lower part of the glass will rotate first in the direction in which the glass is turned, and then will move back in the opposite direction. When this happens, stop the watch. The interval between the starting and stopping of the watch is the setting time of the jelly, usually referred to as the setting time of the pectin. Rapid setting pectins will gel

in less than 2 min, while slow setting pectins require more than 3 min. References

1. Doesburg, J.J. & G. Grevers, Food Res., 25, 634 (1960). 2. Hinton, C.L., J. Sci. Food Agr., 1, 300 (1950).

3. Harvey, H.G., J. Sci. Food Agr., 1, 307 (1950). 4. Owens, H.S. ef al., ibid. 5. Olsen, A.G., R.F. Stuwer, E.R. Fehlberg & N.M. Beach, Ind. Eng. Chem., 31, 1015 (1939).

6. Joseph, G.H. & W.E. Baier, Food Technol. (Chicago), 3, 18 (1949). 7. Pilnik, W., Fructsaft Ind., 5, 277 (1964).

FRACTIONATION

OF PECTIC

SUBSTANCES

Water-Soluble, Oxalate-Soluble and Acid-Soluble

Pectic Substances!

Water-soluble, oxalate-soluble and acid-soluble pectic substances are of high molecular weight and colloidal in nature as compared to low molecular weight degraded pectic substances which do not contribute to consistency or viscosity.

Water-soluble fraction includes pectin and those colloidal pectinic acids of sufficiently high methyl ester content which are located largely in the liquid phase of a food product. They contribute to consistency and serum viscosity. In addition, they act as colloid stabilizers and play an important role in preventing the settling or flocculation of the dispersed solid phase in products such as tomato and citrus juices. Oxalate-soluble fraction includes pectic acid and those colloidal pectinic acids of sufficiently low methyl ester content to be insoluble in water and form gels or precipitates with polyvalent metal ions. They may occur naturally in small amounts or may be formed in larger amounts through the action of the enzyme, pectin methylesterase (pectase, pectinesterase) on watersoluble pectic substances. Acid-soluble fraction (protopectin) is an ill-defined water-insoluble substance in plant: tissue which on-testricted hydrolysis (viz. warm, dilute acid) yields pectinic acids. Over extended period ‘of storage, slow hydrolysis: of this

Pectin

61

material may cause an increase in the pectin or pectinic acid content of the product. Protopectin may act as a large insoluble water-binder and in this manner may contribute to consistency. PROCEDURE

Extraction: Pipette 25 ml or sufficient: sample to contain 25-100 mg of pectic substances. Add two volumes of 95% ethanol and one or two drops of conc HCI to increase the solubility ofiinorganic salts. Bring to a boil to inactivate any pectic enzymes present, and cool rapidly, If necessary, samples car. be stored for some time in this ethanol mixture. Filter the supernatant liquid using sharkskin or nylon cloth and wash the residue several times with a mixture of two parts of 65% ethanol to one part of water and containing sufficient conc HCl to make the mixture 0.05 N with respect to HCl. The washing removes additional interfering materials. Water-soluble pectic substances: ‘Transfer the residue to a 100-ml centrifuge tube with 40 ml of distilled

water, and let it stand for 2 hr at 30° C

with

frequent mixing. Centrifuge and decant the supernatant liquid to a 100-ml volumetric flask. Make a second extraction of the residue. Decant the supernatant liquid to the same volumetric flask. Dilute to mark with water. ‘This contains water-soluble pectic substances. Oxalate-soluble pectic substances: 'To the residue in the centrifuge tube, add 40 ml of 0.2% ammonium oxalate solution. Make two extractions for 2 hr each at 30° C, similar to the water extraction. Combine the centrifugates and make to 100 ml. This constitutes oxalate-soluble pectic substances. Acid-soluble pectic substances: Extract the residue from the oxalate extraction. with 40 ml of 0.05 N HCl. Extract twice for 2 hr each as described earlier, maintaining a temperature of 80—90° C during the extractions. The resulting combined extracts contain acid-soluble pectic substances. Make up the volume to 100 ml. EsTIMATION

Estimate the pectin content in each of the above extracts by any of the procedures described below: 1. Colorimetric estimation: Pipette aliquots containing 4 to 6 mg of pectin of each of the above extracts to separate 100-ml volumetric flasks. Add 5 ml 1.0 INN NaOH to each, and dilute to volume with water. Mix and let stand for

at least 15 min. Analyse 2.0 ml aliquots of each of the above extracts by the colorimetric procedure for the estimation of pectic substances (see page 40). 2. By precipitation: Transfer pectin extracts mentioned above to 500-ml conical flasks. Precipitate the pectin by addition of two volumes of 95% ethanol containing sufficient conc HCI to make the mixture 0.05 N. Filter off the supernatant ethanol-water mixture and wash the precipitated pectic substances at least three times on the paper 1with 65% ethanol containing 0.05 N HCI. Transfer the precipitate to. a tared flat dish and dry in a vacuum oven

62 — Analysis of Fruit and Vegetable Products

at 75° C for 16 hr. Cool in a desiccator and weigh. The value so obtained will give an approximate indication of the pectin content. Reference

1.

McColloch,

R.J.,

Tomato Products,

Determination of Pectic

Substances and Pectic

Enzymes

Fruit and Vegetable Chemistry Laboratory,

in Citrus and

Pasadena, California,

AIC 337 (1952). Water-Soluble, Hexametaphosphate-Soluble and Alkali-Soluble Pectic Substances in Citrus Juices? Pectic substances ate naturally occurring colloidal stabilizers that give citrus juices a viscosity or consistency. When a citrus juice lacks these colloidal pectic substances, the suspended cellular + :zierial settles rapidly and the juice is clear rather than cloudy. In concen yates, the problems of gelation and olarification are related to demethylation of pectin by the enzyme pectinesterase. The water-soluble pectic substances ate sufficiently high methoxyl pectins. In citrus juices, these high methoxy] -pectins give the juice its body or consistency and serve as colloidal stabilizers for the insoluble suspended solids. Sodium hexametaphosphate-soluble |pectic substances are the low methoxyl pectinates of the polyvalent cations, magnesium and calcium. The sequestering effect of the polyphosphates on calcium and magnesium renders the low methoxyl pectinates soluble. The presence of low methoxyl pectins causes gelation and clarification in the frozen concentrates. Sodium hydroxide-soluble pectic substances include the protopectin, and the calcium and magnesium pectates not removed by the sodium hexametaphosphate extractions. Calcium and magnesium pectates of citrus juices are -insoluble ‘compounds formed as end products of chemical or enzymic demethylation of pectin. Protopectin is the water-insoluble parent pectic substance which yields pectinic acids upon acid hydrolysis in citrus juices. During extended storage, hydrolysis of protopectin might increase the water-soluble pectinic acid content of a citrus juice. Based on the solubility, the pectic substances of citrus juices may be classified into three fractions by progressive extractions with ‘distilled water, 0.4% sodium hexametaphosphate, and 0.05 N NaOH. PROCEDURE

Blend the concentrate in a blender for 3 min. Pipette 5 ml of the concentrate to a tared 50-ml graduated centrifuge tube and weigh accurately. Add 5 ml distilled water and 30 ml of 95% ethyl alcohol. In the case of canned or fresh juice, pipette 20 ml of sample into a 100-ml centrifuge tube and preci- pitate thé pectic material with 60 ml of 95% ethyl alcohol. This results in a 70% alcoholic mixture. Heat the alcoholic mixture for 10 min in a water

Pectin

bath at 85°+2°C,

with

occasional

stirring.

Allow the

63

solution to boil

gently during the last 5 min of the heating period. Centrifuge the tubes at . 2,100 r.p.m. (1000 x gravity) for 10 min. Decant and discard the supernatant alcoholic solution. Add 40 ml of 60% alcohol to the precipitate and mix

thoroughly.

Heat

for 10 min in a water

bath

at 85°C,

centrifuge,

decant and discard the supernatant liquid as before. Add 40 ml of distilled water to the centrifuge tube and stir until the mixture is evenly dispersed. Allow the mixture to stand for 10 min at room temperature, and stir again. Centrifuge as before and decant into a 100-ml volumetric flask. Repeat the water extraction using 40 ml of distilled water, centrifuge and decant into the same volumetric flask. Add 5 ml of 1 N NaOH to the water extract to deesterify the pectins to sodium polygalacturonate. Dilute to volume with water. Let it stand for 30 min and estimate the pectin by the

carbazole-hexuronic acid-sulphuric acid colorimetric procedure (see page 42) To the residue in the centrifuge tube, add 40 ml of 0.4% sodium hexametaphosphate solution, stir and allow the solution to stand for 10 min at room temperature. Centrifuge and decant the supernatant liquid to a 100-ml volumetric flask. Make a second extract of the residue similarly and decant into_ the same volumetric flask. Add 5 ml of 1 N NaOH. Allow it to stand for 30

min and estimate the pectin colorimetrically. Extract the residue remainingin the centrifuge tube with 40 ml ot 0.05N NaOH for 15 min ‘at room temperature. Stir occasionally. Centrifuge as before for 10 min and decant the supernatant liquid into a 100-mt volumetric flask. Dilute to volume with water and estimate the pectin by the colorimetric procedure. Reference

1.

Dietz, JH. & A.H. Rouse, Food Res., 18, 169 (1953).

PECTIC ENZYMES!

_

Pectin methylesterase (pectase or pectinesterase) deesterifies pectin and makes it susceptible to precipitation by calcium or other polyvalent cations. Pectinases degrade pectin to galacturonic acid or low molecular weight polymers of it. Measurement of the activities of these two types of enzymes provides valuable clues to the solution of many problems involving jellying, viscosity changes, syneresis and cloud instability. The methods for their measurement ate given below. Pectin Methylesterase Pectinesterase in most plant tissues is rarely in solution but is adsorbed on the insoluble cellular solids. It is, therefore, necessary to extract the

plant material in 0.25 M salt solution and. maintain a pH of 8 for about 1 hr

64

~~ Analysis of Fruit and Vegetable Products

while the enzyme dissolves. below.?.8

The assay for its activity is carried out as given

REAGENTS

1. 0.25 to 1000 2. 1.5 to 1000

M Sodium chloride: Dissolve 14.63 g of NaCl in water and dilute ml. M Sodium chloride: Dissolve 87.75 g of NaCl in water,and ‘dilute ml.

3. 0.02 N Standard sodium sadeels 4, Hinton’s indicator4; Mix together 20 ml of 0.4% bromothymol blue; 60 ml of 0.4% phenol red; 20 ml of 0.4% cresol red and 20 ml of distilled

water. Use sodium salts of the indicators for preparing the solutions. PROCEDURE

Add2 ml of 1.5 M NaCl to 10 ml of 1% pectin solution. Mix by bubbling in carbon dioxide-free air. (To make the air free of carbon dioxide, pass the air through NaOH solution.)iAdd a few drops of Hinton’s indicator and titrate ©

to pH 7.5 with 0.02 N NaOH. Transfer to a constant temperature water bath maintained at 30° C. When the pectin solution has attained the temperature of the bath, add enzyme and water to adjust the volume to 20 ml. Immediately record the time and volume of alkali required to maintain the pH at the constant value. Adjust the concentration of enzyme so as to require about 1 to 3 ml of 0.02 N alkali in 10 min. Express the results in pectin methylesterase units (PE. U/g, the expression for milliequivalents of ester hydrolysed per minute per gram of enzyme) as given below.

PES Ug

ml of 0.02 N NaOH consumed X 3.1 X 1 min

oS: 12 ee are eee seers en ee le ml of Enzyme preparation x Total time of determination in min

References 1.

Owens, H.S. ef al., ibid.

2. Lineweaver, H. & E.F. Jansen, in Advances in Enzymology, Ed. F.F. Nord, Interscience Publishers, Inc., New York, rx, 267 (1951). 3- MacDonnell, L.R., E.F. Jansen & H. Lineweaver, Arch, Biorhem., 6, 389 (1945). 4. Hinton, C., Fruit Pectins, their Chemical Behaviour and Jellying Properties, Chemical Pubji-. shing Co., New York (1940).

Polygalacturonase (PG)?

Polygalacturonase (sometimes considered synonymous with pectinase) seldom occurs in ‘higher plants, and-then only in small amounts. It is produced by many bacteria and fungi and has,been reported in snails. Little information exists

Pectin

65

on extraction of the enzyme from plants, but its assay can be> conducted on plant extracts or macerates as described below. REAGENTS

“1. 1M Sodium carbonate: Dissolve 106 g of anhydrous Na,CO, iin water and make up to 1000 mi. 2. 0.1 N Iodine. 3. 2 M H,SO, solution: Dilute 196.16 g or 120 ml of conc. H,SO, to 1000 ml with water.

4. 0.05 N Standard sodium thiosulphate. 5. Starch. indicator. PROCEDURE

Add 1 ml of enzyme solution of the proper dilution to 99 ml of 0.5% solution of pectic acid at PH 4 and at 25° C. Remove aliquots of 5 ml and add to 0.9 ml of 1 M Na,CO, in a glass-stoppered flask. Add 5 ml of 0.1 N icdine, sti: thoroughly and after 20 min acidify with 2 ml of 2 M H,SO, and titrate the residual iodine with 0.05°N sodium thiosulphate solution. Prepare a

calibration, curve using galacturonic acid monohydrate to obtain the activity. CALCULATION

One milliequivalent of reduced iodine is equal to 0.513 millimole of aldose liberated. Express the activity as PG.U/ml or as millimoles of reducing groups liberated | ay min per millilitre of enzyme solution. References

:

1. Jansen, E.F. and L.R..MacDonnel, Arch. Biochem., 8, 97(1945)

2. Owens, H.S. ef al., ibid.

CHAPTER

3

Polyphenols PoLyPHENOLs confer on fruits, vegetables and other plant foods qualities ‘both desirable and undesirable and are significantly absent in animal foods. These compounds consist of two groups, ##z. flavonoids and cinnamic acid derivatives. Flavonoids form the broad major group and are characterized by the presence of a C,-C,-C, carbon skeleton (Fig 3.1) consisting of the two aromatic rings linked by an aliphatic three carbon chain. This skeleton is made up of two biogenetically distinct fragments: the C,-C, fragment that contains the B-ring; and the C, fragment forming the A-ring.The carbon atoms by convention are numbered as given in the figure. The classification is made on the basis of the state of oxidation of the aliphatic fragment of the basic skeleton. The hydroxylation pattern of A and B nuclei and the degree of polymerization of the C,-C,-C, unit results in a large number of different polyphenolic compounds. The major flavonoids of plants are the anthocyanins, leucoanthocyanins, flavones, and flavonols—widely distributed classes of plant constituents. Accompanying these major flavonoids in certain plant groups are the minor flavonoids, chalcones, flavanones, etc. The cinnamic acid derivatives are not flavonoids in the strict sense. However, they are related to the flavonoids by virtue of the C,-C, (B-ring) unit of the flavonoid compounds. The structures of some of the ee occurring flavonoid and related compounds are given in Fig 3.1.

The effects of the polyphenols on food quality are summarized below: 1, The cause of undesirable astringency in some fruits (e.g. Aaki) and desirable astringency in ciders and wines. 2. In the development of final colour, taste and aroma, in such products as tea and cocoa, oxidation and condensation products of catechins play the ptincipal role. 3. The formation of troublesome haze and precipitates in apple juice, beer and wine has been attributed to the interaction of proteins and phenolic polymers. 4, The development of brown discolouration due to oxidation of phenolic substances (e.g. chlorogenic acids, catechins, .etc.) by the polyphenolase enzymes. 5. Coupled oxidation due to intermediate quinones from enzyme action on other phenolics and production of deeply coloured substances by coupling with amino and sulphydryl group of amino acids. 6, The formation of dark coloured complexes with iron due to sequester-

ing action of the dihydric and trihydric phenolics, which result in undesirable appearance of canned foods.

Polyphenols

67

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68

Analysisof Fruit and Vegetable Products

7. The development of pink to pinkish brown colour in fruit and vegetable products prepared from pear, guava, apple, banana, litchi, broad beans, field beans, black eye peas, cabbage, cauliflower, water chestnut, etc., has been attributed to leucoanthocyanins. Besides the commonly occurring flavonoid compounds, other phenolics have only restricted distribution, but are of very great interest to quality, e.g. thé flavanones naringin and neohesperidin impart bitter taste to citrus juices, anthocyanins in fruits affect aesthetic appeal, etc. It is, therefore, very necessary

to get a comprehensive idea of the phenolic constituents in plant foods.

' ExTRACTION - The phenolic glycosides, catechins and leucoanthocyanins are generally -soluble in water while flavones and flavonols are sparingly ‘soluble. Solvents generally used for extraction of total phenolics are alcohol and acetone. Anthocyanins are stable as their salts and are extracted by acidic alcohol. The extrac-

tion procedure depends on the sbaebscisistibe of the plant product, e.g. seeds * tich in oils, leaves rich in chlorophyll, and resins and waxes have to be separated by an extraction with light. petroleum before fractional extraction of phenolic constituents are undetthken. A general procedure for extraction of phenolic glycosides, flavans and leu‘coanthocyanins is as follows: Prepare a total extract of the material with boiling alcohol and filter. Concentrate the filtrate in a rotary film evaporator under vacuum. Extract the concentrate with light petroleum to remove chlorophyll and waxy matter,if present. Extract the phenolic concentrate with ethyl ether to’ separate catechins and ¢cinnamic acid depsides (chlorogenic acid, etc.). Dry the ether extract by. adding anhydrous sodium sulphate and evaporate the ether. Extract the residual concentrate with ethyl acetate to obtain flavones, flavonols, leucoanthocyanins and some of the lower polymers. Dry the ethyl acetate extract by adding anhydrous sodium sulphate and concentrate under vacuum. ‘Examine the residual aqueous concentrate for polymeric phenolics. Many uncertainties prevail in the complete extraction of leucoanthocyanins because of their unique behaviour. No solvent or combination of solvents has been found which extracts the leucoanthocyanins completely from the tissue. Smathers and Charley! found that 70% acetone in ethanol removed higher proportions of leucoanthocyanins than did either methanol or 95% ethanol. ANALYSIS

Examine the total alcoholic extract (after removal of chlorophyll and waxy matter), the ether extract, the ethyl acetate extract and the residual concentrate

by two dimensional chromatography on paper.

Chromatographic tank and.

ahcillary equipment for the simultaneous development of a large number of chromatograms

are available commercially. For routine work, a Dent

and

Dutta frame, where multiple papers can be used, and a paper 9410 inch square,

Polyphenols

49

have proved very satisfactory. The most general pair of solvents used are. #butanol-acetic acid-water in the proportion of 4: 1:5 or 4:1: 2.2 in the first “direction and 2% acetic acid in the second direction. For special and detailed separations, a fall sized Whatman paper (or equivalent grade) could be used. _ Ascending as well as descending ‘chromatography may also be made use of - Solvent systems suitable for the separation of various polyphenolic compounds are! given in Table 3-1. TABLE

3-1: Solvent Systems for Paper Chromatography of Polyphenols and ‘Sugars. see

Coppeositiga

Layer

Ratio

n-Butanol : acetic acid : water

43135

tia

v/v

Compound

3

det pted

Ref.

Upper

Polyphenols Sugars

Miscible

Polyphenols

3

a

‘2 d

Acids .

do

;

421:2.2 v/v

n-Butanol : 2 N HCl

131

Upper

Anthocyanins

4

n-Butanol : pyridine : water

6:3:1 v/v

“Upper

Sugars

3

Benzene :acetic acid : water

2¢2:1

Upper

Polyphenols

2

Miscible

Polyphenols

6

« Ethyl acetate : acetic acid : water

viv

v/v

9:22:32 v/v

Sugars

Acetic acid : water : conc HC]

30:10:33

Formic acid :conc HCl : : water

52223 v/v

v/v

n-Butanol : benzene : formic acid: water 100:19:10: 25 v/v

n-Pentanol : acetic acid : water

Fi

i

hg

Miscible

Anthocyanidins

Aglycones

8

Upper

Sugars

9

_)

Anthocyanins

Miscible anit

_ Acylated anthocyanins

Chloroform : acetic acid : water

Seo ket

V/V.

Lower

Ethanol : acetic acid : water

g:2:5

viv

Acetic acid : water

; 15385 v/v

Miscible ) Flavonols ; Flavonol glyMiscible J cosides

2:88 v/v

Miscible

Polyphenols

723

viv

Miscible

Acyl groups

and

Identif. ication

do n-Propanol : conc ammonia

Detection

7.

Miscible

10

;

do

ame

12

-

2 - 13

The polyphenols are identified by their fluorescent properties in the ultraviolet (uv) light, colour reactions with specific spray reagents, Ry values and spectral characteristics. Aftér development, allow the chromatograms to air dry and observe visually and under uv light. Expose the chromatogram to ammonia vapour by taking ammonia in a petri dish and: holding the paper slightly above the

70 . Analysis of Fruit and Vegetable Products

dish of ina chamber saturated with ammonia vapour. Observe visually and again under uv light. Colour reactions of polyphenolic compounds are given in Table 3-2. Some of the fluorescent spots observed under uv light may not be polyphenols. Only those spots which give colour reactions with ferric chloride—ferricyanide reagent!” should be considered as polyphenols. Specific spray reagents useful in identification are given below: pray Reagents and Identification of Polyphenolic Compounds 1. Ferric chloride-potassium ferricyanide:!’ Prepare stock solutions of ferric chloride, FeCls.6H2O; and potassium ferricyanide, KgsFe(CN)g,,containing a drop of potassium permanganate separately. Mix solutions ‘freshly and dilute with water so. that the concentration of each is 0.5%. Dip the chromatogram in the solution. Spots develop almost immediately. Rinse the chromatogram with 0.25 N HCl and wash with distilled water to remove excess reagent. o-, p-Dihydroxy, _o-trihydroxy, and some other easily oxidised phenols react to give a prussian " blue (or pale blue) spot on a white background. 2. Ferric reagent:!8 Dissolve 3 g of AR ammonium ferric sulphate, FeNH,(SO,),.12H,O (iron alum), in 100 ml water and use immediately. The reagent gives sharp and distinct blues and greens with pyrogallol and catechol groups respectively. Mono- and meta-hydroxyphenols, if present on ' chromatograms in excessively high concentrations, give weak purple colourations. 3. Ammoniacal silver nitrate reagent:18 Dissolve 14 g of silver nitrate in 100 ml water and add 6 N ammonium hydroxide until the silver oxide formed just dissolves. Spray the chromatograms and wash three times with chloride-free water (or use distilled water) in a large glass trough. Then dip in 0.1% sodium thiosulphate solution in water for 3-4 min and finally wash for 20 min in “running tap water. Carry out all the washings in the dish and air dry the chromatograms. | This reagent gives brown or black spots with compounds containing either pyrogallol or catechol nuclei. In the cold, the ammoniacal silver nitrate is instantly reduced by vicinal phenolic hydroxy groups, e.g. ortho-dihydroxy and ortho-trihydroxy groups, while mono- and meta-hydroxyphenols reduce the silver nitrate very slowly. Colourations produced depend on the nature of the phenolic compounds present. Examples are given below: Metallic grey—Catechol, protocatechuic acid and some other catechol containing units when present in sufficiently high concentrations. Black—Pyrogallol containing units present in sufficiently high concentrations. Brown—Pyrogallol containing units in low concentrations. 4. Bis-diazotised benzidine:!8 (a) Weigh 5 g of benzidine or 6 g of benzidine hydrochloride, add to 14 ml of cone HCl, stir and dissolve the suspension in 980 ml of water. (b) Prepare a 10% solution of sodium nitrite.

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The pigments ,are extracted from the material using acetone-hexane as solvent, carotenes separated from other pigments on magnesium oxideSupercel adsorption column, and measured at 436 nm.

Plant Pigments

87 -

REAGENTS 1. Acetone: Treat acetone with anhydrous Na,SO,. Filter, add a few pieces of granular (10 mesh) zinc and distil. 2. Hexane, b.p. 60-70°C. 3. Adsorbent: Magnesium oxide and Supercel (1-+ 1). APPARATUS

1. Adsorption tube: To a 17 cm glass tube of 2 cm diameter, constricted at™ ~ one end, attach a narrow (0.8-1.0 cm diameter) glass tube 6-8 cm long. 2. Plunger: A 25 cm long glass rod flattened at one end to fit into thé adsorption tube. ; EXTRACTION

i. Dried Fruigs and Vegetables:

Grind sample to pass No.40 sieve.

Weigh

accurately 1 to 4 g into thimble and place in Soxhlet ‘extractor. Add 30 ml acetone-commercial hexane mixture (3 + 7) to flask, reflux for 1 hr or more at the rate of 1-3 drops per sec till no more colour is extracted, and cool to room temperature. Alternatively, add solvent to the ground material and let it stand overnight in dark at room temperature. Decant or filter the extract into a 100-ml volumetric flask, wash the residue

and make to volume

with hexane. This solution now contains 9% acetone.

ii. Fresh or Processed Fruits and Vegetables:

Grind the sample in a blender.

In case of fresh material, if analysis cannot be made immediately, blanch in boiling water for 5-10 min and store in frozen condition. Extract 5-10 g ina blender for 5 min with 40 ml acetone, 60 ml hexane, and 0.1 g magnesium carbonate. Allow the residue to settle and decant into separating funnel. Wash the residue twice with 25 ml portions of acetone, then with 25 ml hexane, and combine the extracts. Separate and remove the acetone from theextract by repeated washing with water..Transfer the upper layer to-a 100-ml volumetric flask containing 9 ml acetone, and dilute to volume with hexane: If desired, alcohol may be used instead of acetone for extraction. CHROMATOGRAPHIC SEPARATION OF PIGMENTS

Prepare chromatographic column with 1 + 1 mixture of bodvated magnesia and diatomaceous earth (Supercel), as described in the previous method. Place 1 cm layer of anhydrous sodium sulphate above adsorbent. With vacuum _continuously applied to flask, transfer 50 /ml of acetone-hexane extract of pigment ' into column. Keep the top of column covered with a layer of the solvent during

entire operation. Carotenes pass rapidly through the column. Bands of xanthophylls, carotene oxidation products, and chlorophylls remain adsorbed on the column. Collect the eluate. If the colour is too light, conceritrate under reduced pressure and | make up to a known volume with 9% acetone in hexane, Measure the colour at 436 nm, setting the instrument to 100%, transmittance with 9% acetone in

88.

Analysis of Fruit and Vegetable Products

hexane. Note the concentration of the carotene in the sample from the standard curve. STANDARD CURVE -Prepare standard solution of B-carotene as in previous method, but using 9% acetone in hexane for making up the volume. Calibrate the colorimeter f-carotene.

of pure

using known concentrations

Plot

absorbence

against

concentration (€452= 13.9 X 10°). . CALCULATION

,

pg of carotene per ml mg of f-carotene

=

x Dilution x 100

as read from the curve

Pst 100 g

Wt of sample x 1000

Reference 1.

Official Methods of Analysis, Association of Official Analytical Chemists, Washington,

D.C., 11th edn., p. 769 (1970).

LYCOPENE

The red colour‘of tomato is due to the pigment lycopene (CygH,,). Tomatoes contain other carotenoid pigments besides lycopene, but in fully ripe fruit, the latter predominates: Unripe green and yellow fruits, however, contain no lycopene, but mainly chlorophyll and other carotenoid pigments. Therefore, estimation of lycopene. is a good index of the quality of fruit used in the manvufacture of tomato products. Lycopene is also present in pink fleshed guava, papaya, grape fruit (C. grandis), orange (C. aurantium), etc.

PRINCIPLE Lycopene has absorption maxima at 473 nm and 503 nm. The molecular extinction coefficient for all /rans-lycopene at 473 nm is 18.6 x 104 and at 503 nm, 17.2 x 104. A rapid method for the estimation of lycopene in tomato products is based on the measurement of absorption of the petroleum ether extract of the total carotenoids at 503 nm.! The errors involved in the method is very small (2-5%), since the carotene has a comparatively negligible absorbence, while lycopene has a large absorbence at 503 nm. A more accurate method would be to measure the total absorbence at 473 nm and, after chroma-

‘tographic separation, apply correction to the absorbence due to carotene. Method REAGENTS

1. 2.

Acetone Petroleum ether

3. Magnesium oxide A. Supercel

I

Plant Pigments

89

APPARATUS

Chromatographic column and Buchner flask as used for B-carotene, EXTRACTION

Weigh 5—10 g of the juice, puree, or ketchup. Extract repeatedly with acetone in a pestle and mortar or a blender until the residue is colourless. Transfer the acetone extract to a separating funnel containing 10 to 15 ml of petroleum ether and mix gently. Take up the carotenoid pigments into the petroleum ethkr by diluting the acetone (lower phase). with water or water containing 5%, Na,SO,. Transfer the lower phase to another separating funnel and the petroleum ether extract containing the carotenoid pigments to an amber coloured bottle.. Repeat extraction of the acetone phase similarly with petroleum ether until it is. colourless. Discard the acetone phase. To the petroleum ether extract, add a small quantity of anhydrous Na,SO,, transfer to a 50 ml volymetric flask and dilute to mark with petroleum ether. Dilute an aliquot (5 ml, if the test sample is tomato juice or ketchup and 2 ml, if puree) to 50 ml with petroleum ether and measure the colour in a 1 cm cell at 503 nm in a spectrophotometer using petroleum ether as blank. CALCULATION

Calculate the lycopene content of the sample as given below using the relationship that an optical. density (OD) of 1.0 = 3.1206 wg of lycopene per ml.

Bt Bais.

mg of lycopene

per 100 g

we:

GE

sample

Caner

made up

“iigdod CNS

1.x Wt of sample x 1000

To illustrate the calculations involved, the following example is given: The petroleum ether extract from 10 gof tomato juice was made up to 50 ml. _ Ten ml was diluted to 50 ml for colour measurement. The optical density at 503 nm using a 1 cm cell was 0.405. E Mole icm

of lycopene 17.2

at different wavelengths are as given below. Chlorophyll 2

660 nm 642.5 nm 600 nm

102.0 16.3 9.95

Chlorophyll 2

4.5 SiS 9.95

For determining the total chlorophyll, the concentration of chlorophyll (C) is first computed from the equation given below, after measuring the OD at 600 nm.

Gites otek OD ad Now, if V is the volume of solution in litres from which the sample was withdrawn for measurement, then the weight of chlorophyll (W) is given by the expression W = VC, ExTRACTION

Select a representative portion of the sample. Grind the dry sample and mix throughly. Weigh accurately2 to 10 g of the sample and add a small amount of calcium or magnesium carbonate to it. Extract with acetone in a blender or in a pestle ard mortar using purified quartz sand. Decant the supernatant liquid. Repeat extraction with acetone till the residue is colourless. In extracting the dry material and/or in making second and subsequent extractions of the wet sample, use 85% acetone. Filter the extract toa 250- or 500-ml volumetric flask, wash the filter paper and make up to mark with acetone. Take 50 ml of ether in a separating funnel. Pipette 25 to 50 ml of acetone extract into this. Add water from the sides of the separating funnel until the water layer is apparently free of all the fat-soluble pigments. Drain off the water layer. Wash the ether layer 5 to 10 times with 10-ml portions of distilled water or until the ether layer is free of acetone. Transfer the ether extract to a 100-ml volumetric flask, dilute to volume with ether and mix.

Transfer the solution from the volumetric flask to 100-ml amber coloured reagent bottle and add 3 to 5 g of anhydrous Na,SO,. Wait till the solution _ becomes clear. Pipette an aliquot of this solution into another dry bottle and dilute. with ether such that the OD of the colour at 660 nm is between 0.2 and 0.8 and’ preferably near about 0.6.” . MEASUREMENT

Take two clean matched cuvettes and ensure that they give the same reading with ether. Use the same cells daily. In one, take ether as blank, and in the other take the diluted ether extract of chlorophyll. Take readings between

Plant Pigments

93

658-665 nm. Adjust the instrument till the maximum absorption is at 660 nm. Note the readings at 660 nm and 642.5 nm for each unknown solution. CALCULATION

Calculate the total chlorophyll and chlorophyll a and b concentrations by substituting the readings in the following equations. 1. Total chlorophyll, mg/litre = (7.12

x OD

at 660 nm) + (16.8xOD at 642.5 nm) 2. Chlorophyll 2, mg/litre = (9.93 x OD at 660 nm) — (0.777 x OD at 642.5 nm) 3. Chlorophyll 4, mg/litre = (17.6 x OD at 642.5 nm) — (2.81 x OD

at 660 nm) -

References

_ 1. 2.

Comar, C.L. & F.P. Zscheile, Plant Physiol., 17, 198 (1942). Comar, C.L., Ind. Eng. Chem., Anal. Edn., 14, 877 (1942).

Total Chlorophyll

Colorimetric Determination” The method involves the extraction of chlorophyll using acetone and measurement of colour at 660 nm. It also involves the determination of total chlorophyll content in the extract using a spectrophotometer and calibration ‘of the colorimeter by making a series of dilutions of the extract. The concentration determined by spectrophotometer is used for noting the concentration in the dilutions. ‘Fhe concentration: may also be determined using a calibration curve prepared from pure chlorophyll. PROCEDURE

Weigh accurately 4 to 5 g of the sample. Adda small quantity (0.1 to 0.2 g) of calcium or sodium carbonate. Macerate in a mortar (having a pointed spout) with purified quartz sand for a short time. Add 85% acetone, a little at a time and continue grinding. Decant the acetone extract. Repeat extraction with 85% acetone till the residue is colourless. Filter the coloured acetone extract

to a 250-ml yolumetric’flask. Wash the filter paper and dilute to mark using 85% acetone. Dilute further, if necessary, and measure the colour at 660 nm. STANDARD CuRVE

Uncertainties are involved in the use of chlorophyll preparations as standards, for colorimeter. Use of a simple plant extract and spectrophotometric standard for calibration purposes are suggested. __ Weigh accurately 4 to 5 g of fresh leaf. Extract with 85% acetone and dilute the filtered extract to volume as in the case of sample. Let this be the stock solu-

94

Analysis of Fruit and Vegetable Products

tion. Make a series of dilutions of the'stock solution with 85% acetone. Measure the colour at 660 nm. Pipette 25 or 50 ml to a separating funnel and trans-

fer the chlorophyll to ethyl ether. Measure the colour at 660 and 642.5 nm in a spectrophotometer as given earlier (see spectrophotometric method). Calculate the absolute chlorophyll content in the dilutions of the acetone extract. From this, calculate the chlorophyll content in each of the dilutions of the acetone extract. Plot absorbence of each dilution against concentration ona graph paper. From the curve so obtained, read the concentration of chlorophyll in the sample. References

1. Petering, H.G., W. Wolman & R.P. Hibbard., Ind. Eng. Chem., Anal. Edn., 12, 148 (1940; 2.

Comar, C.L., E.J. Benne & E.K. Buteyn., Ind. Eng. Chem., Anal. Edn., 15, 524 (1943).

ANTHOCYANINS -- Colour of grapes, plums, cherries, berries, etc., and their products is due to

the presence of anthocyanin pigments. The total anthocyanin content and the nature of the pigment vary considerably. Four different methods have been described below. In the first method, procedure for extraction, purification,

isolation and the measurement of absorption coefficients of individual pigments are given. Procedure for the estimation of total anthocyanin content is given in the second method. The total, as well as the extent of degradation,may be estimated by the third method. Procedure for separation and estimation of individual anthocyanins are given in the fourth. The last three methods are based on procedures developed by Fuleki and Francis! for cranberries. These methods may be applied to other fruits and vegetables with modifications. Method

I: Extraction, Purification, Separation and Measurement of . Absorption of Anthocyanin Pigments

Procedure developed for extraction, purification and separation of anthocyanin pigments from cranberries, cherries, boysenberries and grapes are given below. These procedures may be used to separate anthocyanins from other plant materials. | Cranberries.” Blend 500g of cranberries with 400 ml of n-butanol and filter after letting the blend stand overnight. Extract the residue twice more, filter and combine the filtrates. Add petroleum ether twice the volume of #-butanol. A lower aqueous phase containing the red pigments separates. Add the red solution to Amberlite CG-50 resin column (see Method IV for preparing resin column). Wash the resin column with minimum volume of distilled water. Elute the pigments from the column with 95% ethyl alcohol and evaporate -to dryness in a rotary flash evaporator. Take up again with 2% conc HCl in

‘Plant Pigments

95

methanol. Evaporate to dryness (if necessary, the final few ml in a vacuum desiccator) and store,the powder in sealed tubes. As an alternative method, blend 500 g of cranberries with

i litre of 1°

HCl in methyl] alcohol and filter. Extract the residue six times and filter. Combine the filtrates and evaporate at 40° C to 1 litre in a rotary evaporator. Add saturated lead acetate (see Method IV), centrifuge, discard the supernatant Hauid and wash the precipitate with 500 ml of ethanol. Dissolve the precipitate in 5° HCl in methanol, filter off the lead chloride precipitate and evaporate the eit. to dryness. Dissolve in 100 ml of methyl alcohol, and precipitate the pigments by adding 500 ml of anhydrous ether. Repeat the process of dissolving in methyl alcohol and precipitation with ether five times. Dry the final powder under vacuum.?:3 Prepare a polyamide (Ultramid EM, Badische Anilin and Soda Fabrik, Germany) column by dissolving 20 g of polyamide in 200 ml of formic acid at 86-90° C, cool to 60° C, add 400 ml of ethyl alcohol and 40 g of Celite filter aid (100—200 mesh). Add two additional 200-ml portions of ethyl alcohol with stirring. Remove the free formic acid by decantation with distilled water. Pack the slurry into a column. Dissolve the pigment powder in the upper phase of a butanol-2 N HC] mixture containing 0-5°, methanol, and add to the column. Develop with butanol-2 N HCl and evaporate the eluate to dryness. Separation of pigments: Dissolve the crude pigment obtained from either of the methods described above (the second method is preferable) in 19% HCl in methanol, add dry cellulose powder (4 g powder for 35-100 mg iagiehe and dry the mixture under vacuum. Prepare silicic acid (100 mesh) by mixing 300 g of dry silicic acid with 193 ml of distilled water. Pack the semi-dry powder

into a 5 X 38 cm column. Place the cellulose powder containing the pigment on the silicic acid. Add a layer of silicic acid above the thin cellulose-pigment layer and develop the column for 2-3 hr with the upper phase of n-butanolacetic acid-water-benzene (4: 1:5: 2) mixture. Extrude the column, section the bands and elute the pigments frorn each section separately with 1% HCl in methyl alcohol. Evaporate the eluates to dryness, dissolve in 2% HCl in methyl alcohol or in 2 N HCl and allow to crystallize in the cold. Wash the crystals with 2 N HCl, dry over phosphorus pentoxide and store in sealed glass tubes. Cherries, Remove the pits from 100 g of thawed (or fresh) cherries and blend for 90 sec at 0° C with 100 ml of 1% HCl in methanol under nitrogen gas. Filter through a Whatman No. 1 filter paper in a Buchner funnel under suction. Extract the residue with the same solvent. Extract the pigments from the pulp adhering to the pits with the same solvent. Combine the extracts from the pits and the pulp, filter, and concentrate to a small volume in a flash eva-

porator. Note’ the volume of the concentrate. To the concentrate, add 5 volumes of distilled water and 2 volumes of z-butanol in a separating funnel and shake. Collect the upper #-butanol layer. Extract the lower phase 8 times with n-butanol. Combine the upper #-butanol extracts. Add 50 ml of diethyl

96

Analysis of Fruit and Vegetable Products

ether to the combined #-butanol extract. A new lower layer containing most of the anthocyanins separates. Extract the lower layer four times, each time with 1 ml of distilled water. Dry the anthocyanins in the water layer in a va' cuum desiccator over phosphorus pentoxide at room temperature. Redissolve the pigments with a small amount of 0.02% HCl in methanol and separate chromatographically in ascending direction on Whatman No. 3 paper at 20° C for 12 hr using acetic acid-conc HCl-water (15 :3: 85 v/v) as solvent. Cut the spots containing the pigments and elute with 0.05% HCl in methanol before the paper is dry. Evaporate the eluates separately to a small volume in a flash evaporator under vacuum. Chromatograph once more with the same solvent. Cut the pigment spots, elute, concentrate and store at 0° C under a nitrogen atmosphere for crystallization. In such cases when the pigment concentration is very small, concentrate the eluate to dryness in a flash evaporator. Dissolve the pigment in a small volume of methyl alcohol containing 0.01% anhydrous HCl and then precipitate with diethyl ether. Wash the precipitate with dry ether, dry and keep in the dark under nitrogen in a vacuum, desiccator. A similar procedure may be used to separate anthocyanin pigments in boysenberries.®

Grapes.’ Mix 200g

of frozen (or fresh) grapes with 600 ml of 0.2% HCI in

methanol. Blend for 5 min in a blender undef nitrogen atmosphere. Filter the mixture through two layers of Whatman No. 1filter paper in a Buchner funnel under suction. Mix the filtrate with approximately 200g of Dowex 50WX4 cation exchange resin (100-200 mesh, Dow Chemical Co., Midland,

Michi-

gan) in the hydrogen form. After 1-2 hr, place the resin in a Buchner funnel containing 2 layers of Whatman No. 1 paper. Wash thoroughly with pure. methanol to remove organic residues and then with distilled water to remove

free sugars. Elute the pigments from the resin by successive extractions with 0.1, 0.5 and 1% methanolic HCl. Combine the extracts and evaporate to dyness in vacuum flash evaporator. Redissolve the residue in 10 ml of 0.01% methanolic HCl. Store the pigment mixture at —26° C (—16" F) in the dark under nitrogen atmosphere. Spot the pigments on Whatman No. 3 paper. Separate the pigments by 2dimensional chromatography, using #-butanol-acetic acid-water (4: 1:5) (equilibrated for 3 days) followed by acetic acid-water-conc HCl (15: 82:3). Run the chromatograms in the descending direction in both of the solvents. Run the chromatogram on.a number of sheets (25 to 30). Apply the mixture to the paper with a micropipette in a spot 0.5 cm in diameter. Air dry the chromatograms, cut like spots from the papers and combine. Elute the pigments 4 times with 0.01% methanolic HCl, transfer with the help of a pipette to a small vial, and allow to dry in a light proof vacuum desiccator. Measurement of absorption coefficients. Weigh the crystals of each pigment which have been dried over phosphorus pentoxide and dissolve in 95% ethanol-0.1 N HCI (85:15) or 95%, ethanol-1.5 N HCl (85:15). Take the absorption spectra and calculate the absorption coeffidtent iin terms of 1% solution in 1 cm cell

7

Plant Pigments

at the wavelength of maximum absorption. Absorption maxima of the anthocyanin pigments and their extinction coefficients reported in the literature have been tabulated by Fulcki and Francis‘ and is reproduced in Table 4-1 TABLE 4-1: Molecular Extinction Coefficients of Anthocyanins and Anthocyanidine ~. Mol.

Pigment*

Wwe

Medium

Absorp-

€ max

tion max. (104) nm

Pelargonidin Pl-3-Gl

324.5 486.5 340.5 ;

0.1% HClin EtOH 1.0% HCl in water

Cy-3-Ga

502.5

0.1% HCl in MeOH o.1 N HCI-EtOH (15:85)

Cy-3-Ar Cy-3-RhGI Cy-3,5-G] Peonidin

472.5 650.5 664.5 354.5

Pn-3-GL Pn-3-Ga

516.5 516.5

Pn-3-Ar Delphinidin

486.5 356.5

Cyanidin

0.1% HCl in EtOH

0.1% HCl in EtOH

0.1 N'HCLEtOH (15:85)

Dp-3-GL Petunidin Pt-3-Gl

518.5 370.5 532.5

Malvidin

400.5

M-3-Gl

562.5

M-3,5-Gl

724.5

M-3-p-coumaroyl-G]

718.5

a.

o.1 INHCI-EtOH (15:85) 1.0% HCl in water 0.1% HCl in EtOH 0.1% HClin EtOH o.1 INHCI-EtOH (15:85) 0.1% HCl inMeOH o.1 N HCI-EtOH (15:85) o.1 N HCI-EtOH (15:85) o.1 N HCI-EtOH (15:85) 0.1% HCl in EtOH PPht: 1% HCl in MeOH PPht 0.1% HCl in MeOH 0.1% HCl in EtOH 0.1% HCl in MeOH PPht 0.1% HCl in MeOH 0.1% HCl in MeOH 0.1% HCl in EtOH 0.1% HCl in EtOH 0.1% HC! in MeOH

504.5

1.78

496.0 510.5

2.73° 2.46

547.0 530.0 535-0

3-47 3-43 4.49

535-0

4.62

538.0

4:44

512.0

535-0

2.824

1.25

511.0

Sayz

532.0

4.08

536.0

1.13

532.0. 532.0

4.84 4.84

532.0

4.61

522.5

3-47

$47.0

3.244

543.0

2.90

549-0

3.464

546.0

1.29

520.0 547.0

3.72 3.16

551.0 546.0

3.714 1.39

538.0

2.95

519.0 545.0 536.0

1.07 1.03 3-02

Pl=pelargonidin, Cy=cyanidin, Pn=peonidin, =delphinidin, Pt=petunidin, Mv=malvidin, Gl= glucoside, Ga=galactoside, eee, Rh=rhamnoside.

b.

Since most of the determinations were carried out in a HCl-containing media,» the mol¢cular weights of the chlorides are given and they include one molecule of water of crystallization. c. The authors expressed doubts in regard to the or of the anthocyanin used in the determination of extinction values. d. The authots used the organic phase of freshly distilled phenol (Wg), toluene (W/2 . ml) and 10% phosphoric acid (W ml) as solvent and the extinction coefficient was determined at $50 om instead of the maximum. (Courtesy: Institute of Food Technologists, Copyright ©)

.

98

Analysis of Fruit and Vegetable Products

Method II:

Estimation of Total Anthocyanins

The procedure involves extraction of the anthocyanins with ethanolic HCl and measurement of colour at the wavelength of maximum absorption. The total anthocyanin content can be calculated by making use of the e max (molecular extinction coefficient) values given in Table 4-1 or by establishing the average extinction coefficient for the pigments present. Procedure for deter‘mination of total anthocyanins in cranberry! is given below. Similar procedure may be used with suitable modifications, if necessary, for other plant materials.

REAGENT Ethanolic HCl: 95% ethanol-1.5

N HCl! (85: 15)

PROCEDURE

Extraction: Blend 100 g of the fruit with 100 ml of ethanolic HCl in a blender at full speed. Transfer to a 500-m1 glass-stoppered bottle using approximately 50 ml of ethanolic HCl for washing the blender jar. Store overnight in a refrigerator at 4° C. Filter on a Whatman No. 1 paper using a Buchner funnel. Wash the bottle and the residue on the filter paper repeatedly with ethanolic HCl until approximately 450 ml of extract is collected. Transfer the extract to a 500-ml volumetric flask and make to volume. To prepare the extract for spectrophotometric measurement, filter about 25 ml through a fine porosity, sintered glass funnel or a polyvinylchloride (Polyvic) millipore filter. Determination: Dilute a small aliquot of the filtrate with ethanolic HCl to yield optical density (OD) measurements within the optimum range of the instrument. Store in dark for 2 hr and measure the colour at the absorption maximum (533 nm in the case of cranberry extract). Establishment of the extinction coeficient:; Extract the pigments from the

plant material, purify, separate into individual pigments chromatographically and crystallize or precipitate with ether by following any one of the procedures given in Method I. Prepare stock solutions of the pigments in ethanolic HCl, dilute aliquots of the stock solution to 25 ml or suitable volume with ethanolic HCl, allow to

stand for 2 hr in the dark and measure the colour in triplicate at the wavelength of maximum absorption. Alternative procedure for determination: Identify the anthocyanins present in the extract by the procedure given for grapes in Method I or by Method IV. From Table ’'4-1, note the extinction coefficient of the pigments present and find the average (see Table 4-2). Extract, as before, a known weight of the plant material with the solvent used for determining extinction coefficient, make

to a known volume, find the absorption, maxima of the extract and note the optical density at the wavelength of maximum absorption. CALCULATION

In ethanolic medium, the peak absorption of cranberry anthocyanins are as follows;

Plant Pigments

Cyanidin glycosides Peonidin glycosides

.. ..

_ 99

535 and 536 nm 532 nm

Values at 535 nm are used in the calculations of total anthocyanin content. The results are given in Table 4-2. TABLE 4-2: Extinction Coefficients for Cranberry Anthocyanins in Alcoholic Media

at 535 nm! 9

aay

:poh

Cyanidin-3-galactoside Cyanidin-3-arabinoside Peonidin-3-galactoside Peonidin-3-arabinoside Average

(x08)

958 1002 987 981 982

4.81 4-73 5-09 4-77 4.85

Following example is given to illustrate the calculation: 100 g of berry was macerated, filtered, washed and made to.500 ml as given under extraction. Two ml of the extract was made to 100 ml with ethanolic. HCl, the absorbence of which was 0.4 at 535 nm.

Volume made up of A ee ten the extracts used for x a Total OD per rc a colour measurement “7° 100 g of berry ml of the extract Gage Bok ‘of sample used taken Total absorbence

_

0.4 x 100°x 500 x 100 _ 10.000

per 100 g of berry The Evalue for 1% 982 (Table 4-2).

x 100

2x

100

hae

se

solution (ic. 10 mg per 1 ml) at 535 nm is equal to

Therefore, the absorbence of a solution containing 1 mg per ml is equal to 98.2.

Total anthocyanin content in 10,000 aig (i mg/100 g of berry

BE.

DBI2a YY -

Method IIE Determination of Total Anthocyanins and Degradation Index Frequently, a simple and direct total anthocyanin: determination cannot be made because of interference due to chlorophyll or its degradation products as in ripening fruits or brown coloured degradation products formed either from browning reactions or from the degradation of anthocyanins. In such cases, indirect methods are used. The methods involve bleaching of colour using sodium sulphite’ or oxidation with hydrogen peroxide® or determination

100

= Analysis of Fruit and Vegetable Products

of absorption ratio (i.e. colour at the region of maximum absorption/colour at the region of brownish degradation products, i.e. 400 to 440 nm). The method developed by Fuleki and Francis! in cranberry juice involves measuring of absorbence in samples diluted with pH 1.0 and 4.5 buffers and calculating the degradation index. The calculation is based on the principle that the measuremesit ‘obtained at pH 1 will include the absorption due to the degraded ‘ as well as the nondegraded anthocyanins, while the difference in absorption between pH 1 and 4.5 media will be due only to the nondegraded products. REAGENTS

1. pH 1 buffers 0.2 N KCI-0.2 N HCl (25:67) 2. pH 4.5 buffer: 1 N sodium acetate-1 N HCl-water

(100:60:90)

.

3. 0.1 N HCl PROCEDURE

Procedure developed by Fuleki and Francis! for cranberry juice is given below. It can be used for other juices and wines without difficulty. Note the absorption maximum of the juice. Dilute 200 ml of cranberry juice to 1 litre with 0.1 N HCl. Dilute 25 ml of this to 50 ml with water. Allow to

equilibrate in the dark for 1 hr. Find the absorption maximum (510 nm in the case of cranberry juice) using a spectrophotometer. The pH digerential method for the determination of total unthocyanins. Dilute 10 ml aliquots of juice to 250 ml and 50 ml with the pH 1.0 and 4.5 buffers respectively. Allow the diluted samples to equilibrate in the dark at room temperature for 2 hr. Note the absorbence at 510 nm, using distilled water blank. The pH values selected for the pH differential method should satisfy the following.

1. The difference in absorbence should be the greatest possible, between the two pH values at which the measurements are made. 2. Small variations in pH around the values selected for measurements should cause only slight changes in absorbence. 3. The anthocyanins should be stable at the PH values at which the measurements are made.

Establishment of Extinction Coegicient In the determination of total anthocyanins, alcoholic HCl is used as medium for measurement. In the presentmethod aqueous medium is used. This results in a hypsochromic shift of 19-24 nm at pH 1.0 in the visible region. It is, therefore, essential to establish the extinction coefficients in the aqueous medium. The anthocyanins present in cranberry juice are given in Table 4-3. Establish the extinction coefficient similarly for the anthocyanins present in the test

material.

Plant Pigments

101

TABLE 4-3: Extinction Coefficients for Cranberry Anthocyanins:in Aqueous Medium" Buffer pH r.0

0% Pigment

0

"

the

Buffer pH 4.5

to

pt%

pe

Absorp-

“1 cm

Absorp-

I cm

tion max

ats1o

tionmax

at 510

nm

nm

Cyanidin-3 -galactoside

512

851

517

(86 ©

Cyanidin-3-arabinoside

516

895

521

122

Peonidin-3-galactoside ”

512

886

517

88

Peonidin-3-arabinoside

512

858 ~~.

512

95

Average

873

am

:

item ,at 510

-

nm . 765 773 |

=.

-798

. 763

~- 98

775,

Absorbence of solution containing 1 mg/ml

87.3

9.8 ~

77.5

CALCULATION

In juices with no degraded pigment, total anthocyanins may be determined by measuring at PH 1.0 or by following Method I.,Measure the absorbence — at 510 nm on a sample diluted with pH 1.0 buffer. Calculate the'total absorbence per 100 g of juice as given in Method I and then calculate the total anthocyanin content as follows : Total anthocyanin

_

Total absorbence

in mg/100 ml»

Average absorbence 1 mg/ml (OD of ,87.3)

In cases where degradation products are gig. cinuse: the pH method and calculate as follows: 1. Calculate the total absorbence per 100 ml of the gilice at pH \ 2. Find the difference in total absorbence (AA) between the 1.0 and 4.5 using the expression

‘differential

1 and 4,%; juite at pH cies }

AA per 100 ml

_—

Total absorbence

oO fume

at PH AO. a:1,

Total absorbence at,

pH 4.5.

3. Calculate the total anthocyanin content in mg/100 ml ofsaied using the expression — Total anthocyanin

inmg/1U00ml

—

AA per 100 ml of dps

77.5

.

4. Calculate the degradation index using the expression 7

Degradation index

J

Total anthocyanin in mg/100 fl at pH 1.0

"1 - Total| anthocyanin by the pH diffeténtial method

_

102

~ Analysis‘ of: Fruit and Vegetable Products

Method IV: Purification, Chromatographic Separation and Estimation of Individual Anthocyanins Separation of’ individual anthocyanins is necessary prior to quantitative determination of each anthocyanin. With fresh or frozen fruits, the crude pigment may be separated without any ‘purification. However, with sweetened products, preliminary purification is essential to remove or reduce the sugar content and concentrate the anthocyanins present. The anthocyanins may be

purified using basic lead acetate or Amberlite CG-50 ion exchange resin. The pigments may be separated by multiple ascending chromatography on Whatman No. 1 paper with #-butanol-benzene-formic acid-water (100:19: 10 : 25) and densitometric measurement of individual bands. The method as developed by Fuleki and Francis!1-12 for sepatating and estimating ir dividual cranberry anthocyanins is given below. Similar procedure may be used for separating anthocyanins from other materials. PROCEDURE

For precipitation of cranberry juice with basic lead acetate [basic lead subacetate Pb(C,H,O,)..2Pb(OH),], prepare a saturated solution of basic lead acetate (pH 8.4). To the juice, add sufficient amount of lead acetate solution drop-wise with vigorous stirring to precipitate the anthocyanins. The juice turns to a green slurry when the reaction is complete. Adjust the pH to 7.0-7.1 with drop: wise addition of approximately 0.5 N NaOH and vigorous stirring of the slurry with a magnetic stirrer. The precipitate may stick to the electrode and poison it. Wash electrodes after every measurement with 2 N HCI followed by generous rinsing with distilled water. Store the electrodes overnight in dil HCI solution to dissolve any adsorbed lead salt. Filter the precipitate with suction on a medium porosity fritted glass funnel using a thin layer of filter aid. Check the completeness of precipitation by adding a few drops of approximately 2 N HCl to the bottom of the beaker in which the filtrate is collected before starting filtration. The appearance of a pink colour indicates the presence

of anthocyanins in the filtrate. Transfer the precipitate to a centrifuge tube. Add 5% HCl in methanol, stir and centrifuge. Collect the supernatant liquid and make up to a known volume. Any degraded

anthocyanins present would

be further degraded when alkali is added. Jon exchange resin

column chromatography.

Hydrate

the Amberlite

CG-50,

a weak acidic cation exchange ‘resin of 100-200 mesh (other resins which have been tried aré IR-4B of Rohm and Hass Co., Rexyn 102 of Fisher Scientific Co., AG-50 of Biorad Lab., Zeo-Karb, Permutit Q and H-70 of Permutit Co.) by soaking the resin in water and occasionally decanting the silky _ Supernatant liquid to remove the undersize particles. Make use of an all-glass chromatographic column (300 x 10 mm) fitted with fritted’ glass filter at’

one end. To prevent small particles from passing through the filter, fit a polyvinyl chloride (Polyvic) filter dis¢ on top of the glass filter. Pour a sufficient

Plant Pigments

103

amount of resin slurry into the glass column to form a 15-18 cm column after

the resin settles. Pipette 5-10 ml of the juice on the settled column and allow to adsorb. Wash sugar down from the column with distilled water (7 ml is sufficient). Note the completeness of washing of sugars with Abbe refractometer. Elute the anthocyanins with methanol containing 0.259% HCl. Add methanolic HCl to the column in small increments. Collect the dense part of the anthocyanin band eluted with methanol and make up to a known volume for the determination of individual anthocyanins by chromatography.

Chromatographic Separation of Individual Anthocyanins

The upper phase of -butanol-benzene-formic. acid-water (100: 19: 10 : 25) mixture, aged 3 days, should be used as solvent. Quantitative separation: Apply with the help of a micro-pipette a known volume of the anthocyanin extract separated by any one of the above methods or direct extract from fresh or frozen fruit on a Whatman No.1 paper as 4 cm long streaks in such a way that the direction of development is perpendicular to the machine direction of the paper. Equilibrate the chromatograms with the lower phase in a tank for 12 hr. Transfer to another tank (30 x 30 x 60 cm) and develop: ascendingly with the upper phase. Saturate the atmosphere in the jar with #hfupper phase of the solvent prior to the development by lining the side of #H€:jag with filter paper and soaking it with the developing solvent. Develop: the chromatograms until the solvent front reaches the area within 1 to 2 cm from the top of the paper. Air-dry the chromatograms for 20 min and repeat the development for a second time. Densitometric measurement: Cut the paper into suitable strips. Intensify the attenuation of the anthocyanins by fuming the chromatograms with 6 N HCl for 5 min. Carry out the fuming by suspending the strips from a glass frame in a chromatography tank over the acid. By scanning the chromatogram with different filters in the densitometer, find the filter which gives the greatest response (525 nm in the case of cranberry anthocyanins). Find the slit width of the instrument which gives best response. In the densitometer, scan the chromatogram. Measure the area under each peak. A planimeter may be used for the purpose. Colorimetric measurement: 'The anthocyanins from the individual spots may be extracted with a suitable solvent (see procedure for grapes in Method I) and measured colorimetrically. However, the densitometric method should be preferred.

CALCULATION

1. Find the total anthocyanin content

in mg/100 g (or ml) by Me-

thod I. 2. Find the ratio of individual anthocyanins from the densitometric readings. using the following expression:

104.

Analysis of Fruit and Vegetable Products

Densitometric peak area for No. 1 anthocyanin x 100 No? i dithdepaiin 9p Be sol ee ee ea ae 8 Densitometric peak area for all the pigments No. 1 anthocyanin content _Total anthocyanin in mg x No. 1 anthocyanin 'mg/100 g (ml) 100

The content of each of the anthocyanins may be calculated similarly.

References

1. Fuleki, T. & F.J. Francis, J.Food Sci., 33, 72 (1968). Zapsalis,C. & FJ. Francis, J. Food Sci., 30, 396 (1965).

Chandler, B.V. & K.A. Harper, Aust. J. Chem., 14, 856 (1961); 15, 114 (1962). Spaeth, E.C. & D.H. Rosenblatt, Anal. Chem., 22, 1321 (1950). Lynn, D.Y.C. & B.S. Luh, J. Food Sci., 29, 735 (1964).

Luh, B.S., K. Stachowicz & C.L..Hsia, J. Food Sci., 30, 300 (1965). Smith, R.M. & B.S. Luh, J. Food Sci., 30, 995 (1965).

Dickinson, D. & J.H. Gawler, J. Sci. Food Agr., 7, 699 (1956). Swain, T. & W.E. LP ey AY Set

Hillis, J. Sci. Food Agr., 10, 63 (1959).

Fuleki, T. & F.J. Francis, J. Food Sci., 33, 78 (1968). 4 se & ~ N

Fuleki, T. & F.J. Francis, J. Food Sci., 33, 266 (1968).

, Fuleki, T. & F.J. Francis, J. Food Sci., 33, 471 (1968).

CHAPTER

5

Vitamins ASCORBIC ACID Fruits and vegetables are important sources of ascorbic acid. The most satisfactory chemical methods of estimation are based on the reduction of 2,6-dichlorophenol indophenol by ascorbic acid and those based on the reaction of dehydroascorbic acid with 2,4-dinitrophenylhydrazine.

2,6-Dichlorophenol-Indophenol Visual Titration Method)? _ The dye,which is blue in alkaline solution and red in acid solution, is reduced by ascctbic acid to a colourless form. The reaction is quantitative and practically specific for ascorbic acid in solutions in the pH range 1~3.5. REAGENTS

1. 3°, Metaphosphoric acid (HPO,): Prepare by dissolving the sticks or pellets of HPO, in glass distilled water. 2. Ascorbic acid standard: Weigh accurately 100 mg of tascodiig acid and make up to 100 ml with 39% HPO,. Dilute 10 ml to 100 ml with 3% HPO, (1 ml = 0.1 mg of ascorbic aciay: 3. Dye solution: Dissolve 50 mg of the sodium salt of 2,6-dichlorophenolindophenol in approximately 150 ml of hot glass distilled water containing 42 mg of sodium bicarbonate. Cool and dilute with glass distilled water to 200 ml. Store in a refrigerator and standardize every day. PROCEDURE

Standardization of Dye Take 5 ml of standard ascorbic acid solution and add 5 ml of HPOs, Fill a muicroburette with the dye. Titrate with the dye solution to a pink colour, which should persist for 15 sec. Determine the dye factor, i.e. mg of ascorbic acid per ml of the dye, using the formula:

Dye

factor =

0.5 titre

Preparation of Sample Fruit juices: ‘Take 10 to 20 ml of sample and make up to 100 ml with 3% HPO,. Filter or centrifuge. Solid or semi-solid food: Take 10 g of sample, blend with 3% HPO, and make up to 100 ml with HPO,. Filter or centrifuge.

106

= Analysis of Fruit and Vegetable Products

Assay of Extract

Take an aliquot (2-10 ml) of the HPO, extract of the sample and titrate with

the standard dye to a pink end-point which should persist for at least 15 sec.

Titrate rapidly and make a preliminary determination of the titre. In the next determination, add most of the dye required and then titrate accurately. The aliquot of sample taken should be such that the titre should not exceed 3 to 5 ml.

Elimination of Interference due to Sulphur Dioxide - Sulphur dioxide, when present in sample, reduces the indophenol dye and thus interferes in ascorbic acid analysis. If the sample contains SO,, eliminate the interference by following the formaldehyde condensation procedure given below. To 10 ml of the filtrate in a test tube, add 1 ml of 40% formaldehyde and 0.1 ml of HCl, keep for 10 min and titrate as before. CALCULATION

.

Calculate the ascorbic acid content of the sample from the following formula: mg of ascorbic acid Titre x Dye factor x Volume made up x 100 per 100 gorml ~— Aliquot of extract ¥ Wt or volume of sample taken for estimation

taken for estimation

References

1.

Methods of Vitamin Assay, The Association of Vitamin Chemists, Interscience Publishers, New York, 3rd edn., p. 287 (1966).

2.

Johnson,

B.C., Methods

of Vitamin

Determination,

Butgess

Publishing

Co., Minnea

polis, p. 98 (1948). The Direct Colorimetric

Determination

The direct colorimetric method is based on measurement of the extent to which a 2,6-dichlorophenol-indophenol solution is decolorized by ascorbic acid in sample extracts and in standard ascorbic acid solutions.! Since interfering substances reduce the dye slowly, rapid determination would be measuring mainly the ascorbic acid. REAGENTS

1. 2°, Metaphosphoric acid in glass distilled water. 2. Dye solution: Dissolve 100 mg of 2,6-dichlorophenol-indophenol dye and 84 mg of sodium bicarbonate in hot (85-95° C) distilled water, cool and make up to 100 ml. Filter and dilute 25 ml to 500 ml with distilled water. 3. Standard ascorbic acid solution: Accurately weigh 100 mg of ascorbic acid and make up to 100 ml with 2% HPO,. Dilute 4 ml of this solution to 100 ml with 2% HPO, (1 ml = 40 yg of ascorbic acid).

Vitamins

107

PROCEDURE

Preparation of the sample: Prepare the sample as in visual dye titration method, but using 2% HPO,. If the sample is solid or semi-solid, to get a representative sample, it would be advisable to blend 50 to 100 g of the sample with an

equal weight of 6% HPO, and make up an aliquot of the macerate to 100 ml. Standard curve: To dry cuvettes or test tubes, pipette the requisite volume of standard ascorbic acid solution—1, 2, 2.5, 3, 4 and 5 ml and make up to

5 ml with the requisite amount of 2°, HPO . Add 10 ml of delivery pipette, shake and take the reading within 15 to 20 trument to 100% transmission using a blank consisting of 5 solution and 10 ml of water. Measure the red colour at 518 nm nearest to the required wavelength using a suitable filter.

dye with a rapid sec. Set the insml of 2% HPO, or a wavelength Plot absorbence

against concentration.

Sample: Place 5 ml of the extract (or less:made to 5 ml with HPO,) in a dry cuvette or test tube, add 10 ml of dye and measure as in standard. CALCULATION

Note the concentration of ascorbic acid from the standard curve and calculate the ascorbic acid content in the sample as given below: ,

,

mg ob ascorbic ACK. pets signee 100 g or ml of sample

Ascorbic acid content

ee

a

ml of solution

Volume . made u 100

ie

ae

x 1000 x

taken for estimation

Wt or volume

of sample taken

Reference

1. Loeffler, J.H and J.D. Ponting, Ind. Eng. Chem., Anal. Edn., 14, 846 (1942).

Xylene Extraction Method

The method is similar to that of direct colorimetric method, except that the excess of dye is taken up in xylene and the colour measured. The method is particularly suitable in such fresh materials and stored products where there are considerable interfering substances. It enables the determination of true ascorbic acid. REAGENTS 1. Acetate buffer—pH 4: Mix 1 litre of 50% sodium acetate (CH,COONa. 3H,O), with 1 litre. of glacial acetic acid. 2. Dye: Dissolve

125 mg of 2,6-dichlorophenol-indophenol

in warm

dis-

tilled water, cool, make up to 100 ml and filter. Dilute 18 ml to 100 ml with

108

Analysis of Fruit and Vegetable Products

water (1 ml of dye should be equal to 0.1 mg of ascorbic acid). The stock ‘ solution of dye may be stored in refrigerator for about a week. in HPO; of pellets 3. Metaphosphoric acid: Dissolve 30 g of sticks or distilled water and dilute to 1000 ml. 4. Xylene: Use redistilled xylene. Xylene used in the method may be recovered by shaking with a 20% solution of NaOH to neutralize acetic acid, followed by distillation. 5. Standard ascorbic acid solution: Weigh exactly 100 mg of ascorbic acid and make up to 100 ml with 3% HPO,. Dilute 10 ml to 100 ml (1 ml =:0.1 mg of ascorbic acid). (OF 275 peroxide: Dilute 30% hydrogen peroxide 10 times. 7. Formaldehyde. EXTRACTION

Macerate 100 g of sample with 100 g of 3% HPO, ina blender. Weigha portion of the macerate containing 10 to 15 mg of ascorbic acid (10 to 20g of macerate), transfer to a 100-ml volumetric flask, make up to mark with 3% HPO,, and filter. STANDARD CURVE

Pipette into 50-m] stoppered conical flasks 0.0, 0.5, 0.75, 1, 1.5 and 2 ml of standard ascorbic acid solution and make up to 2 ml with 3°4 HPO,. Add 2 ml of acetate buffer, 3 ml of dye and 15 ml of xylene in rapid succession. Stopper the flasks and shake vigorously for 10 sec to extract the. excess of dye into the xylene. Allow to separate. Pipette out the water layer below the xylene layer and discard. To the xylene layer, adda few crystals of anhydrous Na,SO, to remove traces of moisture. Measure the colour at 520 nm setting the instrument to 100° transmittance using xylene as blank. Plot absorbence against concentration to get the standard curve. PROCEDURE

Basic methed: Take 2 ml of filtrate in a stoppered conical flask, add 2 ml of buffer, 3 ml of the dye and 15 ml of xylene in rapid succession. Stopper and shake for 10 to 15 sec. Pipette out the water layer, add anhydrous Na,SO, to the xylene and measure the colour as in the case of the standard curve. Since ascorbic acid is quite unstable after addition of buffer ( pH 4) and as other reducing substances may react with the dye, buffer, dye and xylene should be added in rapid succession. Formaldehyde modification: lf the material has undergone extensive heat treatment or long storage, apply the formaldehyde condensation procedure to correct for the non-ascorbic acid reducing substances as given below,

*

Vitamins

Conical Flask No. 1

109

Conical Flask No. 2

Total Ascorbic Acid (A) (Basic method)

Non-ascorbic Acid Reducing Substances (Formaldehyde condensation)

2.0 ml sample 2.0 ml buffer

(B)

2.0 ml sample 2.0 ml buffer

1.0 ml water

1.0 ml formaldehyde (40%)

3.0 ml dye 15.0 ml xylene Add in rapid succession, stopper, and shake.

Allow to stand for 10 min. Then 3 ml of dye and 15 ml of xylene. Stopper and shake.

add

Measure the colour in the xylene layer as given in the basic method. Peroxide modification: If the product has been sulphite treated or if it has been stored for a considerable period in a metal container, use peroxide modification to correct for interference by sulphite or iron. Follow the procedure given below.

Sample Buffer 3% Hydrogen peroxide Water Dye Xylene

Conical Flask No. 1

Conical Flask

Total Ascorbic Acid (A) (Basic method)

Non-ascorbic acid reducing substances (C) (Peroxide: modification)

2.0 2.0 a 2.0 3.0 15.0

ml ml

2.0 2.0 2.0 .. 3.0 15.0

ml ml ml

No.

2

ml ml ml ml ml

Add in rapid succession, stopper and shake. Measure the colour in the xylene layer as given in the basic method. CALCULATION

Find the ascorbic acid concentration from the standard as below. Ascorbic acid

Total ascorbic acid mg per 100 gorml

—

curve and calculate

Volume

100 in sample “ made up " ml of solution e Wt or. volume of taken sample taken

Calculate non-ascorbic acid reducing substances in the formaldehyde or

110

Analysis of Fruit and Vegetable Products

hydrogen peroxide modification procedure as given in the case of total ascorbic acid. True ascorbic

acid

_—‘ Total ascorbic

~

acid (A)

Non-ascorbic acid reduc-

cing substances (B or C)

Reference

1. Robinson, W.B. and_E. Stotz, J. Biol. Chem., 160, 217 (1945).

Ascorbic Acid, Dehydroascorbic Acid and Diketogulonic Acid The reaction of the oxidation products of ascorbic acid with 2,4-dinitrophenylhydrazine to form osazone has been made use of by Roe and his co-workers!~4 to estimate ascorbic acid as well as its oxidation products—dehydroascorbic acid and 2,3-diketogulonic acid. 2,4-Dinitrophenylhydrazine forms osazone with dehydroascorbic acid and diketogulonic acid which dissolves in conc H,SO, forming an intense red solution, and can be measured colorimetrically at "520 nm. Ascorbic acid can be estimated by oxidation with bromine to dehydroascorbic acid and subsequent formation of osazone with the dinitrophenylhydrazine. The method has been described in detail by the Association of Vitamin Chemists.5 REAGENTS

1. 9 N H,SO,: Dilute 250 ml H,SO, to 1 litre with water. 2. 2% 2,4-Dinitrophenylhydrazine: Dissolve 2 g of 2,4-dinitrophenylhydrazine in 100 ml of 9 NH,SO,. Store in amber coloured bottle ina refrigerator. Filter before use. The solution is stable for 2 weeks. 3. 20% Metaphosphoric acid: Dissolve 100 g of HPO, in 500 ml of distilled water. Store in refrigerator. Stable for 2 weeks. Preferably prepare fresh. Prepare 5 or 10% solutions of HPO, from the stock solution by dilution with water. 4. 2% Thiourea in 5° metaphosphoric acid: Dissolve 10 g of thiourea in 500 ml of 5% HPO,. 5. 85% H,SO,: Dilute 900 ml of H,SO, to 1000 ml with water. 6. Bromine. 7. Ascorbic acid standard: Dissolve 100 mg ascorbic acid in 100 ml of

5% HPO, (1 ml = 1 mg of ascorbic acid). 8. Hydrogen sulphide (Kipps). 9. Carbon dioxide (Kipps) 10. Stannous chloride. PROCEDURE

1. Total ascorbic acid: Blend 100 g of sample and 100 g of 10% HPO, in a blender to a uniform slurry. Weigh a portion of the slurry estimated to contain

Vitamins

111

1 to 2 mg of ascorbic acid. Transfer to ~ 100-ml volumetric flask and dilute to volume with 5%, HPO,. Mix and filter or centrifuge to get a clear liquid. To the filtrate, add 1 or 2 drops of bromine and shake gently until the solution becomes yellow. If the solution does not become yellow, add another drop and shake. Remove the excess of bromine by passing air or nitrogen (saturated with water) through the solution until it is colourless. Measure the volume and add thiourea to give a concentration of 1% (0.4 g for 40 ml of the extract). Proceed with osazone formation as given later.

2: Dehydroascorbic acid and diketogulonic acid: Weigh a portion of the sample containing 5 to 10 yg of ascorbic acid per ml. Assuming that the sample contains 50 mg/1C0 g ascorbic acid and the final volume to be made up to is 250 ml, weigh 2.5 to 5.0 g of the sample, add 1.25 g of stannous chloride and 12.5 ml.of HPO,, grind the sample and make up to 250 ml with 5% HPO,.

Filter and use the filtrate for osazone formation as described later. 3. Diketogulonic acid: Pipette 100 ml of the filtrate from 2 into a gas washing bottle. Pass H,S for 15 min. To minimize odour, pass the exhaust H,S through NaOH inion To 40 ml of the H,S-saturated solution, add 0.4 g of thiourea to give a concentration of 1%. Shake until dissolved and filter. Bubble CO, into the filtrate for 5 min. The H,S reduces dehydroascorbic acid to ascorbic acid which does not react with 2,4-dinitrophenylhydrazine. Proceed for osazone formation as described below. In heated and/or stored samples, the resulting solution is coloured and it is not possible to measure the diketogulonic acid.

Formation of Osazone

Solution 1 gives:

Ascorbic

acid + Dehydroascorbic acid + Diketogulonic acid Solution 2 gives: Dehydroascorbic acid + Diketogulonic acid Solution 3 gives: Diketogulonic acid For each of the above

solutions, take 3 test tubes, mark as 1, 2 and 3, and

form osazone as described below. 1 Blank

Sample 2% 2,4-dinitropheny]hydrazine reagent

2 Sample

3 Sample

4.0 ml

4.0 ml

4.0 ml

=

i

1.0 ml

|

ml

{

|

_ Incubate at 37°C + 0.5° C for exactly 6 hr in a water

bath

Remove from the water bath and keep in an ice bath

85% H,SO,

{ 5.0 ml

4 5.0 ml

1 5.0 ml

112.

Analysis of Fruit and Vegetable Products

Add H,SO, drop by drop with thorough mixing, and allow to cool

*

\

2%, 2,4-dinitrophenylhydrazine reagent

|

}

eS bt 1.0 ml { Remove the tubes from ice bath and allow to stand. at room temperature for 30 min.

Cotour MEASUREMENT Measure the colour at 520 nm setting the instrument to 100%, transmittance with the blank in place in each set. STANDARD CURVE To 50 ml of standard ascorbic acid solution (1.0 ml = 1.0 mg) in a conical flask, add 2 to 3 drops of bromine until the colour becomes yellow and transfer to a gas washing bottle. Pass water saturated air through the solution to_remove unreacred bromine. Pipette 10 ml of this solution into a 500-ml volumetric flask, add 5.0 g of thiourea and make up to volume with 5% HPO, (1 ml =

20 pg). Pipette 5, 10, 20, 25, 30, 40, 50 and 60 ml of the above solution (1 ml =

20 yg) into separate 100-ml volumetric flasks and make up to mark with 5%, HPO, solution containing 1% thiourea to give solutions containing 1, 2, 4, 5, 6, 8, 10 and 12 yg per ml respectively. Form osazone for each of the seven standards as in the sample, and measure the colour at 520 nm. Plot % transmittance as ordinate and concentration of ascorbic acid as abscissa on a semilogarithmic paper or absorbence against concentration on an ordinary graph paper. CALCULATION Read the concentration

of ascorbic acid in each of the solutions

1, 2 and

3 from the standard curve, and calculate using the following expression.

mg/100g

ug as read from the graph x Volume made up x 100 ml of sample taken x Wt of sample x 1000

Determine ascorbic acid, dehydroascorbic using the following relationship: Diketogulonic acid saved Dehydroascorbic acid = 2—3 Ascorbic acid a (12

acid and

diketogulonic

acid

References Roe, J.H., M.B. Mills, MJ. Osterling & C.M. Dameron, J, Biol. Chem.,17'4,201 (1949).

Roe, J.H. & M.J. Osterling, [. Biof. Chem.,.152, 511. (1944).

Roe, J.H., J. Bioh Chem.,116, 609 (1936).

Koe, ).H. & C.A. Kuether, J. Biol. Chem., 147, 339 (1943). Soa Methods of Vitamin’ Assay, The Association af Vitamin Chemists, Interscience Publishers, Spe

-New York, 3rd edn., p. 328 (1966).

.

Vitamins

113: :

THIAMINF PRINCIPLE

—

_ Thiamine is extracted from the food material using hot dilute acid, and the extract is treated with phosphatase to release the thiamine from the bound form. The extract is then passed through base exchange silicate column to remove-the interfering substances. Thiamine is eluted from the column, and oxidised by alkaline ferricyanide to thiochrome which is measured fluorimetrically. REAGENTS

-1. 0.2. N HCI: Dilute 18 hl of conc HCI (sp. gr. 1.18) with water ‘to 1 litre. a 0.1 N H2SQy,: Dilute 2.8 ml of conc H2SO, (sp. gr. 1.84) to 1 litre with water. “3. 3% Acetic acid: Dilute 30 ml of glacial acetic acid (sp. gr. 1.05) to 1 litre with water. 4. 25% (w/v) Potassium chloride: Dissolve 250 g of KCI in water, and make up to 1 litre using water. 5. Acid potassium chloride solution: Dilute 8.5 ml of HCI (sp. gr. 1.18) to 1 litre with 25% KCI. 6. 15% (w/v) NaOH. 7. Alkaline potassium ferricyanide: Dilute 3 ml of 1% potassium ferricyanide solution to 100 ml with 15% NaOH. Prepare immediately before use. 8. 2.5 M Sodium acetate: Dissolve 205 g of anhydrous or 340 g of hydrated sodium acetate in water, and dilute to 1 litre. 9. Iso-Butanol: Redistil zso-butanol in all-glass apparatus. Add distilled water, and shake until saturated. . 10. Suspend 6 g of ‘a suitable source of phosphatase in 2.5 M sodium acetate solution, and dilute to 100 ml with sodium acetate. Taka diastase of Parke Davis. and Co., Clarase of Takamine Laboratories, Polidase of Schwarz Laboratories or

Mylase of Wallerstein Laboratories may be used as source of phosphatase. 11. Activated base exchange silicate: Zeolite 60-90 mesh and Decalso (60-80 mesh) (also called Permutit T) are suitable for the purpose. Activate the base

exchanger as follows: To 100g

of the material, add 200-250 ml of hot 3% acetic

acid. After 10-15 min, transfer to a Buchner funnel, and apply mild vacuum to drain off solution. Repeat the acid wash. Thereafter, wash with about 250 ml of hot 25% KCI solution followed by hot acetic acid. Finally, wash the material with distilled water till the washings are free from chloride (test the washing for chloride with 1% AgNO, ). Dry it at room temperature or any temperature

* below 100°C. 12. Thiamine stock solution: Dissolve 50 mg of thiaminehydrochloride in 0.2 N HCI, and dilute to 500 ml with 0.2 N HCl (1 ml = 100 ug).

13. Thiamine working standard solution: Dilute 5 ml of the stock solution to 100 ml with 0.2 N HCl. Pipette 5 ml of this solution into a volumetric flask ~ containing 400 ml of acid potassium chloride, and dilute to 500 ml with distilled water (1 ml = 0.05 pg).

PROCEDURE Extraction: Take a known weight of the prepared sample estimated to contain

114

=Analysis of Fruit and Vegetable Products

10 to 30 wg of thiamine. Add 75 ml of 0.2 N HCI, and heat for 30 min ina boiling water bath with occasional shaking. Cool the extract to room temperature, add 5 ml of freshly prepared enzyme solution, and mix. Incubate at 37° C overnight. Transfer the extract to a 100-ml flask, and dilute to 100 ml with water. Mix

thoroughly and filter. Purify the filtrate by passing through the base exchanger column as described below: Use a glass tube column of 10 mm internal diameter and 20cm length with a stop cock. Plug the bottom of the column (above the stop cock) with glass wool, and fill with 6 g of base exchange suspendedin water. Allow the water to drain to within 2 mm of the surface of the column bed. Add 25 ml of the filtrate on to the column, and allow to pass through. Was the column by passing three successive 10 ml portions of hot water, and discard the washings. Thereafter, place 10 ml of acid KCI on the column, and allow to pass through. Collect the effluent solution in a 25-ml volumetric flask. Add another 10 ml portion of acid KCI solution, and collect the effluent in the same 25-ml volumetric flask. Dilute the contents to the mark with acid KCI solution, and mix well.

Pipette 5 ml of the acid potassium chloride eluate into a 50-ml glass stoppered conical flask. Add 3 ml of alkaline ferricyanide solution, and mix gently. Add 15 ml of iso-butyl alcohol, and shake vigorously for 90 seconds. Allow the two phases to separate. Remove the lower aqueous layer using a pipette, and discard it. Add 2-3 g anhydrous sodium sulphate to the iso-butanol phase, and shake well. Into a second 50-ml glass stoppered conical flask, pipette 5 ml of working standard thiamine solution. Carry out the oxidation, and subsequent extraction of thiochrome, exactly as described above in the case of sample. Prepare a sample and standard blank by taking, 5. ml of the sample and standard solution, and treating as described above except for adding 3 ml of 15% NaOH instead of alkaline ferricyanide. Measurement of fluorescence Set the fluorimeter to an excitation wavelength of 360 nm and emission wavelength of 435 nm, or use thiamine primary and secondary filters in the case of filter fluorimeter. Adjust the instrument to zero deflection against 0.1 N H2SO, and:100 against the standard. Now pour the sample into a third fluorimeter cell and measure the fluorescence. Read the fluorescence in the sample and standard blanks. CALCULATION

The content of thiamine per gram of the sample if yg is given by the formula: A—B. 100 1 _ 25 =X XS XK SS By, C—D° 5 * V * wr. of the sample ss H8/8

where A B C D V

= = = = =

reading reading reading reading volume

of the of the of the of the of the

sample sohition sample blank standard standard blank solution used for adsorption on the column

Vitamins

115°

References 1. Bechtel, W.G. and C.M. Hollenbedt! Cereal Chem., 35,,1 (1958).

2. McRoberts, L.-H., J. Assoc. Offic. Agr.Chem., 43, 47 (1960). 3. Conner, R.T. and GJ. Straub, Ind. Eng. Chem., Anal Edn., 13, 380 (1941). 4. Wang, Y.L. and LJ. Harris, Biochem. J., 33, 1050 (1941).

RIBOFLAVIN

PRINCIPLE Riboflavin fluoresces in light of wavelength 440 to 500 nm, and the intensity of fluorescence is proportional to the concentration of riboflavin within a certain range. The vitamin is extracted from the material with hot dilute. acids, interference by other substances eliminated by treating with potassium permanganate, and the intensity of fluorescence of the resulting solution is measured. In fruit and vegetable products, it is necessary to remove the interfering fluorescing substances using adsorbant florisil. REAGENTS

1. 0.2 N HCI: Dilute 18 ml of HCI (sp. gr. 1.18) with water to 1 litre. 2. 0:1 N HeSOx4: Dilute 28 ml of H2SOx, (sp. gr. 1.84) with water to 1 litre. 3. 1 N HCI: Dilute 90 ml of HCl with water to 1 litre.

4. 40% NaOH: Dissolve 40 g of NaOH in water, and dilute to 100 ml. 5. Glacial acetic acid.

6. 3% Potassium permanganate: Dissolve 3g of potassium permanganate

in

distilled water, and dilute to 100 ml.

7. 3% Hydrogen peroxide.

8. Sodium hydrosulfite (Dithionite). 9. Riboflavin stock standard solution: Dissolve 25 mg of riboflavimin 3 ml of. glacial acetic acid, warm if necessary, and dilute to 1 litre with water (1 ml = 25 ug).

10. Riboflavin working standard solution: Dilute 4 ml of OU

NI stock

standard solution to 1 litre with water (1 ml= 0.1‘g).

PROCEDURE Weigh a sample estimated to contain 5-10 mg of riboflavin into a 100-ml conical flask. Add 50 ml of 0.2 N HCL, and keep in a boiling water bath for 1 hr. Cool the sample. Adjust the pH to 6.0 using NaOH with constant swirling during the addition of alkali to prevent local areas of high pH. Add 1 N HCI to bring the pH to 4.5. Transfer the extract toa 100-ml volumetric flask, make up the volume with

water, and filter. Oxidation of interfering substances and measurement of fluorescence : Prepare duplicate tubes as follows: Tube No. Filtrate Water Riboflavin working 1

2

(ml) 10

10

(ml) 1.0

—

standard (ml) —

10" }

116.

Analysis of Fruit and Vegetable Products

Now, add 1 ml of glacial acetic acid to the above tubes, and mix. Add 0.5 ml of 3% .

potassium permanganate to each tube, mix, and allow to stand for exactly 2 min. Then add 0.5 ml of 3%, hydrogen peroxide, and mix. Adjust the fluorimeter to an excitation wavelength of 470 nm and emission

wavelength of 525 nm, or use riboflavin primary and secondary filters if a filter fluorimeter is used. Adjust the fluorimeter to a deflection of zero against 0.1 N .H2S0, and 100 against the contents of tube No. 2. Now measure the fluorescence of the solution in tube No. 1. Add about 20 mg of sodium dithionite to each tube, mix well, and measure the fluorescence within 10 seconds. Readings obtained after

the addition of sodium dithionite serve as the blank value. CALCULATION Calculate the riboflavin content of the sample using the following equation:

2 ARP en sy Bolla vide phys (C—D)-(A-B)

Lith ADO

10 X W

where W = weight of the sample (g) 100 = volume of the extract (ml)

10 -A B C D

= = = = =

aliquot taken for estimation (ml) reading of the sample (tube 1) reading of the sample blank reading of sample + standard (tube 2) reading of sample + standard blank

NOTEs: 1. Riboflavin is light sensitive, and hence avoid exposure of riboflavin containing solutions to strong light. 2. For the analysis of riboflavin in highly coloured samples, use the following adsorption and elution step for purification.

‘In a glass adsorption column (16 cm X 1.5 cm), puta wad of glass wool, and.add fluorésil to a height of about 8-10 cm. Wash the column with about 25 ml of 2% acetic acid followed by about 15 ml of water. After extraction and filtration, pipette a known volume of the filtrate containing 8-12 yg of riboflavinonto the column. After the sample has passed through the column, wash the column with two 15 ml portions of water. Elute the riboflavin from the column with 25 ml of 20% pyridine in 2% acetic acid, and collect the eluate in a 50-ml volumetric flask. Add additional portions of 20% pyridine in 2% acetic acid, collect the eluate in the

same-volumetric flask, and make upto volume. Use this solution for permanganate oxidation and subsequent measurement of fluorescence. References 1. Hodson, A.Z. & L.C. Norris, J, Biol. Chem., 13 1, 621 (1939). 2. Conner, R.T. & GJ. Straub, Ind. Eng. Chem., Anal. Edn.,13, 385 (1941).

3. Kemmerer, A.R., J. Assoc. Offic. Agr. Chemists, 25, 459 (1942).

4. Peterson, W J., D.E. Brady & A.O, Shaw, Ind: Eng. Chem., Anal. Edn., 15 634 (1943). 5. Loy, H.W. Jr., J. Assoc. Offic. Agr. Chemists, 32,461 (1949).

Vitamins

117

FOLIC ACID PRINCIPLE Folic acid is extracted from food samples using mild alkaline buffer, oxidised with permanganate, and the resulting amine is diazotized. The diazotized compound is coupled with N-(1-naphthyl) ethylenediamine, and the cin developed is determined at 550 nm. ' Reagents

1. Dibasic potassium edie solution: Dissolve 60.61 g KeHPO, |in wager and make up to 2 litre. 2. Potassium permanganate solution: Take 0.4 g of KMnOQ, in a 100-mi | volumetric flask, and dilute to volume with water. 3. Sodium*nitrite solution: Take 2 g of NaNO; ina 100-ml: volumetric flask, and

make up to volume with water. 4. 5 N HCl. 5. Ammonium sulphamate solution: Dissolve 5 g (NH4)NHe2SO; in water, aud

make up the volume to 100 ml. 6. N-(1-naphthy!) ethylenediamine dihydrochloride solution: Place 0.1 gof the substance in a 100-ml volumetric flask, and dilute to volume with water. 7. Sodium chloride 8. iso-Butyl alcohol 9. Stock folic acid standard solution: Dissolve 50 mg of folic acid in water with the help of 2 ml of NHs, and make up the volume to 100 ml with water (1 ml = 500

Hg). 10. Working folic acid standard solution: Dilute an aliquot of iisstockstandard solution with K,HPO, solution (Reagent 1) to give a concentration of 10 ug/ml.

PROCEDURE Place a known amount of the sample containinga a cet 100 mg. of folic acid in a 100-ml flask. Add about 50 ml of KzZHPO, solution, and heat the mixture to a

temperature not above 60° C with swirling until the sample is propetly dispersed. Cool to room temperature, and make up the volume to 100 ml with KzgHPO,

solution. Filter or centrifuge the solution. Transfer an aliquot of the clear solution containing about 1 mg of folic acid to a 100-ml volumetric flask. Dilute to volume with K,HPQ, solution. Use this solution for colour development and estimation. Prepare duplicate assay tubes as follows:

Tube No.

1

,

Sample Working Standard Wacer (ml) (ml) extik¢ml) pA! ia —_— | —

Potassium permanganate solution (ml) — 1.0

1.0

2

5.0

2.0

—

3 4 .

5.0 5.0

— 2.0

1.0

=

1.0

—

Add:‘1 ml sodium nitrite solution and 1 ml of 5 N HCI to all the tubes. Mix and allow to stand for 2 min. Add 1 ml of ammonium sulphamate solution, and mix

|

118

Analysis of Fruit and Vegetable Products

with swirling. Now, add 1 ml of N-(1-naphthy!) ethylenediamine dihydrochloride solution, mix, and allow to stand for 10 min. Add 1 g of sodium chloride and 10 ml

of iso-butyl alcohol. Shake vigorously for 2-3 min. Separate the iso-butyl alcohol layer by centrifugation, and remove about 9 ml of the clear supernatant Inyer. Read the colour of the iso-butyl alcohol at 550 nm within 25 min using iso-buty] alcohol as the blank. CALCULATION

Calculate the quantity of folic acid in the saspple pespare tian in mg/ml using the expression: c

A 1-43

04 C2 A, aie Gina where C = concent: signof the working standard of folic acid in mg/ml, and A, Ad, As, and Ag ate-the absorbence of tubes 1, 2, 3 and 4 respectively. References 1. Hulchings, B.L., E.L.R. Stokstad, J.H. Booth, J.H. Mowar, O. W. Wallen R.B. Angier, J. Semb & Y Subba Row, J. Biol. Chem., 168, 705 (1947). 2. Kaselis, R.A., W. Leibermann, W. Seaman, J.P. Sickels, E.1. Stearns & J.T. Woods, Anal Chem., 23,

746 (1951). 3. Schiaffino, S.S., J.M.Webb, H.W. Loy & O.C. Kline, J.Am. Pharm. Assoc., 48, 236 (1959).

CHAPTER

6

Minerals The Destruction of Organic Matter! Fruits and vegetables, and their products like any other foods contain organic matter which must be destroyed prior to the estimation of minerals. Dry ashing or wet digestion is generally used for the destruction of organic matter. The choice of the procedure depends on the nature of the organic material, the nature of any inorganic constituent present, the metal. to be determined and the sensitivity of the method used for the determination. Dry ashing is applicable to most common minerals with the exception of mercury and arsenic. It requires less attention and large amounts of material can be handled more conveniently than by wet digestion. The procedure is particularly suitable when the use of H,SO, (as in the estimation of lead in materials containing alkaline earths whose sulphates occlude lead sulphate) is objectionable. Calcium, phosphorus and iron generally estimated in fruits and vegetables may safely be ashed by this procedure. The loss of potassium — may occur if the temperature is too high, and it is usual to avoid a temperature* of over 480° C if all the potassium is to be retained. A temperature of 450° C should not be exceeded if zinc is to be determined. Excessive heating may also make certain metallic compounds insoluble as in the case of tin. The wet digestion method offers certain advantages. The temperature cannot exceed the boiling point of the mixture and generally carbon is destroyed more quickly than in dry ashing. The wet digestion procedure depends primarily on the use of nitric acid for the destruction of organic matter at a fairly low temperature to avoid loss by evaporation, and in the later stages of diges‘tion, the process is sometimes considerably speeded up by the action of perchloric acid or hydrogen peroxide. The wet digestion method is generally employed when it is required to estimate arsenic, copper, lead, tin and zinc.

Wet Digestion ApPpARATUS

Use long necked 300-ml Kjeldahl digestion flask with B24 ground glass ' joint. Connect this with an extension (Fig. 6.1) to condense the fumes into an acid fume condenser and carrying a side-tap funnel for introducing the reagents. For digestion, make use of a mild steel rack (Fig. 6.2) with asbestos top, having circular holes for supporting the flasks. The diameter of the holes: ‘should be such that a flask receives no direct heat from the burner above the-

120

= Analysis of Fruit and Vegetable Products

Fume condenser

Fig. 6.1: Modified Kjeldahl flask (open type). Dimensions are for a flask of 150 ml capacity. (Reprinted with permission of the Society for Analytical Chemistry, U. K.)

Fig. 6.2:. Kjeldahl digestion flask and acid fume condenser. A water inlet; B connection

to pump; C outlet to waste; D tap with outlet to prevent clogging with solid matter. (Reprinted with permission of the Society for Analytical Chemistry, U. K.)

;

level of the acid. The neck of the flask should have a support on the side of the digestion stand. The extension should dip into the fume condenser. Keep a current of water flowing through the condenser. Connect the upper outlet to a suction (water) pump to facilitate the removal of acid fumes. No clamping is necessary. When it is required to handle the flask to control fumes, etc. use specially made nickel tongs having jaws covered with asbestos string. REAGENTS Use analytical grade (AR) reagents and distilled (preferably glass distilled) water of suitably low metal content

Even used 1. 2. 3. 4,-

throughout the estimation of minerals.

then carry out reagent blank using the same quantities of reagents as in the tests. ) Cone HNO, Conc H,SO, Perchloric acid Hydrogen peroxide

Minerals

121

Reagents 3 and 4 are used to speed. up the digestion and hence reduce the use of nitric acid. PROCEDURE

From among the methods suggested, make use of a suitable method. The weight of the sample to be taken is dependent on a number of factors. If it is desired to determine only one instead of a number of minerals, smaller sample may be taken. The level of mineral content of the material and the sensitivity of the method to be used for determination have also to be considered. Sample containing not more than 5 g of solids (20 to 50 g of fresh sample) may be! taken initially. With experience, each analyst will have to determine. the. quantity of the sample required. Wet digestion using nitric and sulphuric acids:; Weigh out an amount wo) sample, sufficient to contain 5 to 10 g of solids into the digestion flask. Add a few glass beads, 10 ml of H,SO, and 10 ml or more HNO, to keep the sample’ fluid. Heat very gently until the liquid appreciably darkens in colour; avoid excessive frothing. Add HNO, in small proportions (1 to 2 ml) and continue heating until darkening again takes place. Continue addition of acid and heating to fuming for 5 to 10 min until the solution fails to darken. When all the organic matter has been oxidized, allow the solution to cool, add 10 ml of distilled water (solution is either colourless or pale yellow when iron is present) and boil gently to fuming. Allow the solution to cool again, add further 5‘ml of water and boil gently to fuming. Finally cool, and make up the digest to a known volume. Nore: 1. Charting, cither initially or later, should be avoided as arsenic, if present, may volatilize. 2. H.SO,.

If wet sample is taken, boil down to a smal] bulk with HNO, before adding

Thereafter, continue digestion as for solid samples.

Wet digestion using sulphuric, nitric and perchloric acids: Weigh the sample into the Kjeldahl digestion flask. Add a few glass beads, 4 ml perchloric acid and sufficient HNO, to ensure complete oxidation of organic matter (about 7 ml for every gram of material taken). Then add 5 ml of H,SO, and mix gently. Apply very low heat slowly for 5 to 10 min till dense fumes appear. Remove the burner for 5 min and allow to cool. Replace the burner and continue digestion slowly at low heat for 5 to 10 min after the appearance of dense white fumes of H,SO,. Increase the heat and continue digestion for 1 or 2 min. The liquid at thisstage should be colourless or pale yellow, if iron is present. If any carbon is present, add 1 to 2 ml of HNO,, digest again, cool and make up toa known volume. Nore: Use of perchloric acid in the digestion has caused some explosions. Use of nitric, perchloric and sulphuric acids represent the most vigorous, and therefore potentially

the most hazardous method of wet decomposition. Full precautions must

be taken

against possible injury in the event of such explosion. Carry out digestion in an isolated digestion room under fuming cupboard. Use a face-mask. Never raise the temperature or increas¢, the burner till the vigorous oxidation of organic matter by nitric

122.

= Analysis of Fruit and Vegetable Products and sulphuric acids subside. Then only increase the temperature for the perchloric acid to react. In the course of digestion, do not allow to dry. At least 2 to 3 ml of H,SO, should always be present to ensure sufficiency of acid of high boiling point to prevent overheating of the digest in the later stages after the HNO has gone. In the absence of H,SO,, local heating of the digest may lead to decomposition of ammonium perchlorate with explosive violence.

Wet digestion using sulphuric acid, nitric acid and hydrogen peroxide : Digest the sample as stated under wet digestion using HNO, and H,SO, until the digest on cooling is pale yellow or light brown in colour. Add at a time 2 to 3 ml - of 30% (100 volume) AR grade hydrogen peroxide and a few drops of HNO3. Heat to fuming after each addition of hydrogen peroxide until the residue is colourless or no further reduction of the pale yellow colour can be obtained. Cool the solution, dilute with 10 ml of distilled water, and evaporate to fuming. Again dilute the solution with 5 ml of distilled water and evaporate to fuming. Finally, dilute the solution to volume with water.

Dry Ashing Weigh accurately a suitable quantity of the well mixed sample in a tared silica dish. Sample used for the determination of moisture may be taken for ashing. Heat first over a low bunsen flame to volatilize as much of the organic matter (until no more of smoke is given out by the material) as possible. Trans_ fer the dish to a temperature controlled muffle furnace. Keep the muffle at about 300°C until all the carbon has ceased to glow and then raise the temperature to 420°C. Most materials may be ashed in a reasonable time at a temperature as low as 420° C if heated overnight and such low temperatures are to be preferred. Generally, ashing is done at 450°C. The time required at this teniperature will depend on the nature of the material to be ashed. Generally 5 to7 hr are sufficient to ash most of the fruits or vegetables ortheir products. If it is suspected that all the carbon has not been oxidized, remove the dish from the muffle, allow to cool, add 1 to 2 ml of conc HNOg, evaporate to dryness, and heat in the muffle for another hour or so. Remove from the muffle furnace, allow to cool and, if required, note the weight of the ash.

Cover the dish with watch (1+1) with the help of a spattering. Heat over a water Continue heating for another

glass and add gently 40-50 ml of dilute HCl pipette. The watch glass is used to prevent bath for 30 min, remove the cover and rinse. 30 min to dehydrate silica. Add another 10

ml of HCl (1+1) and water to dissolve

soluble salts. Filter into a 100-ml

- volumetric flask using No. 44 Whatman filter paper. Wash the residue basin once or twice using dilute HCl. Wash the residue on the filter with HCl. Make up to volume with water. Return the filter paper dish, ignite, place in muffle for 1 hr at 450°C, cool and weigh. This an approximate estimate of silica. Reference

1.

Analytical Methods Committee, Analyst, 85. $43 (1960).

in the paper to the gives

Minerals »

123

CALCIUM Calcium is precipitated as calcium oxalate. The precipitate is dissolved in hot dilute H,SO, and titrated with standard potassium permanganate. REAGENTS

1. Ammonium oxalate — saturated solution. 2. Methyl red indicator: Dissolve 0.5 g methyl red in UR ml of 95% alcohol. 3. Dilute acetic acid —1+4. 4. Dilute ammonium hydroxide — 1+4. 5. Dilute sulphuric acid —1+4; Add acid to water slowly and with constant stirting. Cool and make up to volume. 6. 0.1 N Potassium permanganate (KMnQO,). 7. 0.01 N Potassium permanganate — working standard; Dilute 10 ml of 0.1 N KMnO, solution to 100 ml with water (1 ml = 0.2 mg of Ca). Prepare fresh epluton before using. PROCEDURE-

Pipette an aliquot (20 to 100 ml) of the ash solution obtained by dry ashing toa 250-ml } beaker. Add 25 to 50 ml of water, if necessary. Add 10 ml of saturated ammonium oxalate solution and 2 drops of methyl red indicator. Make the solution slightly alkaline by the addition of dilute ammonia and then slightly acid with a few drops of acetic acid until the colour is faint pink (pH 5.0). Heat the solution to the boiling point. Allow to stand at room temperature for at least 4 hr or preferably overnight. Filter through Whatman No. 42 paper and wash with water, till the filtrate is oxalate free. (Since HCl has been used for preparing the original ash solution, it is convenient to test for the absence of nae om AgNO.) Break the point of the filter paper with platinum wire or pointed glass rod. Wash the precipitate first using hot dilute H,SO, (1+4) from wash bottle into’the beaker in which the calcium was precipitated. Then wash with hot water and titrate while still hot (temperature 70 to 80° C) with 0.01 N KMnO, to the first permanent pink colour. Finally, add filter paper to solution and complete the titration: CALCULATION

Titre x 0.2x Total volume of ash oaien . “Calcium:

mg/100g

seisdhct sitsigibeisnen Af SEO" binait snsscqra

-—» Wolume taken for x estimation

We of sample. taken for ashing

If the KMnO, standard. solution is not exactly 0.01 N, use the following expression.

124

= Analysis of Fruit and Vegetable Products

Titre x Calcium mg/100 g

=

Normality

Total volume

of KMnO, * 7 ™ of ash solution *

ml of ash solution taken for estimation

00

Wt of the sample taken for ashing

MAGNESIUM

In an alkaline solution from which calcium and iron have been removed, magnesium is precipitated as magnesium ammonium phosphate. The precipitate is dissolved in acid and the amount of phosphorus is determined colorimetrically.1~’ Magnesium is then calculated. REAGENTS

1. Ammonium oxalate (NH,),C,O,. H,O — saturated solution. 2. Methyl red indicator: Dissolve 0.5 g methyl red in 100 mlnat ethyl alcohol. ,

3. 2 Ammonium phosphate [(NH,), HPO,] sida! por © 4. 10% Ammonium hydroxide (NH,OH) (v/v) solution. 5. 0.1 N Hydrochloric acid. .6. Molybdic acid solution :Dissolve 25 g ammonium ‘molybdate in 300 ml of water without heating. Dilute 37 ml H,SO, to 200 ml with water and add to the ammonium molybdate solution. Store in brown bottle. 7. 24 Hydroquinone solution: Add 1 drop H,SO, for each 100 ml. Discard when solution starts to become brown. . 8.

9.

10% Sodium

sulphite (NazgSO3) solution: Prepare fresh, weekly.

Potassium dihydrogen phosphate (KH,PO,). Nore:

Test reagents 6, 7,8, when mixed, no blue or green colour should appear.

PROCEDURE

Measure 10 ml of ash solution into a 15-ml graduated centrifuge tube. Add 1 drop of methyl red indicator. Neutralize solution with NH,OH. Add 1 ml of ammonium oxalate and make the solution to a volume of 13 ml with water. Mix and allow to stand overnight. Centrifuge for 10 min and discard the precipitate. Measure 1 ml of the supernatant liquid from above into a 15-ml centrifuge tube. Add 3 ml of water, 1 ml of ammonium phosphate and 2 ml of NH,OH. Mix and allow to stand overnight. Centrifuge for 7 min, discard the supernatant liquid, mix with 5 ml of dilute NH,OH, centrifuge for 7 min and discard the supernatant liquid. Dry the precipitate by placing the tube in a

container of hot water. — Add 1 ml of dilute HCl and 5 ml of water to dissolve the precipitate. Add 1 ml of molybdic acid solution, 0.5 ml hydroquinone and 0.5 ml sodium sulphite solution. Mix and allow to stand for 30 min. Transfer the solution to a

_Minerals

125

colorimeter tube and read the absorbence, in a colorimeter using a No. 66 ted filter. Set the instrument scale at zero with water.

STANDARD CURVE :.Dissolve 0.4389 g potassium

dihydrogen phosphate in water and make to a volume of 1 litre (1 ml = 0.1 mg of P = 0.0784 mg of Mg). To preparea standard curve, use aliquots of the standard solution from 0.1 to 0.5 ml_ Treat each-aliquot as directed in the last paragraph appearing under procedure een above. References 1. Holzapfel, C.R., Onderstepoort J. vet. Sci. Anim. Ind., 2, 115 (1934). 2.

McCance, R.A. & H.L. Shipp, The Chemistry of Fresh Foods and their Losses on Gooking>,

3.

Ward, G.M, & F. B. Johnston, Chemical Methods of: Plant Analysis, Canada Department of

Med. Res. Council Canada, Spec. Rept. Setics No.

187 (193 3).

\

Agriculture, Publication 1064, p. 28 (1962).

PHOSPHORUS Phosphorus reacts with molybdic acid to form a phosphomolybdate complex. It is then reduced with aminonaphtholsulphonic acid to the complex molybdenum blue which is measured colorimetrically.

REAGENTS 1. Molybdate solution: Dissolve 25 g of ammonium molybdate in 400 tml of water. Add 300 ml of 10 N H,SO, and make up the volume to 1 litre with water. 2 Buiihokesesbilabiphosia acid solution; Dissolve in water 0.5 g 1amino-2-naphthol-4-sulphonic acid, 30 g sodium bisulphite (NaHSO,) and 6 g sodium sulphite (Na,SO,). Make up to 250 ml. Allow to stand overnight and filter. Prepare fresh solution every two weeks. 3.. Standard phosphate- solution: Dissolve 0.4389 g of potassium dihydrogen phosphate (KH,PO,) in water, add'10 ml of 10 N H,SO, and make up to 1 litre with water (1 ml = 0.1 mg P). Add 1 ml of chloroform as preservative.

PROCEDURE To. 5 ml of ash solution obtained by dry ashing, add 5 ml of molybdate reagent and mix. Add 2.0 ml of aminonaphtholsulphonic acid solution, mix, and make up the volume to 50 ml. Prepare similarly a blank using water in place of the sample. Allow to stand for 10 min and measure the colour at 650 nm setting the blank at 100% transmission. | STANDARD CURVE Dilute 10 ml standard potassium phosphate solution to 50 ml with water

(1 ml=0.02 mg P). Pipette aliquots of this solution trom 5 to 40 ml into —

126

Analysis of Fruit and Vegetable Products

50-ml volumetric flasks. Add 5.0 ml of molybdate reagent and mix. Then add _ 2.0 ml of aminonaphtholsulphonic

acid reagent, mix,

make up the volume

to 50.0 ml and measure the colour as in sample. Plot concentration against absorbence.

CALCULATION Read the phosphorus content from the calibration curve. Phosphorus

mgper100g

mg of P in the aliquot of ash _ Total volume : “eat , solution taken for estimation

ml of ash solution taken for estimation

ash solution

+

of

x 100

Wt of sample taken for ashing

References 1. 2.

Fiske, C.H. & Y. Subba Row, J. Biol. Chem., 66, 375 (1925). King, E.J., Biochem. J.. 26, 292 (1932).

IRON The iron in foods is determined by converting the iron to ferric form using oxidizing agents like potassium persulphate or hydrogen peroxide and treating thereafter with potassium thiocyanate to form the red ferric thiocyanate which is measured colorimetrically at 480 nm.! REAGENTS

1. Conc H,SO, (iron-free). 2. Saturated potassium persulphate (K,S,O0,) solution: Shake 7 to 8 g of reagent grade iron free potassium persulphate with 100 ml of water in a glass stoppered bottle. The undissolved excess settles to the bottom and compensates for loss by decomposition. Shake briefly before using. Keep the reagent in the refrigerator. 3. 3 N Potassium thiocyanate (KSCN) solution:. Dissolve 146 g of reagent grade potassium thiocyanate in water and dilute to 500 ml. Filter if turbid. Add 20 ml of pure acetone to improve the keeping quality. 4. Standard iron solution :Dissolve 0.702 g of reagent grade-crystalline ferrous ammonium sulphate [FeSO,.(NH,), SO,.6H,O] in 100 ml of water. Add 5 ml of conc H,SO,, warm slightly, and add conc potassium permanganate solution drop by drop until one drop produces a permanent colour. Transfer to a one-litre volumetric flask, rinse with water and make up to volume. This solution contains 0.1 mg of ferric iron per ml and is stable indefinitely. PROCEDURE

Use the ash solution of the sample prepared by dry ashing for colour deyelopment.

Minerals

127

Colour Development Into three separate stoppered measuring cylinders, pipette the solutions as given below. Blank (ml)

Standard (ml) | Sample (ml)

Standard iron solution 0.0

1.0

Sample ash solution

(1 ml = o.1 mg of Fe)

0.0

0.0

5.0

Water

5.0

4.0

0.0

Conc H,SO,

0.5

0.5

0.5

Potassium persulphate

1.0

1.0

1.0

Potassium

2.0

2.0

2.0

thiocyanate

0.0

In each of the above cases, make up the volume to 15 ml with water. Measure the colour at 480 nm setting the blank at 100% transmission. CALCULATION OD of Iron sample -mg/100g° =—s_—-OD off

sacgiae &

Total volume of x 100 ash solution ee We of ‘sample

standard CALIBRATION

taken for ashing

CuRVE

If it is desired to construct a calibration curve, pipette 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 ml of standard iron solution to stoppered measuring cylinders, add 0.5 ml of conc H,SO,, 1.0 ml of potassium persulphate, 2.0 ml of potassium thiocyanate and make up the volume to 15.0 ml. Measure colour as before. Plot absorbence against the concentration. The concentration of iron in the aliquot of the sample can then be read directly from the calibration curve. Reference

1. Wong, S.Y., J. Biol. Chem., 77, 409 (1928).

POTASSIUM

Flame Photometric Method Potassium in solution is atomized into an oxyhydrogen or oxyacetylene flame. The flame excites atoms of potassium causing them to emit radiations at specific wavelengths.

The amount of radiation

emitted

is

measured

spectrophotometer. Under standard conditions, it is proportional concentration of-potassium in solution.

on

a

to the

128

Analysis of Fruit and Vegetable Products

REAGENTS

1. Potassium chloride (KCI) stock solutions: Dissolve 1.909 g of AR potassium chloride in glass distilled water and make up to 1 litre (1.0 mg K per ml or 1000 ppm). ; 2. Standard solution: Measure 150 ml stock standard solution (containing 150 ppm of potassium) and 5 ml HCl into a flask and make solution to 1 litre. In order to compensate for minute interferences produced by other ions in

the flame photometric determination of potassium, it is recommended that the standard solution be augmented with approximately equivalent concen‘trations of those ions that occur in highest proportions in the sample.being analysed. A background of emission spectra roughly similar to what might be found in an average plant extract is obtained when the standard.contains 150 ppm calcium, 75 ppm magnesium and 15 ppm phosphorus. Dilute aliquots, of the standard solution from 0 to 150 ml making each aliquot to a volume of 150 ml with 0.59% HCl. Atomize as described under procedure given below, setting the top standard at 100°/ transmittance. Note the luminosity of the flame for each concentration. Draw a standard curve by plotting concentration on abscissa and the °4 luminosity on the ordinate. Procepure

_ Dilute an aliquot of ash solutionso that it contains less than 150 ppm potassium. Add sufficient HCI so that the concentration of acid is the same as that in the standard solution. Atomize the diluted extract in a calibrated flame photometer with the wavelength dial set at 768 nm and the transmittance set at 100° for the top standard solution of potassium. Check the instrument periodically with the top standard solution. From the standard curve note the concentration. |

CALCULATION ' Potassium

ppm found from x standard curve

mg/100 g ~ Reference

Volume made up

Dilutions, : x 100 if any

Wt of sample x 1000 Y

1. Ward, G.M. & F.B: Johnston, Chemical Methods ofPlant Analysis, Canada Department ofAgriculture, Publication 1064, p. 19 (1962).

Volumetric

Method

In acid solution, potassium is precipitated as the yellow double cobaltnitrite salt which is dissolved in hot dilute acid and titrated with standard potassium permanganate. Since'the end point is indeterminate, a standard quantity of sodium ardlett.iis added at the end of the titration to produce a giarp end point.}.2

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129

REAGENTS

1. 40% Sodium acetate [NaC,H,O,. 3H,O] solution. 2. i.

Sodium cobalt nitrite solution [Na,Co(NO,)] Dissolve 25 g cobalt nitrate [Co(NO,),. GH,O] in 50 ml water and add 12.5 ml glacial acetic acid. ii. Dissolve 140g sodium nitrite [NaNO,] in 210 ml water. Slowly add the solution to (i). Bubble air through the solution for 3 hr. Filter and Store in refrigerator. Aerate solution for 15 min and filter, if necessary, before using. 3. 12% Sulphuric acid (v/v); Add acid to water slowly, stirring constantly, cool, and make up to volume.

4.

Acetone-water mixture. 300 ml water + 100 ml acetone.

5. Dry acetone: Add anhydrous sodium carbonate (Na,CO,) to acetone in the proportion of 10 g per litre and'store in this condition. 6. 0.01 N Sodium oxalate solution (Na,C,O,). Heat the sodium oxalate overnight at 105° C and cool in a desiccator. Dissolve -67 g in water and dilute to 1 litre. 7. Potassium permanganate (KMnO,). 0.1.N Stock solution: Dissolve 3.16 g in water and make to a volume of 1 litre. Store in a dark-coloured bottle. 0.01 N Standard solution: Dilute 10 ml of stock solution to 100 ml with water. Prepare fresh before using. Standardize by titrating against sodium oxalate solution acidified with 15 ml dil H,SO,. Heat to 80° C before-titrating.

Normality of KMnO,

g of Na,C,O,

|

= ml KMnO, x 0.067 |re ml Na,C,O, x Normality of Na,C,O, ml of KMnO,_

One ml of 0.01 N KMnO, = 0.07 mg K approximately. ait vary slightly with conditions iof experiment. Factor should be determined with standard

potassium solutions.) PROCEDURE Measure 1 ml of ash solution into a 15-ml centrifuge tube. Add 3 ml of water, 1 ml sodium acetate solution and 1 ml sodium cobalt nitrite solution,

the last being added drop: by drop. Mix and allow, to stand for 2 hr at 5° C. Centrifuge at. 1000g for15 min and decant the supernatant liquid. Add 5 ml of acetone-water mixture, mix, centrifuge for 15 min and decant the wash solution. Repeat the washing procedure using acetone. Evaporate the acetone by allowing the precipitate to staid for a few minutes. Add a little ‘standard potassium permanganate from a burette, then.add 2 ml o{dil H,SO,. Complete the reaction by adding permanganate from the burette sith the tube set in a container of ‘boiling water and with constant ‘shaking, always maintaining an excess of permanganate. When the precipitate is completely dis-.

130 ~ Analysis of Fruit and Vegetable Prod:

, obtained, add 2 ml of sodium oxalate: solved:and a permanent pink cc’ solution and titrate to an er. point. True titration _ ml of 0.01 NKMnO, _ ml of 0.01 N Na,C,O, value ty solution solution References

1. 2.

;

:

Kramer, B. & F.K. Tisdall, J. Biol. Chem., 46, 339 (1921). Ward, G.M. & F.B. Johnston, Chemical Methods of Plant Analysis,Canada Department of Agriculture, Publication No. 1064, p. 17 (1962).

SODIUM Flame Photometric Met hod

Sodium in solution is atomized into an oxyhydrogen or oxyacetylene flame. The flame excites atoms of sodium, causing them to emit radiations at specific wavelengths. The amount of radiation emitted is measured on a spectrophotometeti: Under standard conditions it is proportional to the concentration of sodium in solution. REAGENTS 1.. Sodium chloride stock solution: Dissolve 2.5418g of AR sodium chloridé in 1 litre of glass distilled water in a’ volumetric Harte,(1 ml] == 1.0 mg Na). 2. Standard solution: Measure 10 ml of stock standard solution (containing 10 mg of sodium) and 5 ml of HCI into a 1 litre volumetric flask and make to volume with water (this solution contains 10 ppm of Na). In order to compensate for minute interference produced by other ions in the flame photometric determination of sodium, precautions identical to the corresponding case in potassium should be taken.

STANDARD CuRVE Adopt the same procedure as in the case of potassium to draw the standard curve between concentration and per cent luminosity of sodium. PROCEDURE Dilute an aliquot of plant extract

so 'that it contains less than 10 ppm

of

sodium. Add sufficient HCl so that the concentration of acid is the same as that in the standard solution. Atomize the diluted extract in a calibrated flame photometer with the wavelength dial set at 589 nm and the transmit-

tance at 100%, for the top standard solution of sodium. periodically with the top standard solution.

Check the instrument

CALCULATION ‘ -ppm

tf

Sodium mg/100

g

oO

found from the

Volume

made u

standard curve aeteieatee Soonaeeg ee

aay

Cs Dilution x 109 eee

Wt ofsample x 1000

pb

Minerals

a 431

Reference

1. Ward, G.M. & F.B. Johnston, Chemical Methods of Plant Analysis, Canada Department of Agriculture, Publication No. 1064,p. 20 (1962).

COPPER

Copper is isolated and determined colorimetrically as copper diethyldithiocarbamate at pH 8.5 in the presence of ethylenediaminetetraacetic acid (EDTA) as chelating agent. Copper reacts with sodium diethyldithiocarbamate in alkaline solution producing a yellow to brown colour depending on the amount of metal present. The colour is soluble in organic solvents and is extracted from the aqueous solution using carbon tetrachloride and measured colorimetrically.1:?

REAGENTS 1. Sodium diethyldithiocarbamate [(C,H,;);NCS,Na]:, Dissolve 1 g of the salt in water, dilute to 100 ml and filter. Store in refrigerator and ptepare weekly. es Citta tomethy laneddiastinedstctectie acid (EDTA) -solution:. Dissolve 20 g of dibasic ammonium citrate [(NH,),HC,H,;O,] and 5 g of EDTA disodium salt in water and dilute to 100 ml. Remove traces of copper by adding 0.1 ml of carbamate solution and extracting with 10 ml of carbon tetrachloride. Repeat extraction until carbon tetrachloride extract is colourless. 3. 6 N Ammonium hydroxide: Dilute 349.7 ml of ammonia (not less than 279% NH; by weight, sp. gr. = 0.9) to 1 litre with water. 4. 0.1% Thymol blue: Dissolve 0.1 g in water and add enough 0.1 N NaOH to change the colour of the dye to blue and dilute to 100 ml. 5. 2 N Sulphuric acid: Dilute 56.8 ml of AR H,SO, ‘to bus ml. 6. Redistilled carbon tetrachloride (CCl,). 7. Standard copper solution: Dissolve 0.25 g of pure copper wire or foil in 15 ml of HNO, ina conical flask. Cover the flask with watch glass and warm to complete en Boil to expel fumes, cool and dilute to ‘250 ml (solution A). Dilute 25 ml of solution A to 250 ml (1 ml = 0.1 mg) (solution B). To prepare working standard, dilute 5 ml of solution B to 250 ml with 2 N H,SO,

(1 ml = 2.0 pg). As an alternative, dissolve 0.3928 g of copper sulphate (CuSO,.5H,O) in water and make up to mark in a 1-litre volumetric flask. Dilute 2 ml to 100 ml before use (1 ml =2 wg of Cu).

PROCEDURE Pipette 25 ml (or an aliquot of the sample diluted to 25 ml with 2 N H,SO,) from wet digested solution into a 250-ml short stem ‘separating funnel. The ash solution from any of the ashing procedures described earlier may be used. Add 10 ml of citrate-EDTA reagent. Then add 2 drops of thymol

132

Analysis of Fruit and Vegetable Products

Blue indicator and 6 'N NH,OH dropwise until colour turns green or blue _ green. Cool and add 1 ml of carbamate solution and 15 ml of CCl,. Shake vigorously for 2 min, allow the layers to separate and drain off CCl, layer through cotton wad into a glass stoppered tube or flask. Sample blank. Follow the same procedure as that of sample for the blank using a similar aliquot from the blank digest. Measure the colour at 400 nm (or 42 green filter) in a colorimeter setting the blank at 100% transmission.

STANDARD CURVE _ Pipette into separating funnels 0, 1, 2, 5, 10, 20. ait 25 ml of standard copper solution (2 g/ml). Add 2N H,SO, to make up the volume to 25 ml. Extract and develop the colour as in the sample. Plot absorbence against concentration. Since thereis usually some deviation from linearity, read sample values from smoothed curve. CALCULATION et

aS

FePr a mg Yo

=

pg of Cu in . Total volume of the aliquot . digest oI ml of digest taken | Wt of sample taken x 100 for extraction * for digestion

Copper ppm = mg per 100g x 10 References

1. 2.

Ramsey, L.L., J. Assoc. Offic. Agric. Chem., 43, 605 (1960). Callan, T. & J.A.R. Henderson, Analyst, 54, 650 (1929).

TIN

The determination of tin involves reduction of tin in an aliquot of the digest with nascent hydrogen in an atmosphere of CO, (to exclude oxygen) to the stannous form and titration of the stannous tin formed with potassium iodate in the presence of potassium iodide.!>3 The method is suitable for determining tin in amounts of 0-5-1 mg with an accuracy of about 3%. Copper up to 20 ppm or iron up to 200 ppm do not significantly interfere with the process. REAGENTS

1. 3 .N AR Hydrochloric acid: Dilute 294.6 ml of cone HCl (32% HCl) to 1 litre with water,’ 2. Aluminium foil: Purest grade available in pieces of about 1 cm square.

3. 4. 5. agent

5% AR Sodium bicarbonate solution. Starch indicator: 1° soluble starch in 20°, NaCl solution.

Potassium iodide (KI)*: In 100 ml of boiled water contained in a rebottle, dissolve 0.2 g of AR potassium iodide with 3 g AR sodium bicar-

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= 133

bonate. Add a few drops of HCI and shake. When effervescence ceases, insert stopper. 6. 0.1 N Potassium iodate (KIO,) stock goniidpi: Dissolve 5.3505 g afd make up to 1 litre with boiled and cooled water..To prepare working standard (0.005 NN), dilute 10 ml to 200 ml.” 7. Antimony trichloride (SbCl) solution: Dissolve 1.5 g of reagent grade

SbCl; in 50 ml HCl and dilute to 100 ml. 8. Standard tin solution: Dissolve 0.5 g of pure tin in 250 ml conc HCl containing 2 drops of antimony trichloride solution and’ dilute to 500 ml (1 ml = 1.0 mg tin) 3 *NorE; The potassium iodide and iodate’ reagents should be freshly prepared or inf ii daily wheain constant use. PROCEDURE

Remove the product from the can immediately elds opening to ace“rapid . . pick up of tin in thepresence of atmospheric oxygen. Avoid further contamina-. tion during sampling. Macerate the whole contents of can into-a homogeneous puree in a blender. If the drained solids and the liquor are to be analysed separately, the solids should be macerated in a blender. Digest the sample (50 g) as given under wet digestion. Continue digestion until the digest on cooling is pale yellow or light brown in colour. Add 10 ml of hydrogen per- | oxide (30%) dropwise and again heat to fuming. Repeat -the- addition of hydrogen peroxide until the digest is colourless like water. If a white crystal“line deposit of calcium sulphate appéars on cooling the digest, tedissolve it by adding distilled water and warming. Make up the digest to 50 ml with distilled water. Pipette 20 ml of digest into a 150-ml conical Bask with a B24 ;neck. Add1 drop of antimony trichloride solution, 30 ml of 3 N HCl and an excess (about 0.3 g) of aluminium foil. Connect the flask by means of a B24 ground joint and a capillary (2 mm internal diameter) to a “‘suck-back” test tube containing sodium bicarbonate solution (Fig. 6.3). Sodium bicarbonate drawn back

Fig. 6.3:

Apparatus for the determination of tin.

134

Analysis of Fruit and Vegetable Products

into the flask lowers the acidity and generates CO, to maintain the inert . atmosphere. Heat the flask gently until evolution of gas commences and then withdraw heat. When the aluminium is almost completely dissolved, heat again. Disperse any metal particles at the liquid surfaces by careful agitation. Boil until the liquid is clear. Cool the flask in ice water to below room temperature, while still connected to the guard tube. Disconnect the flask and wash down the sides with about 4 ml of potassium iodide solution ‘run in from a 5-ml pipette. Add a few drops of starch indicator and titrate rapidly with 0.005 N potassium iodate solution to a blue end-point stable for several seconds. Make a blank determination on the reagents at the same time which should not exceed 0.2 ml of 0.005 N potassium iodate. CALCULATION

:

pernettin

Titre* x

Normality of _ Total volume x 59.35 x 1000 x KIO, of digest ml taken for estimation x Wt of sample

*Titre = Sample — Blank

Some departure from stoichiometric relations in the reaction between potassium iodate and tin may occur at very low concentrations.’ Therefore, run a series of standards covering the complete range of tin concentrations expected

to be found in the samples and calculate conversion factors for each level of tin. References

1. 2.

McKenzie, H.A., J. Coun. Sci. indusir. Res. Aust., 18, 181 (1945). Kefford, J.F., Food Pres. Quart., 18 (1), 15 (1958). Laboratory Manual for Canners and Processors. (National Canners Association, Pub. Co., Westport, USA, Vol. 2, p. 266 (1968).

USA), Avi

ZINC

The procedure is based on the elimination of lead, copper, cadmium, bismuth, antimony,

mercury

and silver from

the digest

as

sulphides,

elimination

of cobalt as complex of alpha-nitroso-beta naphthol, nickel as complex of dimethyl glyoxime with chloroform andextraction of zinc as zinc dithionate which is measured colorimetrically.1-3 REAGENTS

1.

Cong HNO,

2.

Conc H,SO,

3.

Cone

4.

Chloroform

ammonia

(CHCl,) — redistilled,

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135

5. Diphenylthiocarbazone (dithizone) solution: Dissolve 30 mg of dithizone in 2 ml of ammonia and. 100 ml of water, and extract repeatedly. with CCl, until the solvent layer is clear bright green. Discard the solvent layer and filter the aqueous portion through a washed ashless filter paper. Prepare. the solution fresh. 6. Carbon tetrachloride (CCl,)—redistilled. 7. 0.04 N Hydrochloric acid. 8. Standard zinc solution: Dissolve 0.5 g of pure granulated zinc in slight excess of dilute HC] and dilute to 1000 ml.

For use, dilute 10 ml of this

stock solution to 1000 ml with 0.04 N HCl (1 ml = 5 yg of zinc). 9. Copper sulphate solution: Dissolve 8 g of CuSO,.5H,O in water and dilute to 1 litre (1 ml = 2.0 mg of copper). 10. Ammonium citrate solution: Dissolve 225 g of ammonium citrate [(NH,),HC,H,O,] in water. Adda few drops of phenol red (pH 7.4) indicator. Neutralize with ammonia till there is a distinct colour change. Add 75 ml in excess and make up to 2 litres. Extract this solution immediately before use as follows: : Add slight excess of dithizone and extract with CCl, until the solvent layer is clear bright green. Remove the excess of dithizone by repeated extraction with CHCl,, and finally extract once more with CCl, (Remove dithizone completely as otherwise zinc will be lost during elimination of Co and Ni.) 11. Dimethylglyoxime solution: Dissolve 2g of dimethylglyoxime in 10 ml of ammonia and 200—300 ml of water, filtér and dilute to 1 litre.

12. Alpha-Nitroso-beta-naphthol solution: dilute to 500 ml. Nore:

Dissolve 0.25 g in CHCl, and

Use glass distilled water. Clean glassware with hot HNO,.

PROCEDURE To an aliquot of the sample (prepared by wet digestion) containing 25 to 100 ug of zinc, add 2 drops of methyl red indicator and 1 ml of the CuSQ, solution. Neutralize the H,SO, with NH,OH. Add sufficient ¢onc HCl to make the solution approximately 0.15 N with respect to this acid (approximately 0.5 ml of HCl is required for 50 ml of the solution). Pass H,S into the solution until the precipitation is complete. Filter using Whatman No. 42 paper into a 250-ml beaker. Wash the flask and the filter paper 3 or 4 times with ‘small portions of water. Boil the filtrate gently until there is no odour of H,S. Then add 5 ml of saturated bromine water and continue the boiling until bromine has been expelled. Cool, and neutralize with NH,OH using phenol red as indicator. Make it slightly acidic using 5°94 HCl. Add an excess (0.2 ml) of 1:1 HCI. Dilute the resultant solution to a volurne. For optimum conditions of measurement, solution should contain 0.2-1.0 ug of zinc per ml. To eliminate nickel and cobalt, pipette 20 ml into a 125-ml separating funnel. Add 5 ml of the ammonium citrate solution, 2 ml of the dimethylglyoxime

solution and 10 ml of the alpha-nitroso-beta-naphthol solution, and shake for

136

Analysis of Fruit and Vegetable, Products

2 min. Discard the solvent layer and extract the aqueous phase with 10 ml of CHCl, to remove the residual alpha--nitroso-beta-naphthol. Discard the solvent layer. Isolation and estimation ofzine: To the aqueous phase, following the removal of Ni and Co (pH: 8.0-8.2), add 2.0 ml of the dithizone solution and 10 ml of ‘CCl, and shake for 2 min. Allow the phases to separate and remove the aqueous ave as completely as possible. Withdraw the liquid by means of a pipette attached to a vacuum line. Wash down the sides of the sepatating funnel with approximately 25 ml of water and again draw off the aqueous layer without shaking. Then add 25 ml of the 0.04 N HCLand shake for 1 min to transfer the zinc to the acid-aqueous layer. Drain off and discard the solvent. Carefully dislodge and remove the drop that usually floats on the surface to the acid solution containing zinc, add 5.0 ml of the ammonium

citrate solution,

10.0 ml CCl, (pH 8.8-9.0) and 1.5 times the dithizone required to ex‘tract.20 wg of zinc. Shake for 2 min and allow to separate. Draw off the CCl, layer. Dilute 5.0 ml of this with 10.0 ml of CCl, and measure the colour at 540 nm. STANDARD

CURVE

Pipette 4.0 ml of working standard into a separating funnel and dilute to 25 ml with 0.04 N HCl. Add 5.0 ml of citrate buffer and 10 ml of CCl,. Add 0.1 ml of dithizone, shake for 2 min and note the colour. Add a further 0.1 ml

of dithizone and shake. Continue the increment until the colour in the aqueous phase is faint yellow. Note the volume required. To a series of separating funnels, pipette aliquots of standard solution containing 0, 5, 10, 15 and 20 pg of zinc and dilute to 25 ml with 0.04 N HCl. To each, add 5.0 ml of the ammonium citrate buffer, 10 ml of CCl, and 1.5

times the dithizone required to extract 20 ,g of zinc. Shake for 2 min, allow to separate and drain off the CCl, layer. Dilute 5.0 ml of this with 10.0 ml of CCl, and measure the colour at 540 nm. Plot the transmittance in the logarithmic ‘scale against concentration in the linear scale on a semi-log paper and draw a smooth curve through points. Read the concentration of the sample ‘from the curve.

CALCULATION mg of zinc as read Volume made up after Total from the calibration’ x H,S treatment before x volume of x 100 Barifaz to curve Ni and Co elimination digest

100 g

ml of digest 20 x

taken for x MiG estimation

ppmof Zn = mg of Zn per 100 g Xx 10.

W

Hf

*° ample

x 1000

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137

References

. 2. 3.

Alexander, O.R. & L.V. Taylor, J. Assoc. Offic. Agric: Chem. » 27, 325 (1944)

Official Methods of Analysis, Association of Official dat iGeal Chemists, Washington, D.C., 11th ed., p. 423 (1970). Taylor, L.V. & O.R. Alexander, J. Assoc. Offic. Agric. Chem., 28, 211 (1945).

LEAD

When an ammoniacal cyanide solution of dithizone is added to a solution containing lead, a red precipitate of a lead-dithizone complex . soluble in chloroform or carbon tetrachloride is formed. The red colour is measured at 510 nm. The method given below is that of the Analytical Methods Committee!

of the Society for Analytical

Chemists.

Dithizone is, however,

not

a specific reagent for lead as it forms coloured compounds with 14 other metals as shown in Table 6-1. TABLE 6-1.

Separation of Metals using Dithizone in Chloroform?

Metal ions extracted

f

pH of nae: eliion

Noble mctals+ Hg

or

Cu,, Bi, So7 +

bes

Less than 2 2-3

Zn, Cd, Pb, Ti and all the above

a

4-7

All the above. Washing with 0.04N ammonia solution removes Sn*t+. Addition of KCN leaves only Pb, Ti, or Bi, if not previously removed

Cd remains, Zn is removed

ss

a

7-10

Above

11

For the preliminary separation of lead, two methods ate given. In Method I, lead is extracted with dithizone from an alkaline citrate and hexametaphosphate solution. This method is applicable to fruits and vegetables excluding legumes (peas, beans and pulses) and leafy vegetables, which are not rich in calcium, magnesium and phosphates. Method II requires additional manipulative work and should be used only when Method I will not suffice, particularly in samples (peas, beans and leafy vegetables) that have so high a content of calcium, magnesium and phosphate that sodium hexametaphosphate and ammonium citrate as stated in Method I will not allow the quantitative extraction of lead with dithizone under the alkaline ‘conditions. Therefore, a preliminary extraction of lead from acid solution with a solution. of diethylammonium diethyldithiocarbamate in chloroform has been substituted for the hexametaphosphate procedure.

The Method II is suitable for lead contents up to 5 ppm, but the range can be extended by using a suitable amount of sample.. The method has wide : applicability. Bismuth, however, is liable to interfere. Measure the optical

Analysis of Fruit and Vegetable Products

138

density (OD) at 490 nm and at 520 nm. With a solution of pure bismuth dithizonate, the OD at 490 nm is approximately 1.20 times that at 520 nm. When

considerable bismuth interference is indicated, the modified procedure’ given under “Interference of Bismuth” (see page 140) should be used.

REAGENTS

1. 1% Dilute nitric acid. 2. 5M Hydrochloric acid: Dilute 442.25 ml of conc HCl (35 % by weight, Sp. gr. = 1.1778 at 15°C) to 1 litre with water. 3. Ammonium hydroxide, sp. gr. 0.880. 4, 25°4 Ammonium citrate [((NH,),HC,H,O,] solution w/v in water. 5. 10°4 Sodium hexametaphosphate solution w/v in water. 6. 10°4 Potassium cyanide (KCN) solution w/v in water: Keep for at least 2 days before use to oxidise traces of sulphides that might be’present.

7. 20°4 Hydroxylamine

hydrochloride

(NH,OH.HCI)

solution

w/v in

water. 8. Chloroform (CHCI,): Shake 250 ml of CHCl, with 25 ml of water containing 1 ml of 10°4 KCN solution and about 20 drops of 5 M NH,OH. Separate and reject the aqueous layer. Wash the CHCl, with water and filter. 9. 0.10% Dithizone diphenylthiocarbazone (C,H;.N:N:CS.NH.NH.C,H;), w/v stock solution in CHCl. Filter and store in a refrigerator. 10. Dithizone working solution: Shake 6 ml of the dithizone stock solution with 9 ml of water and 1 ml of 5 M NH,OH. Separate and reject the lower CHCI, layer. Centrifuge the aqueous layer until clear. Prepare this solution freshly on the day of use. 11. Ammoniacal sulphite-cyanide solution: Mix 340 ml of NH,OH (sp. gr. 0.0880), 75 ml of 2% w/v sodium sulphite solution, 30 ml of 10% w/v KCN solution and 605 ml of water. (The concentrations of these reagents are critical.) 12. Standard lead solution: Dissolve 1.60 g of lead nitrate [Pb(NO,),] in water, add 10 ml of conc HNO,, and dilute to 1 litre. To prepare working standard, dilute 1 ml of lead solution to 100 ml with water as required (1 ml = 10 yg of lead).

13.

0.04°% Thymol blue w/v indicator: Warm 0.1 g of thymol blue with

4.3 ml of 0.95 N NaOH and 5 ml of 90% ethanol. plete, dilute with 20° ethanol to 250 ml. Nore:

When dissolution is com-

Use glass distilled water and analytical. grade lead-free reagents throughout.

Destruction of Organic Matter The organic matter may be destroyed either by wet digestion or by dry ashing. If wet digestion is adopted, use not more than 5 ml of conc H,SO,. If much calcium is present, use perchloric acid in place of H,SO,. After digestion is complete, cool, add water, and transfer to 2 100-ml volumetric flask. Rinse

the digestion flask twice with minimum (1-2 ml) quantity of water. Then add

Minerals

139

10 ml of 5 M HCI to a Kjeldahl flask, boil, and, if insoluble matter is present,

filter through ashless filter paper into the volumetric flask. Rinse the digestion flask with water, pass through filter paper and make up to volume. Dry ashing at a temperature not exceeding 500°C is usually convenient for foods which are relatively chloride-free. If necessary, continue ashing overnight. For Method I, use 2 ml of a solution containing 15% of ammonium nitrate and 15% of potassium sulphate as ash aid. For Method II, use 10 ml of 10% magnesium nitrate solution. When a clean ash of the sample is obtained, add 5 ml of water and 10 ml of 5 M HCI and boil gently for 5 min.

Filter through ashless filter paper into a 100-ml volumetric flask. If residue is present, boil with acid, pass through filter paper, wash with water, cool and make up to volume. Separation of Lead

Method I (for samples in which the concentrations of calcium, magiesium and phosphate are not high.) Pipette an aliquot of the ash solution. Add 5 ml of ammonium citrate solution and 10 ml of sodium hexametaphosphate solution. Add a few drops of thymol blue indicator solution and sufficient NH,OH to give a blue-green colour (pH 9.0 to 9.5). Cool, add 1 ml of potassium cyanide solution, and, if much iron is present, add 1 ml of hydroxylamine hydrochloride solution. Transfer the solution to a 100-ml separating funnel containing 10 ml of chloroform and rinse with a few millilitres of water. The volume of the aqueous layer at this stage should be approximately 50 mi. Add 0.5 ml of dithizone working solution, shake vigorously for 1 min, and allow to separate. Run the chloroform layer into a second separating funnel. To the liquid in the first separating funnel, add 3 ml of chloroform and 0.2 ml of dithizone working solution. Shake vigorously for 30 sec, allow the chloroform layer to separate, and add it to the main chloroform extract. The last chloroform extract should be green. If it is not, further extraction with chloroform and dithizone must be made until the green colour of the final extract indicates that all the lead has been extracted. Reject the aqueous layer. Add 10 ml of dil HNO, to the combined chloroform extracts, and shake vigorously for 1 min. Allow to separate and reject the chloroform layer as completely as possible.

Method II (for samples witha high content of calcium, magnesium and phosphate.) The following additional reagents are required for this method.

1. 2. 3.. 4.

Dil H,SO,(1+1). Perchloric acid, sp. gr. 1.54. 20% Sodium iodide w/v solution in water. 1.25% Sodium metabisulphite w/v solution in water:

solution freshly as required and filter before use.

Prepare this

140

= Analysis of Fruit and Vegetable Products

5. 1%, Diethylammonium diethyldithiocarbamate solution (carbamate reagent): Dissolve 1 g of the pure crystalline reagent in 100 ml of redistilled chloroform, and store in an amber coloured bottle. This solution is not stable

and so discard after 1 week. 6. 0.01% Methyl red indicator; Warm 25 mg of methyl red with 0.95 ml of 0.05 N NaOH and 5 ml of 90% ethanol. When dissolution is complete, make up to 250 ml with 50% ethanol. Pipette an aliquot of the ash solution. Add 2 drops ot methyl red indicator and make just alkaline with NH,OH. Make the solution just acid with 5M HCl and add a further 10 ml. Warm the solution (50° to 70° C), add 2 ml

of sodium iodide solution, and reduce any liberated iodine with 2 ml of sodium metabisulphite solution. Cool the solution, transfer to a separating funnel, and adjust the volume to 50 to 75 ml so that the concentration of the solution is 1 N with respect to HCl. Add 50 ml of carbamate reagent by pipette and shake the funnel vigorously for 50 sec. Allow the layers to separate and transfer the chloroform layer to a 100-ml flask. Wash the aqueous layer twice with small amounts of chloroform without mixing and add these washings to the flask. Repeat the extraction with 10 ml of carbamate reagent and add the second extract to the main extract. Reject the aqueous layer. To the combined extracts, add 2.0 ml of dil HjSO, and evaporate the chloroform. Add 0.5 ml of perchloric acid to the residual solution, and heat until fumes ate evolved and the fuming solution is clear and colourless. Cool the solution, add 10 ml of water and 5 ml of 5 M HCl,

boil for 1 min; cool, and

then add 2 ml of ammonium citrate solution. Add a few drops of thymol blue indicator solution and continue separation of lead as given im Method I. DETERMINATION

To the HNO, layer left in the separating funnel, add 30 ml of ammoniacal sulphate-cyanide solution, exactly 10 ml of chloroform, and 0.5 ml of‘dithizone working solution. Shake vigorously for 1 min and allow to settle. Run off a little of the chloroform layer, insert a plug of cotton-wool into the dry stem of the funnel, and, after rejecting the first few drops, collect in a cuvette.

Measure the colour at 510 nm in a spectrophotometer or colorimeter. Measure the colour of the test and the blank solutions against chloroform. STANDARD

CURVE

Measure 0, 1.0, 2.0, 3.0 and 4.0 ml of working standard lead solution (1 ml = 10 xg of Pb) into separating funnels and dilute each to 10 ml with dil HNO,. Proceed as described under “Determination of lead.” Measure the colour using chloroform as blank. Plot OD against concentration of lead. Interference

of Bismuth

The following additional reagents are required for extraction of bismuth.

1. HCl, sp. gr. ,J.18.

|

re

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141

2. 1%. Diethylammonium diethyldithiocarbamate solution (carbamate reagent): Prepare as described under Method II, Prepare the digest by wet decomposition or by dry ashing. If the organic matter has been destroyed by wet decomposition, add to the contents of the flask 6.0 ml of HCl.and transfer the solution to a 50-ml separating funnel. Rinse the conical flask with several 1-ml portions of water, and add the rinsings to the separating funnel. The volume of the contents of the separating fennel must not exceed 35 ml in order that the HCl concentration may be not less than 3 N (see Note). Extract the acid solution directly in the cold, first with 10 ml and then with 5 ml of carbamate reagent, shaking for 30 sec each time. Separate and discard the lower (chloroform) layer. Finally, shake the acid layer with 5 ml of chloroform for 10 to 15 sec and discard the chloroform layer. Transfer the acid layer to a 100-ml conical flask, rinse the separating funnel with a few millilitres of

water, and add the rinsings to the conical flask. Proceed as in Method I or II. If the organic matter has been destroyed by dry ashing, add 15 ml of HCl (sp. gr. 1.18), transfer to a 50-ml separating funnel, and adjust the volume of the solution to a maximum

of 35 ml in order that the HCl concentration

may be approximately 6 N (see Note).

Continue as described in the previous

paragraph. Nore: After wet digestion, the extraction solution consists of the residual H,SO, to which HCl has been added; the acidity of the solution should not be less than 3 N in either H,SO, or HCl. When the organic matter has been destroyed by dry ashing, the extraction solution consists of HCl alone, and the acidity must be raised to about

6 NW in HCl to

effect quantitative separation of bismuth and other clements from lead.

CALCULATION Leadas Pb _

mg/100g

mg of lead as read Volume,,.0f 14, 100 from standard curve a digest ie

Ss ml of digest taken

x Wt of sample x. 1000

Lead ppm= mg of lead per 100 g x 10 References

1. 2.

Analytical Methods Committce, Analyst, $4, 127 (1959). Hibbard P.L., Ind. Eng. Chem,, Anal. Edn., 9, 127 (1937).

ARSENIC

Arsenic may be determined either by the Gutziet method or by the colorimetric molybdenum—blue method. The Association of Official Analytical Chemists: (AOAC) in the USA and the Analytical Methods Committee (AMC) in the UK: have described procedures for both the methods. Minor variations exist with regard to apparatus and the procedures described. The Gutziet method described es the AMC involves preliminary distitlation of the digest

142

Analysis of Fruit and Vegetable Products

which is excluded in the AOAC method. The AOAC method is suitable for routine determination. The preliminary extraction of arsenic by distillation in Gutziet and colorimetric methods recommended by the AMC eliminates most of the interference from other metals.

The colorimetric method involves

considerable manipulative procedures. All the three methods are described below.

Gutziet Method (as modified by AOAC) Arsenic in the wet digest of the sample is reduced to arsene by nascent hydrogen which causes a stain on a strip of mercuric bromide paper. The length of the stain is proportional to the concentration of arsenic in the sample which is estimated by comparing with the length of ,the stain produced by standard arsenic solution of different concentrations. REAGENTS

1. Stannous chloride solution: Dissolve 40 g of arsenic-free SnCl, 2H,O in HCl and make up to 100 ml with HCl. 2. Zinc: Use arsenic-free granulated zinc. 3. Ammonium oxalate [(NH,),C,0,.H,O]—saturated solution. 4. Potassium iodide (KI) solution: Dissolve 15 g of KI in water and dilute to 100 ml. 5. Sand: Clean 30 mesh (through 30 mesh, but not 40 mesh) white seasand: Prepare by washing successively with hot 10 % NaOH solution, hot HNO, and hot water. Dry the clean sand. 6. Standard arsenic solution: Dissolve 1 g of arsenic trioxide (As,O,) in 25 ml of 20% NaOH solution. Saturate the solution with CO, and dilute to 1 litre with recently boiled and cooled water (1 ml = 1 mg of As,O,). Dilute 40 ml of this solution to 1 litre. Make 50 ml of this diluted solution to 1 litre (1 ml = 0.002 mg of As,O,). Use this solution for preparing standard stains. Prepare fresh dilute solutions at frequent intervals. 7. Mercuric bromide (HgBr,) paper: Prepare HgBr, paper by cutting No. 40 Whatman filter paper into strips 2.5 mm wide and 12 cm long. Soak the strips for 1 hr in a fresh 5 % solution of HgBrg in 95 % alcohol. Dry and store in amber coloured bottles covered on the outside with black paper. Use these strips as soon as possible. Handle the sheets by paper attached to either end and cut in half just before use. Strips must be clean and free of any contamination. APPARATUS

Generators and absorption tubes. Set up the generator as shown in Fig. 6.4. Use 2-oz wide-mouthed bottles as generator. Fit a glass tube 1 cm in diamieter and 6 to 7 cm long, with a constricted end to the stopper. Place a small wad of glass wool in constricted bottom end of the tube and add 3.5 to 4.0 g of the sand, taking care to have the same quantity in each tube. Moisten the

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143

Fig. 6.4: Generator used for determination of arsenic in the Gutziet method. (Reprinted from Official Methods of Analysis, 11th edm., Pp. 400, 1970, with permission of the Association of

Official Analytical Chemists, Washington, DC, USA)

sand with 10° lead acetate solution and remove the excess by light suction. Clean the sand, when necessary, without removing the sand from the tube by treatment with HNO,. Rinse with water under suction. Treat with the lead acetate solution. Ifisand has dried, clean and remoisten. Connect the tube by means of a rubber stopper with a narrow glass tube, 2.6~-2.7 mm in internal diameter and 10 to 12 cmrlong. Clean and dry the tube. Introduce clean end of the strip of HgBr, paper into the narrow tube without curling which results in ‘uneven stain and poor end point. DETERMINATION

Destroy the organic matter in the sample by wet digestion. Make up the digest to a known volume with water. Determine the H,SO, present in a definite volume of sample solution by titration. Place aliquots (not exceeding 30 ml) containing 0.01 to 0.03°mg of As,O, in the Gutziet generator. If arsenicin aliavnts taken is found to be outside the limits specified, repeat

determinativu with a proper aliquot.

Add sufficient 25% arsenic free NaOH

solution to exactly neutralize the H,SO, present. Then add 5 ml of HCl and cool, if necessary. Add 5 ml of potassium iodide reagent followed by 4 drops of the stannous chloride solution. Prepare standards cortesponding to 0.01, 0.02 and 0.03 mg of AsO, from the standard arsenic solution. Since standards must contain the same kind and quantities of acid as in samples, add 5 ml of

- HClas in the sample. If the H,SO, has been neutralized, add equivalent quan-

144

— Analysis of Fruit and Vegetable Products

tity of arsenic-free Na,SO, to standards. Mix and allow to stand for 30 min at not less than 25° C or 5 min at 90° C. Dilute with water to 40 ml. Prepare the generator and the centre strip of HgBr, paper as stated earlier. Add to each of the standards and the samples 2 to 5 g of granulated zinc. The same quantity must be added to each generator. If sheets of strips are used, prepare the sample and standard strips from the same strip. Keep at room temperature (20-25° C) or immerse the apparatus to within 1 in. of the top of narrow tube in a water bath (20-25° C) and allow the evolution to proceed for 1.5 hr. Remove the strips and note the average length of stains on both sides of the paper in mm. Plot the length of the stain in mm as ordinate and mg of. As,O, as abscissa. From the graph, note the quantity of arsenic present in the sample: Take smaller or larger aliquots when stain is longer or shorter than the highest or the lowest standard respectively. Report as ppm. Frequent blank should be made, with reagents of suitable quality. Blank should not be more than 0.001 mg of As,O3. CALCULATION

; Pras et

mg of As,O, of the sample as read from graph.

205 mg/1008 = ~~ Of digest taken

*

Total volume of digest

— -*x ‘Wet of sample

ane

As,O, as ppm == mg of As,O, per 100g x 10 Reference

1.

Official Methods of Analysis, Association-of Official-Analytical Chemists, Washington, DC, USA, 11th edn., p. 399 (1979).

Gutziet Method (as modifted by AMC)

After destruction of the organic matter by wet oxidation and distillation as trichloride, the arsenic is reduced by nascent hydrogen to arsene which

produces a stain on the mercuric chloride paper. The extent of stain produced is taken as a measure of arsenic by comparison With the stain produced by known concentrations of arsenic. The method is suitable for arsenic contents from 0.5 to 5 mg (as As) in the sample taken. APPARATUS

Use glassware made of borosilicate or Corning glass. Immediately before use, clean with H,SO, and HNO, and thoroughly wash with water to ensure that it does not yield traces of arsenic. va : Distillation apparatus: This apparatus is described in the “Molybdenumblue.colorimetric method” given in page 147. | — | Gutziet apparatus: Fit a wide-mouthed bottle of 120-ml capacity with a rubber bung havinga hole, Fit into this hole a glass tube of length 200 mm

Mineruts

145

and an internal diameter of exactly 4.0 mm (external diameter about 5.5 mm) drawn out at the lower end to a diameter of about 2 mm (Fig. 6.5). A. hole, Rubber band 4mm

. 5.5 mm

Ground faces

Hole 3 mm dia.

2mm

Fig. 6.5: Device for holding Gutzict test paper. (Reprinted with permission of the Society for Analytical Chemistry, UR)

about 3 mm in diameter is situated at the side of the tube near the constricted part. The constricted end of the tube, inserted into the bottle (containing 70 ml of liquid) through the bung, should be above the surface of the liquid and the hole in the side should be below the bottom of the bung. The tube must have a shallow projection, 0.4 to 0.5 mm thick, at its upper end which must fit into a slight depression in a cap consisting of a short tube of the same internal diameter as the lower tube. The contacting edges of the two tubes must be flanged and ground smooth at right angles to the tube so as to fit closely. The short upper tube must have a grooved top, and the lower tube must have two projections, in order to secure the upper tube to the lower by means of a rubber band.

REAGENTS 1. Chloride-hydrazine-bromide mixture: Grind together intimately 5 parts of NaCl, 0.5 part of hydrazine sulphate, and 0.22 part. of potassium bromide. 2. Conc AR HCl— arsenic-free. 3.

Lead acetate treated cotton-wool:

Moisten

cotton-wool

with a 10%

solution of lead acetate in water and dry. 4, Mercuric chloride paper: Soak Whatman No. 1 filter paper in a saturated solution of AR mercuric chloride in water, press to remove superfluous solution, dry at about 60° Cin the dark, and discard the edges of heor

paper.

Store in the dark in a stoppered bottle until required.

146

Analysis of Fruit and Vegetable Products

5. Stannous chloride solution: Dissolve 65 g of AR stannous chloride, SnClz2H,O, in 180 ml of water and 200 ml of HCl. Boil until the volume has been reduced to 200 ml, cool, and filter through a fine-grained filter paper. Store in a well-stoppered bottle. 6. HCl-stannous chloride solution: Mix 10 ml of stannous chloride solution with 90 ml of HCL. 7. Potassium iodide AR — arsenic-free. 8. Zinc: Use arsenic-free zinc pellets. 9. Standard arsenic solution: Dissolve 0.066 g of arsenious oxide (As,O3) in 50 ml of HCl and dilute to 100 ml with water. Dilute 1 ml of this solution just before assay to 100 ml with water (1 ml = 5 pg of As). PROCEDURE

Destroy the organic matter in the sample and distil the arsenic present in the wet digest as given in the ‘“Molybdenum-blue colorimetric method” (see page 147). Make up the distillate to 50 ml in a volumetric flask using distilled water. Carry out a blank test using the same amounts of-reagents as are used in the test except the sample. ‘DETERMINATION

Preparation of apparatus: Pack the lower glass tube of the apparatus lightly _ with lead acetate treated cotton-wool, so that the upper surface of the cottonwool is 25 to 30 mm below the top of the tube. Place a piece of mercuric chloride paper against the ground end of this tube with the smooth face of the filter paper downwards to form a diaphragm between it and the upper tube, and fasten the tubes together by means of a rubber band. (The paper may also be folded and fastened by a rubber band instead of fastening between two tubes.) Treatment of test and blank solutions, (i) If the expected arsenic content of the test solution is more than 5 wg, mix an aliquot (X ml, where X is not more than 25) of the test solution containing 2 to 5 wg of arsenic with HCl in the proportions X: (8—16X/50) ml. Dilute the mixture to 60 ml with water and add 1 ml of HCl-stannous chloride solution and 1 g of potassium iodide. (ii) If the expected arsenic content of the test solution is 5 yg or less, add to the whole of the solution 1 ml of HCl-stannous chloride solution and 1 g of potassium iodide, and dilute to 60 ml with water. (iii) Treat the blank solution as

in (ii). Transfer the diluted test and blank solutions to separate wide-mouthed bottles, add 10 g of zinc to each, and immediately place the prepared glass tubes in position, protecting them from sunlight throughout the remainder of the test. Allow the reaction to proceed without the application of external heat fot 15 min and transfer the bottles to a water-bath maintained at 35 to 40° C for 30 min. Compare, in normal daylight, the stain produced on the mercuric chloride paper with a series of freshly prepared standard stains,

Minerais

147

Standard stains, Measute known amounts of dilute standard arsenic solution, in the range 0.5 to 5.0 wg of arsenic. Add 1 ml of HCl stannous chloride solution and 1 g of potassium iodide. Dilute to 60 ml with water, and complete the estimation as given for the diluted test sample. From the colours of the stains, estimate the amounts of arsenic in the test

and blank solutions and calculate the amount of arsenic in the sample. Reference 1. Analytical Methods Committee, Analyst, 85, 629 (1960).

Molybdenum-Blue Colorimetric Method (as modified by AMC)! After a preliminary extraction of the digest of the sample with diethy]lammonium diethyldithiocarbamate solution, the arsenic in the extract is converted to the arsenomolybdate complex, which is then reduced by means of hydrazine sulphate to a molybdenum-blue compound which is measured at

840 nm. The method is suitable for determination of arsenic from 1.5 to 15 pg in the sample taken. APPARATUS Use glassware of borosilicate or Corning glass. Thoroughly clean with HaSO, and HNO,,

and wash thoroughly with water

immediately before use.

““Cold-finger” condensers (Fig. 6.6): These consist of smalk test tubes with flanged mouths, fitting loosely into 50-ml conical flasks. When the condenser is in position, the bottom should be about 10 to 15 mm from the bottom of the flask. Distillation apparatus (Fig. 6.7): Connect a Kjeldahl flask of a capacity 100 or 200 ml with a ground-glass (B19 or B24) joint to a 2-bulb or 3-bulb con-

hw 10 to 15 mm

os a

[

Fig. 6.6: Cold-finger condenser.

Fig. 6.7:

Distillation apparatus.

A flask ofooor

(Reprinted with the permission of the

200 ml capacity with a neck length of 7 to 8 inches,

Society for Analytical Chemistry, UK)

B & C bulbs of condensers of 25 mi capacity, D samé as B & Cbut may be omitted in the two-bulb version. (Reprinted with permission of the Society for Analytical Chemistry, UK)

Analysis of Fruit and Vegetable Products |

148

denser carrying a side funnel with a tap. If the wet digestion of the sample is carried out in a Kjeldahl-flask having a ground-glass joint, the arsenic can be

distilled from the’ same flask. REAGENTS 1.

HCl, AR

arsenic-free.

2. Thioglycollic acid (HS.CH,-COOH)

solution: Dilute

12 g of thio-

glycollic acid (90% v/v) to 100 ml with water. Store in an amber coloured — bottle (stable for one month). 3. Potassium iodide-ascorbic acid solution: Dissolve 15 g of potassium igdide and 2.5 g of ascorbic acid in water, and dilute to 100 ml with water. Prepare freshly every 2 or 3 days. 4, Chloroform — Redistilled or AR grade. 5. Dithiocarbamate solution: Dissolve 1 g of AR grade diethylammonium

diethyldithiocarbamate in 100 ml of chloroform. Store in amber coloured glass bottle, and discard after one week.

6. 1.NH,SO,:

Prepare from AR H,SO, and store in a polythene bottle.

7. Acid molybdate solution: Mix exactly 250 ml of 10 N H,SO, (accurately standardized) with exactly 250 ml of a 7% w/v solution of ammonium molybdate [(NHa)gMo7O,,. 4H2O]in water. Filter into a 1-litre volumetric flask, washing the filter paper with water. Add exactly 250 ml of 4 N perchloric acid (accurately standardized) and dilute to mark with water. Store in a polythene

bottle.

m

8. Standard arsenic solution: Dissolve 4.17 g_ of AR sodium arsenate (Nag HAsO,7H,O) in water and dilute to 1 litre with water. Before use dilute 10 ml to 1 litre with water (1 ml = 10 yg of As). 9. 0.03% Hydrazine sulphate (H,NNH,. H,SO,) w/v solution in water. 10. Chloride-hydrazine-bromide mixture: This reagent is required if only the distillation procedure is followed. Mix intimately NaCl, hydrazine sulphate, and potassium bromide in the ratio of 5 : 0.5: 0.02 by weight and grind.

PROCEDURE Decompose the organic matter in the sample by wet digestion. During digestion, do not allow to dry as arsenic would volatilize. Do not use more

than 10 ml of H,SO,. If the sample taken is less than 5 g and if the arsenic is extracted directly from this solution, reduce the amount of H,SO, at the rate

of 20 ml for every 1g’ or less sample taken, but in no case to less than 5 ml. This reduction in the amount of acid will have no deleterious influence on the

subsequent extraction of arsenic. Measure all the reagents used for decomposition and carry out a blank using the same amounts of reagents.

Preliminary Treatment of Digest when Necessary lfthe total heavy metal content of the sample digest exceeds 100 pg, and/ or if there is an excessive amount of insoluble material present in the sample

digest and distil off the arsenic from the sample as described below.

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149

If the H,SO, used in the wet decomposition is less than 8 to 10 ml, supplement at this point. Set up the distillation apparatus as shown in Fig. 6.7. .If the wet decomposition has not been carried out in the Kjeldahl flask, transfer the digest to it. Rinse with the minimum amount of water, evaporate until white fumes are evolved, and allow to cool. Add 7 ml of water, and cool again. Add 5 g of chloride-hydrazine-bromide mixture. Avoid: contamination of the ground portion of the neck of the flask while adding, and fit the condenser. Moisten the joint with water to prevent leakage. Clamp the apparatus so that the condenser is vertical and its tip is just short of the bottom of a 25-ml measuring cylinder containing 15 ml of water. Cool the measuring cylinder in a bath containing ice and water. Add, through the side funnel, 10 ml of HCl and carefully close the tap. Heat the flask with a microburner at a rate such that the contents are brought to boiling in not less than 30 min. After the condenser has become full of steam, continue to heat the flask so that the distillation proceeds smoothly for 3 to 5 min. During the whole of this procedute, particularly at the moment when the steam reaches the cold water in the receiver, take care to prevent a suck back. Do not take distillation too far, i.e. white fumes of sulphur trioxide must not be evolved. When the distillation is complete, open the tap, remove the burner, and disconnect the condenser. Wash down the con-

denser once with a few millilitres of water, collecting the washings in the cylinder. Separation of Arsenic (i) If the distillation of the digest has been carried out, transfer the distillate, without further addition of HCl, to a 100-ml conical flask. Rinse with minimum

amount of water. (ii) If the digest has not been distilled, dilute the digest with 15 ml of water, boil gently for a few minutes, cool to about 70° C, add 10 ml

of HCl and allow to cool. Transfer the solution to a 100-ml conical flask. Filter if the solution is not clear and rinse with minimum amount of water. Warm the solution from (i) or (ii) to about 40°C, add 2 ml of thioglycollic acid solution, mix well, and allow to cool for 15 min. Then cool the solution

more rapidly in a bath of ice and water to room temperature. Add 1 ml of potassium iodide-ascorbic acid solution, wash down the sides of the flask with a few millilitres of water, and mix carefully. Transfer the solution to a 100-ml calibrated separating funnel containing a few millilitres of chloroform. Rinse the conical flask with several small portions of water. The volume of the solution at this stage should be 45 to 50 ml. Add 5 ml of dithiocarbamate solution and shake vigorously for 40 sec. Remove the stopper, wash with a few drops of chloroform, allowing the washings to drain into the separating funnel and to separate. Transfer the lower chloroform layer to a clean 25-m! separating funnel, taking care not to allow any of the aqueous layer to enter the tap of the first separating funnel. Wash the aqueous layer twice with 0.5 ml portions of chloroform, without mixing, and add the washings to the main extract.

150.

Analysis of Fruit and Vegetable Products

Extract the aqueous layer with a further 2-ml portion of dithiocarbamate solution, shake for 30 sec, and allow to separate. Transfer the lower chloroform

layer to the second funnel, washing the aqueous layer twice with 0.5 ml portions of chloroform as before, and adding the washings to the main extract. Discard the aqueous layer. To remove interference from phosphates, add 10 ml of 1 N H,SO, to the combined extracts in the second separating funnel, shake for 5 sec, and allow to separate. Transfer the lower chloroform layer to a 50-ml conical flask. Wash the acid aqueous layer with two small portions of chloroform without mixing, and.add the washings to the chloroform solution in the flask. During this operation, take care not to allow any of the aqueous layer to enter the tap of the funnel.

Add 2.0(-+ 0.02) ml of acid-molybdate solution to the chloroform solution, close the mouth of the flask with a glass bulb, and evaporate off the chloroform

on a boiling water bath. Evaporate slowly to avoid spattering. When the the flask to a hot-plate, and evaporate chloroform has been removed, transfer until fumes of perchloric acid evolve accompanied by a sudden teaction. Continue to heat for another 1 min (not longer). Allow to cool and remove the glass bulb. Wash it with a few drops of water and allow the washings to drain into the flask. Heat, without the glass bulb in position, until white fumes

evolve again. All traces of organic matter should have disappeared by this stage. Insert into the flask a cold-finger condenser, filled almost to the brim with cold water. Ensure that the outside of the condenser is clean and dry. Place the flask on the hot-plate (a hot-plate with a surface temperature of approximately 250° C.is suitable for this operation), heat for 10 min at a temperature such that a blanket of fumes fills about half of the flask and the temperature of the water in the condenser rises to about 90° C (--5° C), and allow to cool. Wash down the condenser and the sides of the flask with 7 ml of 1.N H,SO, and then with 2 ml of water, using pipette for both additions. Close the mouth of the flask with a glass bulb, boil until the total volume is reduced to 6 or 7 ml and free chlorine has been removed, and clear and colourless at this stage. STANDARD

cool. The solution

should be

CuRVE

Transfer 0.2, 0.6, 1.0, 1.5 and 2.0 ml of dilute standard arsenic solution to a series of 50-ml conical flasks. To each add 2 ml of acid molybdate solution

and 7 ml of 1 N H,SO,, mix well, and heat on a hot-plate until the volume is reduced to 5—6 ml. Cool the solutions, add 1 ml of hydrazine sulphate solution to each.mix, and transfer the solutions to a series of 10-ml stoppered cylinders, rinsing with a small amount of water and using the washings to dilute the respective solutions to the 10-ml mark. Mix thoroughly and return each solution to its original 50-ml conical flask. Close the mouth of each flask with a glass bulb, heat ona boiling water bath for 15 min, and allow to cool

Minerals

151

for 30 min. If any fading due to the presence of excess of chlorine is observed at this stage, reboil and repeat reduction with hydrazine sulphate of the test and blank solutions. Measure the colour in a spectrophotometer at 840 nm. Plot OD against the amount of arsenic in micrograms.

Determination of Arsenic in Test and Blank Solutions Add to the solution remaining in the flask (see “Separation of arsenic’) 1 ml of hydrazine sulphate solution and continue as stated under “Standard curve.” Measure the OD of the test solution against the blank. Read from the standard curve the number of micrograms of arsenic in the test solution and calculate the amount of arsenic in the sample. The OD of the blank solution consisting of 2 ml of acid molybdate solution and 8.0 ml of 1 N H,SO, should not be more than 0 to 0.05 at 840 nm. Purification of Reagents Thioglycollic acid solution:; Extract 100 ml, first with 10 ml of dithiocarbamate solution and then with two 5-ml portions of chloroform. Store in a polythene bottle and prepare freshly every 2 or 3 days. Acid molybdate solution: Measure exactly 250 ml of a 794 w/v solution of ammonium molybdate in water and extract first with two 10-ml portions of dithiocarbamate solution and then with three 5-ml portions of chloroform. The final chloroform extract should not be darker than the colour of straw. Some batches of ammonium molybdate give more deeply coloured second extracts; such material will give high blank values and should not be used for the determination of trace quantities of arsenic. Mix the extracted solution with exactly 250 ml of 10 N H,SO, (prepared from AR H,SO, and accurately standardized) and filter into a 1-litre calibrated flask, washing the filter with water. Add exactly 250 ml of 4 N perchloric acid (prepared from perchloric acid, 60% w/v and accurately standardized), and dilute to the mark with water. Store in polythene bottle. Reference 1.

Analytical Methods

Committee,

Analyst, 85, 629 (1960).

ESTIMATION OF MINERALS BY ATOMIC ABSORPTION SPECTROSCOPY Atomic absorption spectroscopy and flame photometry are important analytical techniques used for the detection and determination of metals in foods and other allied products. Atomic absorption spectroscopy and flame photometry are closely related. Normal flames, which are hot enough to achieve dissociation of salt or

element to free radicals, have energy sufficient enough for a small proportion of liberated metal atoms to become excited while the rest remain in the ground state.

152.

Analysis of Fruit and Vegetable Products

Those which are activated will return immediately to the ground state with the emission of radiation. The measurement of this emitted radiation is “flame photometry” which is generally made use of in the estimation of potassium and sodium described earlier. The unexcited atoms forming the major fraction of the sample in the flame can be made to absorb radiations of the correct wavelength from an external source, thus reaching the higher energy level. The measurement of the radiations absorbed is atomic absorption spectroscopy. The total amount of energy absorbed by the sample element to achieve the excited state is equivalent to the number of electrons available — in other words, to the concentration of the

element in the sample. Thus the amount of external energy supplied to achieve such transitions is an indication of the concentration, subject to calibration of the system. This forms the basis of atomic absorption spectroscopy, viz. the measurement of the amount of energy of the correct wavelength necessary to. elevate the metal atoms from ground state to the excited state. The ratio of the number of atoms which become excited without recourse to external energy sources to the number which remain in the ground state is giverbyBoltzmann Distribution. Nx N°

where

Nx = number of atoms in the N° = number of atoms in the A = constant for a particular K = universal constant (1.3805 T = temperature (K)

excited state ground state system X 10° erg/k)

This shows that the higher the temperature of the flame, the greater will be the number of atoms in the excited state. Therefore, for atomic absorption spectroscopy, the temperature of the flame should be kept as low as possible. The processes occurring in the flame are the following:

When the test solution is aspirated into the flame, the solution is broken into drops; the solvent evaporates and the solid is dispersed in the flame. The heat energy from the combustion and the collisions between the melecules produces atoms, ions and free radicals. Neutral atoms require the smallest amount of energy to raise to the excited level. Hollow cathode lamps supply the necessary energy, the wavelength of which is exactly similar to that of atoms in the flame. The excited atoms return to the ground state by losing its energy by molecular collision or giving off a ray of radiation. INSTRUMENTATION

While flame photometer consists of atomiser, monochromator, detector and

electronics, atomic absorption spectrophotometer consists of hollow cathode lamp or electrode-less discharge lamp (source of external energy), chopper, atomiser, monochromator, detector and electronics.

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153

METHOD

The organic material is removed by dry ashing or wet digestion. The residue is

dissolved in dilute acid, and sprayed into the flame of an atomic absorption spectrophotometer. The absorption or emission of the metal to be analysed is measured at a specific wavelength.

REAGENTS 1. 0.3 N, 3.0 N and 6.0 N HCI.

2. 10% w/v Lanthanum chloride solution. 3. Standard solutions of metals: Weigh AR grade chemicals (Table 6-2) which give standard solutions having 1000 mg/litre of the reagent. Dissolve the chemical in 25 ml of 3 N HCl, and dilute to 250 ml with water. Dilute the standard solution

so prepared with water, if the sample has been prepared by wet digestion, or with 0.3 N HCL, if the sample has been prepared by dry ashing, to prepare a series of standards so that the concentrations of the metal is within the working range given in Table 6-2. Add other salts, if necessary, as indicated in Table 6-3.

TABLE 6-2 : Weight of Material to be taken for Preparation of Standard Soiutions

We of reagent g/litre of

Metal

Reagent

Calcium

Calcium carbonate, CaCO3

2.496

Copper

(dry) Copper sulphate, CuSO,4.5H:,O

3.824

Iron

Iron alum,

solution

Fe2(SO4)3(NH,4)2S04.24H2,0

8.632

Lead

Lead nitrate, Pb(NOs)2

Magnesium

Magnesium sulphate, MgSO,4.7H2O

10.120

Manganese Potassium ‘Sodium

1.5985

Manganese sulphate, MnSO,.4H2O

4.060

Potassium chloride*, KCI Sodium chloride*, NaC}

2.544

1.904

Cadmium

Cadmium metal, Cd

1.000

Tin

Tin metal, Sn

1.000

Mercury

Mercuric chloride, HgClo

1.354

Zinc

Zinc sulphate, ZnSO4.7H2O Zinc metal, Zn

4.400 1.000

Arsenic

Arsenious oxide, As203

1.320

“ Dry for 2 hr at 105° Cc.

154

= Analysis of Fruit and Vegetable Products

TABLE 6-3: Recommended Conditions for Metal Analyses using Air-Acetylene Flame wibeetsetel = weeds nele pee bes aye De eth erel Seee b he eee Absorption Limit of detection (A) or Wavelength Salt to Element pg/ml Brie nm be added

ee

range” ug/ml

$$

a

193.7

Arsenic

Calcium

ot

Working

0.5% LaCl

0.26

50.00-200.00

0.01

0.05-5.0

A A

0.002 0.006

0.05-5.0: 0.05-5.0 *

422.7 324.8 248.3

Copper Iron

A

Lead

217.0

0.015

5.0-20.0

Magnesium

285.2

A

0.001

0.02-0.5

1.0 mg Na/ml

279.5 766.5

A A

0.005 0.002

0.02-3.0 0.01-2.0

-do-

766.5

E

0.002

1.00-20.0

1.0 mg K/ml -do-

589.0

A

0.002

0.10-1.0

589.0

E

Manganese Potassium Sodium

0.002

1.00-20.0

Cadmium

228.8

0.0007

0.50-2.0

Tin

224.6

0.03

15.00-60.00

upto 300

Zinc Mercury

213.9 254.0

A

0.015

0.4-1.6 upto 300

* Maximum level indicates the limit of linear working range.

APPARATUS

Atomic absorption spectrophotometer (AAS) with hollow cathode lamp of the specific metal to be estimated which gives the specified resonant wavelength. Thoroughly wash all glass apparatus used in the determination of metals by AAS method with dilute HNO 3. Keep the glassware used for trace metals separately. PROCEDURE Ashing Dry ashing: Unless otherwise stated, organic matter present in the sample is destroyed by dry ashing in a muffle furnace at a temperature not exceeding 550° C. See the procedure described in the beginning of this chapter. To the ash, add 5-10 ml of 6N HC] to wet it completely, and heat to dryness at low temperature on a hot plate. Add 15 ml of 3 N HCL, and heat on a hot plate until the solution just boils. Cool and filter through an hardened ashless filter paper (Whatman 541) which had been washed with 3 N HC] before use. Collect the filtrate in a volumetric flask. Add 10 ml of 3 N HCI to the dish, and heat until the solution just begins to boil,

cool and filter through the same filter paper into the volumetric flask. Wash the

Minerals

155

dish thrice with water, and filter the washings through the filter paper. Wash the filter paper and collect the washings in the flask.

If calcium is to be determined, add 5 ml of lanthanum chloride solution per 100 ml of solution. Cool the contents, and make up to volume with water. Prepare a blank by taking the same amount of reagents as in the case of sample. Wet Digestion: In case of dried products (samples containing less than 10% moisture), take 2 g of the sample. If the sample contains more than 10% moisture, take 5g or more, and transfer toa100-ml Kjeldahl flask. Add 10 ml conc HzSO, and

shake vigorously. Ensure that there are no dry lumps. Add 5 ml of conc HNOs and mix. Heat gently in a fume cupboard until the initial vigorous reaction has subsided. Thereafter, heat vigorously until most of the nitrous fume has ceased to evolve. Add HNO; dropwise, and continue heating until all the organic matter is destroyed and white fumes of H2SO, evolve. Cool the digest, add water, again cool,

transfer to a volumetric flask, and make up the volume with water. Carry out a blank through the operations using the same amount of reagents as in the case of sample. MEASUREMENT Set the apparatus according to the manufacturer’s instructions. Working conditions for lead and copper are given as examples in Table 6-4. Use water (if wet digestion method is used) or 0.3 N HCl (if dry ashing method is used) as blank. Note the readings of the standard solutions, and draw a calibration graph of concentration («g/ml of metal) against absorption or emission. Measure the sample solutions and the reagent blank. While running the samples, ensure periodically that the calibration values remain constant. TABLE 6-4: Working Conditions of Lead and Copper Lead

Copper

Air flow rate

L/hr

500

500

Acetylene flow rate

L/hr

100

100 0.02-0.03

Slit width

0.06

‘Lamp current

5.0

5.0

Sensitivity range

90-100

90-100

Photomultiplier setting Gain Time

4 3-4 4

4 2 2

CALCULATION

mA

sec

.

Metal content, ppm _

a—b) XV

(or mg/1000 g)

WV

Metal content |

_(a—b)X V X 100

mg/100 g

“WX

1000

156

Analysis of Fruit and Vegetable Products

g b V W

where

= concentration =concentration = volume made = weight of the

of the metal in the sample solution, ug/ml of the metal in the sample blank, ug/ml up of the ash solution or digest of the sample, ml sample, g.

AOAC recommended methods for specific metals are given below:

Cadmium

PRINCIPLE Sample is digested using HNOs3, H2SO, and H2O. All metals are extracted from the digest using dithiozone in chloroform at pH 9.0. Cadmium is removed from the extract by acidifying the dithiozone extract with dilute HCl, and is determined by using atomic absorption spectrophotometer at 228.8 nm. REAGENTS 1. 50% HeOz, solution.

2. Dithiozone solution: Weigh 200 mg and dissolve in 200 ml of chloroform (1 ml = 1.0 mg). Dilute 50 ml to 250 ml with chloroform when required for use (1 ml = 0.2 mg). 3. Cadmium standard solution: Dissolve 0.25 g of pure (99.9%) cadmium metal

in 41 ml of conc HCI in a 250-ml volumetric flask, and make up to volume with water (1 ml = 1.0 mg) (stock solution). Dilute 10 ml of this solution to 1000 ml with 2 N HCl just before use (1 ml = 10 wg) (intermediate standard). Prepare working standards by diluting C, 1, 5, 10 and 20 ml of intermediate standard solution to 100 ml with 2 N HCI so that the concentration of cadmium is 0, 0.1, 0.5, 1.0 and 2.0 wg per ml.

4. Thymol blue indicator: Grind 0.1 g of the indicator with 0.05 N NaOH inan agate mortar, and dilute to 200 ml with water. PROCEDURE

To 50 g of sample in 1500 ml beaker, add 100 ml of HNOs, and allow to stand overnight at room temperature. Heat on a burner until nitric oxide fumes begin to evolve. Control frothing by cooling or by adding water from a wash bottle. If the sample contains fat, allow the digest to cool, and filter the supernatant through glass wool. To the residue, add 100 ml of water to rinse the fat, chill and filter. Wash the funnel and the glass wool pad with approximately 20 ml of water. To the HNOs digest or to the filtrate contained in 1500 ml beaker, add 20 ml of conc H2SQ,, and dilute with water to about 300 ml. Evaporate over the flame.

When the charring is excessive, add 1 ml of 50% H2Oz, and heat vigorously to let the SO3 fumes evolve. Continue adding 1 ml of HO. ata time and heating until the

charred remove Carry used in

mass has become clear, and the digest is colourless. Heat vigorously to excess H2Oz, and cool to room temperature. © out a blank through the same operations using aliquots of the reagents = the sample.

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157

EXTRACTION

Add 2 g of citric acid to cooled digest, and cautiously dilute to approximately 25 ml with water. Cool in an ice bath. Add 1 ml of thymol blue as indicator. Add ammonia slowly with mixing and cooling until the solution changes from yellowish green to greenish blue (pH 8.8). Transfer the solution to a 250-ml separating funnel. Wash the beaker with water, and transfer the wash water to the separating funnel, dilute to about 150 ml, and cool. Extract with two 5 ml portions of dithiozone, shaking 1-2 min each time. Transfer the extract to a 125-ml separating funnel. Extract with further 5 ml portion of dithiozone until the extract shows no further change in colour. Wash the combined extract with 50 ml of water. Transfer the solvent layer to another 125-ml separating funnel. Wash the aqueous layer with 5 ml of chloroform, and add this to dithiozone extracts. Add 50 ml of 0.2 N HCl to the combined dithiozone extracts, shake vigorously for 1 min, and allow the layers to separate. Discard the dithiozone layer. Wash the aqueous layer with 5 ml of chloroform, and discard the chloroform layer. Transfer the aqueous layer quantitatively to a 500-ml beaker and ‘evaporate to dryness. When dry, wash the sides of the beaker with 10-20 ml of water and again evaporate to dryness. . Dissolve the dry residue in 5 ml of 2 N HCI, and use for determination.

DETERMINATION Set the instrument to previously established optimum conditions using air-acetylene as oxidising flame, and hollow cathode tube of 228.8 nm resonant wavelength. ' Using 2 N HCI as blank, determine the absorption of the standard solutions. Flush the burner with water between readings. Draw a calibration curve of absorption against micrograms of cadmium per ml. Determine the absorption of sample and that of sample blank. If the concentration of cadmium in the sample is more than 2.0 ug/ml, dilute the solution with 2 N HCl. Determine the cadmium content from the calibration curve. CALCULATION

:

Cadmium, ppm

_ ml of 2 N HCl =

¢

of sample

X pg Cd/ ml.

Lead PRINCIPLE

Samples of food are digested using HNO3, H2SO, and HCIO, (perchloric acid). Lead in the digest is coprecipitated with strontium sulphate (SrSO,). The precipitate is separated ftom the digest, and converted to carbonate by agitation with ammonium carbonate solution. The carbonates are dissolved in 1 N HNOs, and the solution is aspirated into the flame of an atomic absorption spectrophotometer. Light emitted from a lead hollow cathode lamp is passed

158

Analysis of Fruit and Vegetable Products

through the flame. The lead content of the sample solution is determined by atomic absorption at 217 nm or 283.3 nm. REAGENTS 1. 2% Strontium solution: Dissolve 6 g SrCle.6H2O in 100 ml of water. 2. Ternary acid mixture: Add 20 ml H2SO, to 100 ml of water and mix. To this add 100 ml of HNO; and 40 ml of 70% HCIO,, and mix.

3. 2 N HNO. 4. 1 N HCl. 5. Lead standard solution: Stock solution—1 mg/ml. Dissolve 1.5985 g of pure lead nitrate crystals in 1% HNOs, and dilute to 1000 ml with 1% HNOs. Prepare more dilute solution with 1% HNOsz as needed.

Intermediate standard solution: Pipette 2 ml of stock solution into 100-ml volumetric flask, and dilute to volume with 1% HNO; (1 ml = 20 yg) (Solution A).

Pipette 25 ml of solution A into 500-ml volumetric flask, and dilute to volume with 1% HNOsz (1 ml = 1 wg) (Solution B).

Working standard solution: Make up 10, 20, 30 and 40 ml of solution B to 100 ml each with 1% HNOs to get solutions having 0.1, 0.2,0.3 and 0.4 ug/ml respectively. PROCEDURE

Accurately weigh sample containing 10 g of dry matter and 3 pg of lead or more. Place in a 500-ml Kjeldahl flask, and add 1 ml 2% Sr solution and glass beads. Prepare reagent blank, and carry through the same operations as in the case of sample. Add 15 ml of ternary acid mixture for each gram of dry matter, and allow to stand for 2 hr or more. Heat under hood until the flask contains only H2SO, and inorganic salts. (Avoid sample loss from foaming when heat is first applied. Foaming occurs soon after sample chars. Remove heat, and swirl the flask before continuing digestion.) Add HNOs, if necessary. Cool the digest for a few minutes. The digest should be cool enough to add 15 ml of water safely but hot enough to boil when the water is added. Wash, while still hot, into a 40-50 ml

tapered-bottom centrifuge tube, and swirl. Allow to cool, centrifuge for 10 min at 350 X g, and decant the liqu’d into a beaker meant for collecting the water. (Film like precipitate on surface may be discarded.) Dislodge the precipitate by vigorous stirring. To complete the transfer, add 20 ml of water and 1 ml of 1 N H,SO, to

original flask, and heat. Do not omit this step even though the transfer appears to be complete in the first wash. Wash the hot contents of the original digestion flask into the centrifuge tube containing the precipitate. Swirl to mix, cool, centrifuge, and decant the liquid into waste beaker. Dislodge the precipitate by stirring vigorously, add 25 ml of saturated ammonium carbonate solution (approximately 20%), and stir well until all precipitate is dispersed. Allow to stand for one hour, centrifuge, and decant the

liquid into a spare After decanting, liquid. Add 5 ml of blank if more than

beaker. Repeat treatment with ammonium carbonate. invert the centrifuge tube on a filter paper, and drain all the 1 N HNOs. Use larger volume of 1 N HNOs in both sample and 25 yg of lead is expected. Stir vigorously to expel CO2. Allow to

Minerals

159

stand for 30 min, and centrifuge if precipitate remains. Use the same technique for all samples including blank. DETERMINATION Set the instrument to acetylene oxidising flame of sample solution, blank lead/ml. Determine lead

previously established optimum and 217 nm resonant wavelength, and standard lead solutions. Plot content of the sample from the

conditions. Using air determine absorbence absorbence against ug standard curve.

CALCULATION

Pb, ppm = [(ug of Pb/ml) x (ml of 1NHNOs)]/g of sample. ;

Mercury

PRINCIPLE The sample is digested using H2SO,, HNO3 and HCIO, (perchloric acid). The

digest is treated with a reducing solution. Mercury vapours are flushed into the gas flow-through cell of atomic absorption spectrophotometer. Light emitted from mercury hollow cathode lamp is passed through the cell. The mercury content of the sample is determined at 253.7 nm. APPARATUS 1. Atomic absorption spectrophotometer equipped with Hg hollow cathode lamp and gas flow-through cell (Fig. 6.8). 2. Digestion apparatus. 3. Water condenser: Use a condenser of 12-18 mm internal diameter and 400 mm length made of borosilicate glass and having 24/40 standard joint, modified to hold 6 mm Raschig rings. Fill the condenser with Raschig rings to a height of 100 mm, and then place a 20 mm layer of 4 mm diameter glass beads on the top of the rings. 4. Digestion flask: Use a 250-ml flat bottom boiling flask with 24/40 standard joint. 5. Pump: Use a diaphragm pump with internal parts coated with acrylic paint, and having 16 gauge teflon tubing for all connections. 6. Gas inlet adapter having 24/40 standard joint. REAGENTS 1. Reducing solution: Mix 50 ml of H2SO, with 300 ml of water. Cool to room temperature and dissolve 15 g of NaCl, 15 g of hydroxylamine sulphate and 25 g of SnCl, in solution. Dilute to 500 ml.

2. Diluting solution: To 1000 ml volumetric flask containing 300-500 ml of water, add 58 ml of HNOs and 67 ml of H2SO,. Dilute to volume with water.

3. Magnesium perchlorate: It is placed as drying agent in filter flask and is replaced as needed [Caution: Mg(ClO,)2 is explosive when in contact with organic substances].

160

Analysis of Fruit and Vegetable Products

4. 2% Sodium molybdate solution. 5. Mercury standard solution: Dissolve 0.1354 g of HgCle in 100 ml of water (1 ml = 1000 yg). Dilute 1.0 ml of this solution to 1 litre with 1 N H,2SO, before use (1 ml = 1 pg). PROCEDURE

Weigh 5 g of sample into the digestion flask. Add 25 ml of 18 N H2SO,, 20 ml 7 N HNOs, 1 ml of 2% sodium molybdate solution, and 5-6 boiling chips. Connect the condenser to the digestion flask. Circulate water through the condenser. Heat the flask gently for one hour. Remove heat, and allow to stand for 15 min: Add 20 ml of HNO3-HCIO, (1 + 1) mixture through the condenser. Stop the water circulation through the condenser, and boil vigorously until white fumes appear in the flask. Continue heating for 10 min. Cool the flask, and cautiously add 10 ml of water through the condenser while swirling the liquid in the flask. Again boil the solution for 10 min. Remove heat, and wash the condenser with three 15 ml portions of water. Cool the digest to room temperature, and make up to 100 ml in a volumetric flask with water. Transfer 25 ml aliquot to another digestion flask. Adjust the volume to about 100 ml with diluting solution (see reagent 2). Adjust the output of the pump to about 2 litres of air/min by regulating the speed of the pump with a variable transformer. Connect the apparatus as shown in Fig. 6.8 except for gas inlet adapter. With pump working and spectrophotometer

set to zero, add 20 ml of reducing solution to diluted aliquot. Immediately connect the gas inlet adapter, and aerate for about 3 min. (Adjust the aeration time to obtain maximum absorption.) Record the absorption, disconnect the pressure on the ‘out’ side of the pump, and open the vent on filter-flask to flush the system.

16 Gauge tetlon tubing

--adgapter |

115 cm Cell LX

Vent with clamp

__ 125 mi Filtering tlask

10g Mg (CIO,Ip 250 ml Boiling tlask

Fig. 6.8 Apparatus for flameless atomic absorption analysis.

(Reprinted from: Official Methods of Analysis, 12th edition, 1975, with the

permission of the Association of Official Analytical Chemists, Arlington VA 22209)

Minerals

; 161.

Prepare reagent blank and standard curve by adding 0.0, 0.02, 0.5, 1.0, 1.5 and 2.0

“pg of mercury to a series of digestion flasks. To each flask, add 100 ml of diluting solution. Finally add the reducing solution, aerate the standards, as in the case of

sample, and note the absorbence. Plot absorbence against ug of Hg. Determine wg of Hg in the sample from the curve. He, ppm = ug Hg/g of sample.

Zinc The AOAC procedure is similar to the general procedure described. Reference

g

Official Methods of Analysis, Association of the Official Analytical Chemists, P.O. Box 540, Benjamin Franklin Station, Washington, DC 20044, 1980 (13th edn.).

‘CHAPTER 7.

Maturity Indices and Quality Criteria INTRODUCTION

CotouR, flavour and texture determine the quality of fruits and vegetables for processing. In contrast to these requirements, the grower is concerned with ease _of cultivation, disease resistance and yield. A good quality processed product cannot be made from poor quality raw material. Requirements for processing -with respect to size, shape, colour, texture and flavour are more exacting chap for the fresh prodtice market. The stage of harvesting determines the quality. Most of the fruits are harvested when they are mature and allowed to ripen subsequently during transit, storage or when displayed in retail shops. In such cases, the maturity standards should ‘correlate with the quality of the fruit when ripe. On the other hand, the optimal stage ofmaturity of most of the vegetables is transient, and delay in harvesting by a day of two may render them more mature. Consequently, it is difficult to define with precision the maturity indices in relation to the required quality of the horticultural produce. The maturity and the quality are more easily felt than. described or determined. In practice, maturity is determined by one or more of the following methods: a. Computation of days from bloom to harvest. ~'b. Measurement of heat units. c. Visual means — skin colour, persistence or drying of parts of the plant, fullness of fruit, etc.

d. Physical methods — ease of separation, pressure test, density grading, etc. e. Chemical

methods

—

total solids, sugars, acid, sugar-acid

ratio, starch

content, etc.

f. Physiological methods — respiration rate, etc. Each method has its own advantage and limitations. In spite of these limitations, maturity indices for various fruits and vegetables are specified. In USA, the Agriculture Department specifies standards for US Extra Fancy, Fancy, No. 1, No. 2 and commercial grades. The Organization for Economic Cooperation and Development

(OECD)

of some 25 countries in Europe has

developed and published detailed grades and quality standards for a number of fruits and vegetables!. These standards include colour, appearance, juice or soluble solids contents, fruit size and allowable defects. Excellent colour prints are

Provided to illustrate the desirable and undesirable product characteristics.

Maturity Indices and Quality Criteria

- 163

Standards help to specify the type of product the seller delivers, which the consumer receives with certain expectations. Standards must describe characteristics such as maturity, colour, cleanliness, shape, freedom} from av and blemishes, and uniformity of size. ms, In this chapter, maturity indices and quality requirements are given. Procedures for determining maturity by physico-chemical methods are described, in Chapter 18.

The subject matter of quality, maturity indices, etc. are discussed in detail in the works of Hulme’, Ryall and Pentzer* and Pantastico‘ to which the readers amay

refer for detailed information. Harvest indices have been discussed by Pantastico et a/.+ and quality of raw materials for processing by Rodriguez et al. APPLE

a

“The best maturity index is the number of days ror full bloom to harvest',,; Delicious variety, harvested after 135 to 140 days from bloom, ripens with fair dessert quality. Optimum maturity is reached after 140 to 150 days, when the fruit is ideal for prolonged storage. Late maturity is 150 days from full bloom. For other varieties, see Fig. 7.1.

Evaluation of indices of maturity MATURITY

DD petimu Late VARIETY

Williams

Early Red---ZN

McIntosh

ee

eB

Jonathan

Grimes

YAN

Golden

Delicious

-------------------------

Golden

Deliciolus--—

Yellow

Newtown

Rome

|

==

.et meee

a

------+-fY

ae

eas

NS

77 LS

Beauty

Stayman

Winesap------

~------

-------------------

é

Winesap York-Imperial-

.-----------

70

80

w-------------------

90

100

110

Elapsed

Fig. 7.1:

120 130

time

from

140

BY

150 160 170

bloom (days)

Days from full bloom to harvest maturity of apples. (Source: Haller and Smith®)

180

164

Analysis of Fruit and Vegetable Products

Other indices of harvest are the following:

a. Colour of the skin: It is not a good criterion for varieties having Bak skin colour. b. Ease of separation of the fruit stem from the spur of the tree: It is not a good criterion when preharvest sprays have been used to prevent fruit drops. c. Firmness of flesh: Not a good index of maturity. Fruits must be harvested at optimum maturity, if required for long storage. The firmness of apples in relation to pressure test is shown in Fig. 7.2.

Ss aWw Gh “Ni ©&

= oO N

(pounds) test Pressure

Golden

Delicious|Delicious|

Fig. 7.2:

Rome

Beauty

Albemarle

| Stayman

Pippin

Firmness of apples in relation to pressure test. (Source: Haller °)

Apples must be sound, clean, free from decay and physiological diseases such as internal breakdown, scald, soft scald, bitter pit, water core, etc. When purchasing,

select firm, crisp, well coloured fruits. Do not choose overripe apples that are soft, dull coloured or have a mealy textured flesh. Look for soft, decayed spots on the fruit. Brown discolouration on the skin is an indication of scald but the injury is only skin deep. Fruits picked early are susceptible to scald.

APRICOT

Fruits do not mature uniformly. Selective harvesting has to be done. According to US Standards, maturity means fruits which have reached the stage of maturity

Maturity Indices and Quality Criteria

165

that will insure a proper completion of the ripening process. In USA, under Californian conditions, at least three-fourths of outside surface area must have attained a yellowish green colour, and the flesh should be at least half yellow as

described in a standard colour chart. This is a reasonable method though subjective. The number of days from full bloom, and the accumulated heat units above 45° F are used as guides by growers in setting the harvest dates.'°"! The first six weeks after full bloom is critical in respect of temperature effects.'2 High temperature (102-103° F) during 2 or 3 days, late in the growing season, causes- softening a3theflesh near the pit and subsequent browning. Apricots must be tree ripe to attain a pleasing davoaremesencae of the fruit. Do not select fruits with greenish colour that are firm to hard. Select fruits/with* full colour, firm ripe to ripe, but not mushy and overripe. Soft fruits might have decayed. Soft spots indicate the beginning of the decay. “Harvesting should be done, for drying, when full ripe; for canning, when less mature but still firm

- enough to be pitted and processed; and for freezing, when more ripe than for canning. Maturity test of Tilton variety of apricot using Magness-Taylor Pressure Tester with 5/16 inch diameter plunger was as follows.'3

Mature but not ripe Average commercial maturity

Pressure

Consumer

test (Ib) 8.5 11.3

acceptance (%) 85 a0

(hoo) (%) ‘u3 10.0

—-

CITRUS FRUITS Sweet Orange

Maturity standards are largely based on juice yields, soluble solids content and Brix-acid ratio. These vary depending upon the area of cultivation, season, etc. as shown below: ° Brix

Florida, USA

es 10°

Texas, USA

° Brix-acid ratio

10.5 : 1.0 05: 1.0.

8.5

10.0 :1.0

pour

9.0: 10

aWhen the actual °Brix is as given, the Brix-acid should faaich International Standards Organisation (ISO) sets the minimum iuice content for

166

Analysis of Fruit and Vegetable Products

sweet oranges as: Thomson Navel and Washington Navel orange 30%, and other “orange varieties 35%. The quality characteristics of highest quality include firmness, good weight for size, skin and flesh colour, and skin texture. All these characteristics vary with the variety, area of production, etc. Bright orange colour is always preferred by the buyer, but is not a dependable index of the internal quality of the fruit. For example, Californian oranges are bright coloured but Florida and Texas oranges are pale yellowish orange when fully mature. Similarly Mosambi (C. sinensis Osbeck) grown in Andhra Pradesh is bright orange yellow, while Sathgudi (C. sinensis Osbeck) grown in Tamil Nadu is greenish yellow, when fully mature. Some oranges, during early harvest season,contain some chlorophyll in the peel, although meeting all harvest requirements. Similarly, late in the harvest period, when the growth condition of the tree is active, with the ripening of the fruit, additional chlorophyll may accumulate in the peel. This behaviour, termed regreening, does not in any way affect eating quality of the fruit.

Mandarin In Coorg (India), fruits are harvested from December

to January, when the

colour changes from green to orange, the acidity is 0.4% and the TSS range from 12 to 14%. Desirable characteristics of the mandarin group, which includes tangerines and tangelos, are smooth surface, thin skin, good weight in relation to size, high colour for the particular variety, and freedom from physical damage or decay. According to ISO, minimum juice content required is 33%. Grape Fruit |

Good quality grape fruit must have a pleasant blend of sweetness and tartness. Harvest maturity is attained when the fruits have attained acceptable eating quality which is dependent upon juice content of the fruit, total sugars, and the sugar-acid ratio. These characteristics vary with the area of cultivation, season, etc. In US, separate standards have been laid down for each growing area as shown below: Florida, State ° Brix

8.1-9.0

‘Sugar-acid ratio

Jito, I

.

°Bhix

11.0-11.1

Sugar-acid ratio

6.25. to 1

California State

6 to 1 in desert areas

5.5 to 1 in other areas*

®Characteristic yellow colour over 2/3 of the fruit surface..

According to ISO, grape fruits must yield 35% juice. The fruits should be firm, light to medium yellow, smooth skinned, globular to somewhat flattened at stem _ and stylar ends, clean, and have no evidence of physical damage or incipient

Maturity Indices and Quality Criteria

infection. The skinned. Light with extensive sunken brown

167

best fruits are heavy iin relation: fo size, and not Auitiy or loose russeting of the peel does not affect internal quality. Avoid fruits scab or melanose. Fruits shoild be free from surface pitting or areas around the stem.

Lemon

>

Fruits are harvested when the surface colour is still green. The colour develops during storage or prolonged transit. The only standard laid down in USA is the juice content which should be not less than 30%, and in the green fruits for export, not less than 28%. According to ISO standards, the juice yield of lemons should be not less than 25%. Lemons of good quality, irrespective of the size and shape, must be light to medium yellow, firm, smooth skinned, and heavy for their size. They should be free from decay, physical damage, sunburn and oil spots. Internally, they should be juicy and free from discolouration. Bronze colour, dry and hard skin, and black buttons indicate aging. Such fruits may have off-flavour and lack normal juice:

content. Production of citrus fruits and processing are discussed in detail in two volumes édited by Nagy et al.'4 Limes

Ripe limes of Indian, West Indies: and Mexican origin have ‘yellow to slight orange yellow colour. Good quality fruits should be firm, smooth skinned, free from scars, thorn scratches, sunburn, or disease. The flesh should be fine-grained

and greenish yellow. The fruits should be juicy and very acid (6%).

BERRY FRUITS Gooseberries and Currants

Processors must set their own standards. Fresh gooseberries and currants should be firm and bright, with colour characteristic of the variety, and be free

from injury or insect decay.

Raspberries

\

‘Fresh fruits should be firm, bright, well developed“and not soft, broken or

overripe. The whole surface of each berry should show a colour characteristic of _the variety, whether red, black or purple. They should be free of bruising, crushing or mould growth. Berries with adhering cores are usually immature, and should

be avoided.

.

168

Analysis of Fruit and Vegetable Products

Cherries

~ Cherries-are of two kinds — sweet and sour. Sweet cherries should be dark red in colour which indicates that the fruit has been left long enough on the tree to attain sweetness and full flavour. The colour should be bright; the skin glossy; and the fruit, plump with fresh, green stems.'> The fruits do not ripen uniformly. Soluble solids may range from 15 to 22%. Selective picking is not possible, and hence, the fruits are harvested when the average ripeness is of eating quality: Tests for maturity include separation by brine floatation and refractometer reading. US Standards for No.1 sweet cherries specify fruits to be mature (stage that will ensure proper completion of the ripening process), fairly well coloured, _ well formed, clean and free from specified defects. Bing cultivar of California must

have not less than 16% soluble solids. Lamberts must have light red colour. According to US Standards for No.1 grade, red sour cherries must be fairly well coloured; 5/8 inch in diameter; free from decay, worms, pulled pits, attached stems, and from damage caused by bird pecks, nail marks, limb rubs, wind whips and other scars, sun

scald, shrivelling,

foreign

material,

diseases,

insect or

mechanical damage.

DATES Dates vary with variety in texture, contents of invert sugar and sucrose, moisture, and colour. Fresh dates must be plump, smooth, golden brown to almost

black depending on variety and be free from dirt, mould, insect and surface sugar crystals. They may be soft and syrupy, or firm and dry depending on type and end use. FIGS Figs must be fully ripe at harvest to be of good keeping quality. Harvest maturity is based on colour and firmness. Black Mission figs should be light to purple rather than full black, and should yield to slight pressure rather than being soft ripe. Calymyrna figs should be yellowish white to light yellow, and firm to slightly yielding for best harvest maturity. Figs must be harvested when fully mature, if they-are to be sweet, and have good flavour. At this stage of maturity, they are highly perishable and easily bruised. Consequently, the moulds find the soft, sweet flesh an excellent media for growth. In the case of Mission figs, select fully coloured fruits that are soft and ready to eat. Check for moulding spots or any internal sign of damage. Calymyrna figs are large and yellowish. Choose the large well coloured fruits, free from surface mould and internal spoilage. Kadota figs are used for canning. They are firm fruits of medium size and have greenish yellow to yellow colour when ripe. For canning Burnswick or Magnolia fig, pick before it is ripe,i.e., when the fruit has turned f om greentoa pale green, sometimes mottled green before it is soft. For making preserves, pick when fully ripe. |

Maturity Indices and Quality Criteria

169

PEARS

All commercial varieties of pear must be harvested while hard and green in colour. The time of harvest is critical. The desert fruits picked too early will be poor, and if picked too late, the storage life is shortened. Pears are classified as summer and winter pears. Summer pears include Bartlett variety (called Williams Bon Chretien in many countries). It is mostly used for canning. During maturity, the colour changes from dark green to light green or yellowish green. The US Department of Agriculture has a colour chart of 4 shades ranging from green to light yellow. Unlike apple, firmness of pear flesh declines from wellbefore ripeness to eating ripeness. Hence, firmness of flesh has been made use of as a test for measuring the maturity of pears. Bartlett pears when tested with Magness-Taylor Pressure Tester having 5/16 inch plunger on a paired surface must yield the following values: 1. Fresh marke — High t quality: under 20 Jb —Best quality: 19-17 Ib ca8 3. Best canned quality: 17-15 lb The Agricultural Code of California specifies that Bartlett pears are considered mature, if they comply with the following: a. The average pressure test, of not less than 10 representative samples of each commercial size in any lot, does not exceed 23 Ib. b. The soluble solids in a sample of juice, from not less than 10 samples of each

commercial size in any lot, is not less than 13%. c. The colour of the pears corresponds to the yellowish-green colour of the chart.

In USA, the €alifornia Tree Fruit Agreement considers the fruit to be mature if they meet the following standards: Minimum Solids

Soluble

Maximum Pressure (lb) Size (inch)

Zoe. 1]

2.5 and larger

10%

19.0 Ib

20.0 Ib

10% 11% 12%

20.0 Ib 20.5 Ib 21.0 Ib

21.0 Ib 21.5 Ib 22.0 Ib

In lieu of any pressure test, pears are considered mature, if the TSS its 13%, or the colour is yellowish green corresponding to No.3 on the standard colour chart.'6 Winter pears: Anjou, Comice, Bosc, Winternelis and Ester are the important varieties. The firmness of the flesh at best harvest maturity varies with the area,

and must be 10-14Ib for Anjou pears, 9-12.5 Ibfor Comice, 11-18 lb for Bosc, 10-14

~ Ib for Winternelis, and 15-16 lb for Ester using Magness-Taylor Pressure Tester

with 5/16 inch plunger.

170

Analysis of Fruit and Vegetable Products

The time from full bloom to harvest, which ranges from 135-155 days, is also a - good index of maturity. In California, the colour of the skin is also made use of for determining maturity. The fruits should not be allowed to riperi on the tree as the flesh around the core breaks down and develops off-flavour. Pears must be harvested when still firm. The pressure test is one of the indices of maturity. Other indices are skin colour, soluble solids content, and the length of the growing season. Pears harvested too early will not ripen to good quality, and are susceptible to scald. Pears harvested too late are susceptible to core breakdown which is more serious in climates where the summer is cool. Wilted or shrivelled fruits indicate that they have been harvested too early or have remained too long after harvest. Similarly, pears with brown spots on the surface or soft areas in the flesh should not be used. PEACH The fruits might be either freestone or clingstone, and yellow or white fleshed. They do not mature uniformly, and have to be picked as and when they ripen. Colour, size, and suture filling are the indices used for picking the fruit from the tree. The parameters used for measuring the maturity of peach are Magness-Taylor pressure test (5/16 inch plunger) made on both sides of the cheek for firmness of flesh, titratable acidity, and in some, total chlorophyll content in flesh. The pressure test readings at eating ripeness range from 0.5 to 1.5 lb.'’ The firmer a peach is when harvested, the longer it takes to ripen. Peaches harvested with pressure test reading of 10 lb require 7 to 8 days to ripen at 70° F. If such fruits, after harvest, are stored at 42° F for 9 days, thereafter 7 days are required for ripening at 70° F. The longer the peaches are allowed to mature and grow on the tree until ripe before they are picked, the better is their edibility when ripe,'’"® but this is not possible when the fruits have to be transported to long distances. When the pressure test in Magness-Taylor Pressure Tester with 5/16 inch plunger on both pared cheeks exceeds 16 to 17 lb, the fruits may be designated as immature.'’”* Peaches testing above this do nat ripen with acceptable dessert quality, while those testing from 10 lb to the critical firmness for the cultivar, have acceptable quality after ripening, and enough life to withstand transportation and sale.

PLUMS°*® The colcur characteristic of the variety when ripe is a good criterion for determining harvest maturity. The colour of the mature plum may belight green, yellow or red depending upon the variety. Since the colour develops after harvest, the colour of the fruit in retail stores is nota definite indication of maturity of fruit. In purchasing the fruit of unknown variety, select medium to large fruits having a deep colour characteristic of the variety..The fruits should be soft. and ripe.

Maturity Indices and Quality Criteria

171

Overripe fruits will have poor flavour and are often affected with brown rot or other fruit rots. The characteristic colour of some of the important varieties at harvest maturity are as follows: Beauty: 85% yellowish green or traces of red. Burmosa: Yellowish green or 50% red. Santarosa: 40% red colour or full light greenish yellow. Fellenberg: 75% purple. —

STRAWBERRY*® _

Selection of the fruit is on the basis of colour. The fruit should be harvested when ¥% to *4 of the fruit has developed colour. In California, strawberry with 2/3 surface having pink or red colour is considered mature. Fresh strawberries must be ¢lean, bright and firm with attached calyx. Most berries should be full red, and all have at least 3/4 surface red or pink. A dull or shrunken appearance indicates overripeness. The berries should be of uniform size, free from cuts, bruises and mould growth. The US No.1 Grade berry should be 3/4 inch in diameter with a tolerance of 5% for berries below the minimum.

GRAPES

Grapes should be mature and fairly well coloured. Maturity is determined based on the ease with which the berry separates from the bunch. Maturity at harvest is also based on the colour, although the soluble solids content of the berries is important to the processors, and is commonly used to establish the harvest time. Winkler2° examined grapes grown in California under different climatic conditions and found that soluble solids content above 20% assures good quality. When the soluble solids are below 20%, a soluble solids-acid ratio is a better indicator of quality. Thompson Seedless

°Brix a

Brix : acid ratio 5

Cornichon, Muscat, Ohnez, Emperor

and Tokay Red Malaga

17 16

30:1 399: A

Ribier

16

Dial

In Sultana (Thompson seedless) grown in Israel, the sugar-acid ratio is used as a good index for harvest maturity. The Agriculture Code of California defines “harvest maturity at a minimum 16.5% 78s.in the juice, with variants from the standard established foi certain varieties.‘ The stems should not be brown or aa and the berries should not be dull, shrivelled or have mould growth. The TSS at harvest in Anab-e-Shahi (Selection | 7) should be 15.16%; Thompson seedless 18-20%, and in Bangalore Blue (used for juice and wine) 12-14%.

172

Analysis of Fruit and Vegetable Products

Clusters should be well developed, fairly tight, and with fresh, green cluster stems and capstems. The berriés should be firm and plump, and have typical shape and colour of the variety. The berries should be firmly attached to the capstems, and be free from splitting, crushing, decay or shrivelling. Besides the chemical indices, physical characteristics like peel colour, texture of the pulp, easy separation of the berries from the bunch, and development of characteristic aroma subjective.?!

and flavour are also useful. However,

these methods

are

BANANA For transportation to distant places, banana is harvested at about 75 to 80%

maturity with plainly visible angles, and will ripen in about 3 weeks. For interstate shipment, harvesting is done at about 80 to 85% maturity when fruit angles remain still well defined. Such fruits ripen in 1 to 2weeks. For the local market,

fruits should be more than 90% mature. Such fruits ripen in less than a week. Indices used in judging maturity widely vary. Pulp-to-peel ratio, days from the emergence of inflorescence, disappearance of the angularity of fingers, drying of the leaves, and brittleness of leaves are the indices used in India.

Angularity or “fullness of fingers” appears to be the standard index used. The _ following terminologies are used:

a. Three-quarters—fruits at about one-half their maximum

size with clearly

visible angles. b. Full three-quarters—fruits with less prominent angles. c. Full—fruit angles have virtually disappeared. This has the disadvantage in that the terminology used varies considerably. Small growers generally wait for the leaves to dry up considerably before harvesting. This practice is not reliable as most commercial varieties «re susceptible to leaf spot diseases. Dwarf cavendish banana usually takes 99 to 107 days for the fruits to reach the “full three-quarters” stage after fruit set**» This has the pulp-to-peel ratio of 1.35 to 1.40 and is considered ideal for long distance transport.

MANGO Maturity of mangoes grown in India may be designated into four different stages, viz., A, B, C and D.*3

Stage A: The fruits have their shoulders in line with the stem, and the skin colour is olive green. Stage B: The shoulders have outgrown the stem end. This is the best stage for export. Stage C. The colour has lightened towards yellow. Stage D: The fruits are fully ripe with a typical flush developed on the skin. The stem-end and shoulder relationship does not hold true for all the varieties.

However, it applies to many varieties in which a beak is retaiged even at the ripe stage. Stage B is about the best time to pick the fruit for shipment.

Maturity Indices and Quality Criteria

173

Physico-chemical changes durifig ripening of four important varieties of mango i 1 are shown in Table 7-1. As fruits mature , pressure requ ired to pierce the fruit, acidity, and water-insoluble solids decrease, while sugars increase. It is generally . seen that a rise in the sugar by solids ratio to a value approach ing unity mig ht bea criterion of ripeness for Indian mangoes.*4 This, however, does not seem to hold

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TIAVL‘€-01 214

Analysis of Fruit and Vegetable Products

Edible Oils and'Fats

215°

Contaminants

Maximum level % w/w 0.2 0.05 0.005

1. Volatile matter at 105° C 2. Insoluble impurities 3. Soap content 4. Minerals

Virgin oils

Non-virgin oils

mg/kg

mg/kg

a. Iron (Fe) b. Copper (Cu)

5.0 0.4

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0.1

According to PFA specifications, refined vegetable oil is a vegetable oil obtained by expression, neutralized with alkali, bleached with absorbent earth and/or activated carbon, and deodourised with steam. No other chemical agent should be used. The name of the vegetable oil from which the refined oil has been . . manufactured should be clearly specified on the label of the container. Refined oils should not contain more than 0.25% free fatty acids and 0.10% moisture by weight. In addition, they should also conform to the standards prescribed for the edible oils from which they have been prepared. Although not specified, the refined oils should have as light a colour as possible; be odourless; neutral to taste; and should have good keeping quality. Generally, refined oils are free from moisture and enzyme activity. The deterioration in refined oils is either due to rancidity or flavour reversion. The latter may be caused by the action of even en an extremely small amount of oxygen. Solvent extracted vegetable oils are obtained from oil--bearing materials by the process of extraction using a solvent.

PHYSICAL CHARACTERISTICS Specific Gravity Note the tare weight of a clean dry pycnometer filled with recently boiled and cooled distilled water at 20-23° C, insert the stopper, and incubate in a water bath

at 25° + 0.2° C for 30 min. Remove the bottle from the bath, wipe dry and wish, Note the weight of the water. Dry the oil using anhydrous sodium sulphate and filter the oil through filter paper. Cool the sample to 20-23° C and fill the pycnometer to overflowing. Avoid air bubbles. Insert the stopper and incubate in a water bath at 25° + 0.2° C far 30 min. Carefully wipe off any oil on the outer surface, clean, dry thoroughly and weigh. CALCULATION

:

Specific Gravity at 25/25° C =

We of oil

oF Water

216

= Analysis of Fruit and Vegetabie Products

Refractive Index

Make use of an Abbe or Butyro Refractometer with a monochromatic light (tungsten lamp light with compensatoror sodium vapour lamp). Ensure that the instrument gives the correct reading at specified temperature as shown in Table 28-5. Check the readings given by two standards whose refractive indices are within the sample test range. If the readings differ from the stated values, correct using, the instrument key. Adjust the temperature of the refractometer to 40° + 1° C by circulating water _ from a constant temperature water bath. Melt the sample, if not already a liquid, and filter through a filter paper. Smear the sample on the cleaned prism and read the Refractive Index. After taking the reading, clean the prism using luke warm _ water and wipe dry. Temperature corrections: Approxiniate correction may be used using the _ following equation: R = R’+K (T’- T) where ite R_ = refractometer reading adjustedto the specttied temperature

R’ = the reading at T° C T’ = the temperature at which the reading R’ is taken T = the specified temperature (generally 40° C), and K = a constant which is-0.00365 for fats and 0.00385 for oils (if Abbe refractometer is used \ or = 0.55 for fats and 0.58 for oils (if Butyro reffactometer is used). When

- the latter is used, convert the fons to refractive indices (#p) using standard tables.

Fusion, Slip and Melting Point of Fats Fats are complicated mixtures of a large number of components. They do not display a melting point but a melting range. The transition between the various crystal forms varies for different fats and proceeds very slowly. To hasten, the fat is preheated to a prescribed temperature for a certain period. Heat the fat sample to a few degrees above the melting point. Dip fine glass capillary tubes, previously warmed, under the liquid fat and allow the sample to rise up the capillary tube. Remove the tube and quickly push the molten fat farther up the capillary tube using a fine wire. Clean the surface of the tube and rapidly seal the end which is nearest to the fat sample. Repeat the process of filling the fat into ‘the capillary tube but do not seal the end of the tube. ‘Allow the. fat to solidify by chilling in ice. Allow the capillary tubes to remain at 15-20° C_for 24 hr. Attach the capillary tubes to the bulb of a ‘thermometer with a rubber band. Immerse the thermometer bulb and the whole length of the capillary tubes that contain the fat samples in a beaker containing cold water and a glass rod stirrer. Raise the temperature slowly and note the temperature when the following occurs:

Edible Oils and Fats

217

a. In the unsealed capillary tube, when a meniscus forms on the surface, it is the incipient fusion point, and when the fat commences to rise up the tube, it is ’ the slip point. The reproducibility of this method leaves much to be desired. Differences of 3-4° C may occur between the results if the diameter of the capillary or the volume taken is varied. b. In the sealed tube, when the sample completely clears, it indicates the melting point (or clear point) of fat. c. The temperature at which the liquefied fatty acids prepared from fats solidify is called titre. The non-classical method for assessing the melting behaviour of fats is by differential thermal analysis. The temperature of fat as also of a reference compound during heating and cooling is measured continuously. As long as no transitions in the crystalline form of the fat occur, the temperature difference is 0° C. When transition occurs, the temperature difference deviates from 0° C, and

its course is characteristic of the type of fat. Colour

Measure an colour in Lovibohd Tintometer using a 1 inch or 5.25 inch cell. The colour may also be measured in 4 spectrophotometer using carbon tetrachloride as blank at the wavelength of maximum absorption.

CHEMICAL CHARACTERISTICS

~

Saponification Value When a fat is boiled with an excess of alcoholic KOH, the triglycerides hydrolyse, and glycerol and soap are formed. The alkali consumed for this hydrolysis is a measure of the saponification value (S.V.) and is defined as the number of milligrams of KOH required to saponify completely one gram of the oil or fat. Itis also a measure of the mean molecular weight of the fatty acids originally bound as triglycerides. Saponification equivalent of the fat is 56,000/S.V. This value is onethird of the mean molecular weight of the fat. After accounting one-third of the molecular weight of glycerol and the rest into account, the mean molecular weight

(M) of the fatty acids is

eee H

12.67.

Since fat contains unsaponifiable

constituents, an error of about 1% invariably occurs.

REAGENTS

ha

1. 0.5 N Alcoholic KOH: Dissolve 30 g of KOH in.20 ml of distilled water and add sufficient aldehyde-free alcohol to make to 1000 ml. See page 858 for preparing aldehyde-free alcohol. Allow-to stand overnight, decant the clear liquid and store in a bottle with rubber stopper.

2.0.5 N HCl. | PROCEDURE Melt the sample, if not already a liquid, and filter. Ensure that the sample is free of impurities and moisture. Weigh 1 to 2 g of the sample into a 250 ml Conical flask

218

Analysis of Fruit and Vegetable Products

having a ground glass joint. Pipette 25 ml of alcoholic KOH solution and add a few glass beads or pieces of pumice. Connect the flask to an air condenser at least 65 cm (or preferably 202 cm) long. Reflux’the sample on a water bath or a hot plate for not more'than 1 hr. Boil gently but steadily until the sample is completely saponified as indicated by the absence of any oily matter and appearance of clear solution. Allow the flask and condenser to cool. Wash the inside of the condenser with about 10 ml of hot ethyl alcohol neutral to phenolphthalein. Add 1 ml of phenolphthalein indicator and titrate with standard HCl. Conduct a blank determination alongside. When testing samples which give dark coloured soap solution, the observaticn of the end point of the titration may be facilitated either by using (i) thymolphthalein or alkali blue 6B in place of phenolphthalein, or (ii) an indicator containing 1 ml of a 0.1% (w/v) solution of methylene blue in water to reach 100 ml of phenolphthalein (mixed before titration). CALCULATION

SV.

= Blank titre — Sample titre X N of KOH X 56.01

Ane

Wt of sample (g) Unsaturation

Methods made use of for measuring unsaturation are determination of iodine value and thiocyanogen value. These methods are based on the addition of halogen and thiocyanogen respectively at double bonds. Iodine Value (Wijs Procedure)

Iodine value (I.V.) is the number of grams of iodine absorbed per 100-2 of the oil or fat, when determined using Wijs solution. The material is treated in carbon tetrachloride medium with a known excess of iodine monochloride in glacial acetic acid. The excess of iodine monochloride is treated with potassium iodide, and the liberated iodine is estimated by titrating with standard Na2S2O3 solution.

REAGENTS 1. Wijs solution: Dissolve 8 g of iodine trichloride in 200 ml of glacial acetic acid and mix with 9 g of iodine dissolved in 400 ml of glacial acetic acid. 2. 10% Potassium iodide solution: Dissolve-10 g of potassium iodide in 90 ml of water. 3. 0.1 N NaeS2QO3 solution.

4. Starch indicator, freshly prepared. PROCEDURE

The weight of the sample required is 2.5-3.0 g in the case of coconut oil and 0.15

Edible Oils and Fats

219

to 0.6 g in the case of other oils depending upon the iodine value. Weigh accurately by difference, an appropriate quantity of the oil or fat (previously melted) into a clean dry 250-ml glass-stoppered conical flask, and add 10 ml of carbon tetrachloride. Add 25 ml of Wijs solution, replace the stopper after moistening with potassium iodide solution, mix, and store in a dark cupboard for 30-min in the case of non-drying and semi-drying oils and 60 min in the case of drying oils. Add 15 ml of 10% potassium iodide solution and 100 ml of distilled water. Titrate with 0.1 N Na2S203 solution using starch as an indicator near the end point.

Carry out a blank determination alongside without the fat.

LV. =

(Blank titre — Sample titre) X N of Na2S203

— We of sample (g)

X 12.69

Thiocyanogen Value The thiocyanogen (SCN): value (T.V.) is a measure of the unsaturation of fats

and oils, and is expressed as equivalent number of centigrams of iodine absorbed per gram of sample (% iodine absorbed). Thiocyanogen does not add to fatty acids such as linoleic and linolenic in the same proportion as iodine. Hence, it is possible through thiocyanogen and iodine values to calculate fat or fatty acid composition. The method is applicable to all fats which do not contain conjugated systems. REAGENTS

1. Potassium iodide solution: Dissolve 150 g in water and make up to 1000 ml. 2. 0.1 N Potassium dichromate solution. 3. 0.1 N NagS2O3 solution.

4. Dehydrated glacial acetic acid: To 2000 ml of acetic acid in a 3000-ml Florence flask, add 100 mt acetic anhydride. Insert a ‘cold finger’ condenser, circulate cold water through the condenser and boil on a hot plate or oil bath for 3

hr at about 135° C. Cool and store‘in a glass-stoppered bottle. 5. Lead thiocyanate, Pb(SCN)z, solution: Dissolve 250 g of neutral lead acetate in 500 ml of distilled water and 250 g of potassium thiocyanate in another 500 ml of distilled water. Mix the two solutions with continuous stirring. Filter through a Buchner funnel and wash the precipitate with distilled water, and then with ether. Dry the precipitate in a desiccator over P2Os for 8-10 days. The precipitate should be white or only slightly coloured. If it is considerably coloured, it should not be used. 6. 0.2 N Thiocyanogen solution: Suspend 50 g of lead thiocyanate in 500 ml of anhydrous acetic acid:. Dissolve 5.1 ml of bromine in another 500 ml portion of anhydrous acetic acid. Add the bromine solution to the lead thiocyanate suspension in small portions with mixing until the lead thiocyanate solution is colourless. Allow the precipitated lead bromide and excess lead thiocyanate to settle. Filter using a Buchner funnel. Refilter, if need be, to get a clear solution. Store in an amber coloured glass- stoppered bottle at 18° to 21° C.

220

~=Analysig of Fruit and Vegergble Products

PROCEDURE

Clean the glass apparatus thoroughly using cleaning mixture, and dry. Dry oil or melted fat using anhydrous sodium sulphate and filter. Weigh accurately about 0.2 to 1.2 g of the sample depending upon its thiocyanogen value. The higher the value, the lesser the weight of the sample to be taken. Add 25 ml of the thiocyanogen solution, mix and store in a dark place at 18 to 21° C for 24 hr. Make at least 3-blank determinations with.each sample alongside. Titrate one of them immediately after adding potass%xgn iodide and water (see below) and treat the other two exactly in the same way as the sample. Remove the sample after 24 hr, add 1.66 g of dry, powdered potassium iodide, and mix by swirling for 2'min. Mix the blanks for 3 min. Add 30 ml of distilled water. . Titrate with 0.1 N Na2SeO3 using 1 to 2 ml of starch solution as indicator.

If the titre value of the thiocyanogen reagent exceeds by 0.2 ml of 0.1 N Na2S203 between the titrations of the blanks carried out immediately, and after 24 hr, the

reagent is not satisfactory.

,

CALCULATION

T.V. in terms of I.V. =

(Blank titre—Sample titre) x N of NazS203 x 12.69

We of sample (g)

The thiocyanogen method is intended for fats containing oleic acid, linoleic acid and linolenic acid. Apart from these acids, only small percentages of higher unsaturated acids should be present. Conjugated double bonds should be absent: With regard to addition of thiocyanogen, 1 mole of fatty acid with 1 double bond

absorbs 1 mole of SCN while the fatty acid with 2 double bonds absorbs approximately 1, and with 3 double bofds absorbs 2 moles of SCN. Hence, only when there is one double bond, thiocyanogen behaves like iodine. Calculation of Oleic Acid and Linoleic Acid By converting thiocyanogen value (T.V.) to iodine value (I.V.), and combining with the actual iodine value, oleic acid, linoleic acid and linolenic acid contents can be determined as follows:

The constants for fatty acids are: -

Acid Linolenic Linoleic Oleic Saturated and un- °

saponifiable

Expressed ‘in % xX Y

zs

S

_.V.

yaa!)

2/3, 181.1 89.9

167.) 96.7 89.3

0

0*

“The I.V. and the T.V. of unsaponifiable matter are not actually zero, but this is assumed for purposes of calculation.

Edible Oils and Fats

221

CALCULATION

1, When no linolenic acid is present: 181.1 Y +89.9 Z= 100 LV.

96.7 Y + 89.3 Z = 100 T.V. S = 100—(Y+Z) Y = 1.194 LV.— 1.202 T.V. Z = 2.421 T.V.— 1.293 LV.

S = 100 —(Y+Z) 2. When linolenic acid is present: For fats containing linolenic acid in addition to Slee acid and linoleic acid, the saturated fatty acid content must be determined independently so that three equations with three unknowns can be drawn up. 273.7 X + 181.1 Y + 89.9 Z = 100 LV. 167.1 X+ 96.7 Y + 89.3 Z = 100 T.V. xX + Yo Z= 1008S X = 1.5902 T.V. —0.12901 LV. +1.3040 S— 130.40 Y = 1.3565 LV. —3.2048 T.V.—1.6423 S +164.23 Z= 1.6146 T.V. Fh 75 AN 0.6617 Sa- 66.17

This method of calculation is used only when gas chromatographic facilities are

not available for determining the fatty acid make up, and has been included with the permission of AOCS. Reference Official and Tentative Methods, 3rd edn, American Oil Chemists’ Society, South Sixth Street, Champaign, Illinois, 61820, Vol. 1, Cd 2-38 (1975).

Crismer Test

Crismer test determines the miscibility of the sample with a standard reagent. The values obtained are characteristic within certain limits for each kind of oil. According to Codex specifications (CAC/RS 34-1970), the Crismer Value of rapeseed oil should be 80-85. PROCEDURE

Mix equal volumes of ethyl and amy] alcohols, and determine the temperature at which turbidity appears when tested with almond oil as described below. Adjust the alcohol mixture with water, if’ ieed be, after correction for acidity, till it

becomes turbid at 70° C. Use this mixture for further tests. Dry oil or sample of melted fat to be tested using anhydrous sodium sulphate, filter and heat on a water bath. To a test tube (1.3 X 12.7 cm) etched.to indicate 2 and 4 ml capacities, transfer the hot sample to the 2 ml mark. Add amy]l-ethy] alcohal reagent to the 4 ml mark. Fit the test tube with a cork having a centre hole toinggre a ea ightthand a side notch for asmall stirrer. Ensure that the bulb of

222. ~=— Analysis of Fruit and Vegetable Products

the thermometer is covered by the liquid and suspended about equidistant from the sides and bottom. Heat the water and sample slowly, moving the stirrer up and down until the mixture is clear. Continue heating until the temperature is approximately 5° C above the point of clarification. Raise the stirrer from the liquid without removing the cork, stop heating, and allow to cool. Observe the temperature at the first point of definite cloudiness. Repeat the procedure using a new sample. Duplicate readings must agree within 0.5° C. Determine the free fatty acid content as oleic acid by the method described later. Apply the correction to the free fatty acid content by referring to Table 10-4. TABLE 10-4: Corrections for Acidity of Oils

Class of oil

Typical oils

Correction factor for each 1% acidity

Drying

Linseed, sunflower and soybean

2.05

Semi-drying

Cottonseed, sesame and corn Rapeseed

2.03 1.61

Non-drying

Almond, olive and groundnut Coconut and palm kernel Palm Butter fat

2.07 2.01 1.72 1.54

Reprinted with the permission of the American Oil Chemists’Society.

CALCULATION Crismer Value, ery

— =

Turbidity beeen Pe +(%

om acidity x Correction factor)

The corrected Crismer values vary more or less within a narrow range for a given kind of pure oil. Reference Official and Tentative Methods, 3rd edn., American Oil Chemists’ Society, 508, South Sixth Street Champaign, Illinois 61820, Vol. 1, Cb 4-35 (1975). ;

Impurities Volatile, Insoluble and Mineral Matter

Volatile matter consists of moisture and other volatiles. The impurities consist of dirt, meal and other foreign substances insoluble in kerosene and petroleum ether. Soluble mineral matter includes minerals combined with fatty acids in the

formof soaps and in soluble form. These are usually present in the form of lime

~soaps although phosphates or iron may be absent.

Edible Oils and Fats

223

These constituents are impurities in fats and oils. The FAO/WHO Codex Alimentarius Commission has specified the oes maximum levels in all | vegetable oils. Maximum

1. Volatile’ matter at 105° C 2. Insoluble impurities 3. Soap content

Level

0.2% w/w 0.05% w/w 0.005% w/w

Taking one sample, all these determinations could be done in a series. The procedures are given below: Moisture and Volatile Matter

Water tends to settle in fats which

have softened and melted. Hence, mix the

samples thoroughly to distribute the water uniformly. Soften solid fats with gentle heat but do not melt. Mix thoroughly with a mixer. Weigh accurately 5 g of the sample into a tared dish which had been previously dried and cooled in a de ‘ccator. Dry in an oven at 105°C (A.O.CS. recommendation —

101° C .: i° C), remove from the oven, cool in a desiccator

and weigh. Repeat the procedure until the loss in weight does not exceed 0.05% per 30 min drying period. % Moisture and volatile matter

’

:

Loss in weight x 100 - We of sample

= —————=—_

Insoluble Impurities Prepare a Gooch crucible with an approximately 2 mm thick pad of acid washed asbestos using water, alcohol and ether in succession. Dry the prepared Gooch crucible to a constant weight at 101° + 1°.C, cool in a desiccator and weigh. To the residue from the moisture and volatile matter determination or a sample

prepared similarly, add 50 ml of kerosene and heat on a water bath to dissolve the fat. Filter through the prepared Gooch crucible using vacuum. Wash with five 10ml! portions of hot kerosene, allowing each portion to drain before adding the next. Wash thoroughly thereafter with petroleum ether or carbon tetrachloride to remove the residual kerosene. Dry the crucible and the contents to constant weight at 101° + 1° C, cool to room temperature in a desiccator and weigh. % Insoluble ; 3 impurities

_

Gain in wt of crucible ooo Too & 100 Wt of sample taken for moisture determination

Soluble Mineral Matter and Fatty Acids Combined as Mineral Soap Take the combined kerosene filtrate and kerosene washings from the insoluble impurity determination ina dish. Place an ashless filter paper folded in the form of a cone with the apex pointing upwards. Place the dish in a hood or a place free from draft, and light the apex of the cone when the bulk of the kerosene burns

224

Analysis of Fruit and Vegetable Products

ina muffle furnace at 550° C, cool to room temperature in quietly. Ash the residue i a desiccator and weigh. % Soluble mineral matter

_

We of residue X 100 Wt of sample taken for moisture determination

.

When the soluble mineral matter is more than 0.1% and phosphate is absent, multiply the percentage found by 10 and report as per cent fatty acids combined mineral soap. When phosphate is absent in the ash, add a few drops of strong sfleten of ammonium carbonate to ash, evaporate and dry in a hot air oven at 105°+ 2° C, cool and weigh. Calculate the fatty acids combined as mineral soap using the expression: % Fatty acids combined __ Increase in wt of residue X 1280 as mineral soap Wt of sample taken for moisture Reference Official and Tentative Methods, 3rd edn., American Oil Chemists’ Society, 508, South Sixth Street, Champaign, Illinois 61820, Vol. 1, Ca 4-25

(1975).

COMPOSITION Free Fatty Acids (Acid Value)

Acid value is the number of milligrams of KOH required to neutralize the free fatty acids present in one gram of the oil or fat. If the free fatty acid content of coconut or palm kernel oil has to be in percentages by weight, the calculation is based on the molecular weight of lauric acid. REAGENTS

1. Neutral alcohol: Boil ethanol, add a few drops of phenolphthalein and titrate against 0.01 N NaOH. 2. 0.01 and 0.1 N NaOH or KOH PROCEDURE

Weigh 10 g of oil or melted fat. Dissolve the sample in hot 100 ml of neutralized ethanol and titrate using 0.01 or 0.1 N alkali using phenolphthalein as indicator. Shake vigorously during titration and keep the solution warm. When testing oils and fats which give dark coloured solution, use the indicators as stated under determination of saponification value. CALCULATION

Acid value as oleic acid

. mlof alkali X N of alkali X 56.1 wt of sdmple (g)

Edible Oils and Fats

225

The acid factors for 0.01 N alkali are lauric acid = 0.002; palmitic acid = 0.00256;

and oleic acid = 0.00282. toexpress % free fatty acids in terms of the predominant acid in the sample. To convert % free fatty acids (as oleic) to acid value, ‘multiply the former by 1.99.

Unsaponifiable Matter Unsaponifiable matter is that fraction of substances in oils and fats:which is not saponified by caustic alkali, but is soluble in ordinary fat solvents. The material is completely saponified with alcoholic KOH and extracted with petroleum ether. The ether extract is first washed with aqueous alcohol, and then with water. The washed ether extract is evaporated. The weight of the residue minus the fatty acid present in it gives the unsaponifiable matter. In place of petroleum ether, ethyl] ether is also made use of.The latter‘acts more rapidly and the solubility of unsaponifiable matter is better. Consequently the values found are higher. Owing to various sources of error, the accuracy of the method is not great and is often unsatisfactory. REAGENTS

1. Alcoholic KOH solution: Dissolve 70-80 g of KOH in 70-80 ml of water and make to 1000 ml with 95% aldehyde-free alcohol. See page 869:for preparing aldehyde-free alcohol. 2. 0.02 N NaOH.

3. Acetone free from residue. PROCEDURE

Weigh 5 g of the sample into a reflux flask. Saponify by boiling under reflux with 50 ml of alcoholic KOH solution for 1 hr. Wash the condenser with about 10 mlof ethyl alcohol. Cool the mixture and transfer'the contents to,a separating funnel using not more than 50 ml of petroleum ether. Some ire aa the use of ethyl ether instead. Transfer the lower layer to another separating funnel. Repeat the extraction thrice. If any emulsion forms, add a small quantity of \ ethyl alcohol or alcoholic KOH solution. Collect all the ether extracts in a separating funnel. Wash the extract thrice with 25 ml portions of 0.5 N aqueous KOH and twice with distilled water. Transfer the ether layer to a tared flask containing a few pieces of pumice stone, and evaporate to dryness on‘a water bath. Add small amounts of acetone to hasten evaporation. To remove the last traces of ether, dry in a hot air oven at 80° C for about an hour.

Note the weight of the residue after cooling. Dissolve the residue in 50 ml of neutral ethyl alcohol and titrate with 0.1 N NaOH using phénolphthalein as indicator.

CALCULATION

Fatty acid content in the | ether extract as oleic acid in ane = ml of NaOH x N ofINaOH x» 0.282

.226

Analysis of Fruit: and Vegetable Products |

% Unsaponifiable _ Wt of residue—We of fatty acids in the residue’ matter Wt of sample taken vce

Reichert-Meissl, Polenske and Kirschner Values

a

These methods involve the determination of volatile fatty acids. ReichertMeissl (R.M.) value is the number of millilitres of 0.1 N NaOH solution required

to neutralize the steam volatile, water-soluble fatty acids distilled from 5 g of an oil or fat under the precise conditions specified in the method. It is a measure of water-

soluble steam-volatile fatty acids, chiefly butyric and caproic acids. The R.M. fatty

acids can be further split into fatty acids which form water-soluble silver salts, and fatty acids which form water-insoluble silver salts. Kirschner value is a measure of the steam-volatile, water-soluble fatty acids forming water-soluble silver salts (butyric acid). In recent years, this analysis is not usually done. Polenske value differs from the R:M. value in that it is a measure of steam_ volatile but of water-insoluble fatty acids like caprylic, capric and lauric acids present in oils and fats. APPARATUS

- Standard R.M.-Polenske-Kirschner apparatus consisting of flat-bottom boiling . flask, still head (10.7 cm wide and 18 cm high), condenser (52 cm long with 30 cm cooling length and 7 cm entry tube) anda receiver (with graduations at 100 ml and

110 ml).

'

REAGENTS bs

. Analytical grade glycerol. . 50% w/w NaOH solution in water. Protect from COx. Use the clear portion. . Pumice powder: 1.4 - 2.0 mm in diameter,

. Dilute H2SO,4: 25 ml of HgSO,4 made to 1000 ml with water. . Finely powdeted silver sulphate. 90% Neutral ethyl alcohol (v/v). 0.05 N Barium hydroxide. ONAYVHRWN . 0.1 N NaOH (not to be used, if Krischner value is to be determined). PROCEDURE

Heat the sample until the fat sepatates but do not allow the temperature to rise above 50° C. Filter through a filter. Paper until the filtrate is clear. Weigh 5 + 0.01 g of the fat sample-into a Polenske flask. Add 20 g of glycerol and 2 ml.of 50% NaOH solution from a burette which is protected from the CO, pick

up. Wet the tip of the burette before adding alkali to free it of carbonate deposit and reject the first 0.5 ml of NaOH. Heat the mixture over a low flame with wire

gauze until the liquid becomes clear and the fat has saponified. Do not overheat at this stage which causes discolouration. When all the fat has saponified, cover the

Edible Oils and Fats

227

flask with a watch glass, and allow to cool. Add 93 ml of boiling distilled water which is free of CO2 and mix. The solution must be completely clear at this stage: and pale yellow in colour. If the solution is not clear which indicates incomplete saponification, or if it is darker which indicates overheating, repeat the procedure with a fresh sample. An old sample of oil or fat may behave similarly. Add 0.1 g of pumice powder and 50 ml of dilute H2SO,. Connect to the distillation apparatus (Fig. 10.1). Warm the mixture until any insoluble material which may be present melts. Increase the heat and distil 110 ml of solution in 19 to 21 min. The distillation is considered to begin when the first drop forms in the

still-head (Fig. 10.2).

Fig. 10.1. Reichert-Meissl distillation apparatus ;

(all diménsions are in millimetres) -

Stop heating soon after 110 ml has distilled over, and replace the graduted flask by a measuring cylinder to collect drippings from the condenser, Close the _ graduated flask with the stopper. Do not mix. Place in a water bath at 15° C for 10 min, and ensure that the 110 ml graduation mark is below the water level. Mix and

228

Analysis of Fruit and Vegetable Products, 107-5 22-5

18045

37-522-50D0

4 t1dHole

2042

Fig. 10.2. Still-Head for Reichert-Meiss! distillation apparatus (all dimensions are in millimetres).

filter through a 9 cm Whatman No.4 filter paper. Reject the first 2 or 3 ml of the filtrate and collect the rest in a dry flask. Wash the condenser, still head and the 110-ml graduated flask with three lots of 15 ml distilled water passing each washing through the measuring cylinder, 110 ml flask and stopper, Filter through the same filter paper ensuring that all the insoluble matter is transferred to the paper. Discard the filtrate. Do not mix with the filtrate of the distillate got in the previous step. The filtrate should be free from water-insoluble fatty acids. In the case of coconut oil, it is not easily achieved. When the filtrate contains insoluble fatty acids, transfer the filtrate to a separating funnel, separate the lower aqueous layer, and add the insoluble acids to the main bulk of the insoluble acids on the filter paper.

Run a blank experiment without the sample. R.M.Value

Pipette 100 ml of the filtrate to a dry 250 ml conical flask and titrate with 0.1 N NaOH using phenolphthalein as indicator. R.M. value=Sample titre — Blank titrexN of NaOHx11 Polenske Value

Dissolve the insoluble fatty acids by three washings of the condenser, the measuring cylinder, the 110 ml flask with stopper and the filter paper containing the main bulk with 3 similar washings as before using 15 ml portions neutral ethy] alcohol. Collect the alcoholic washings (45 ml) in a clean dry flask and titrate with 0.1 N NaOH using phenolphthalein as indicator Polenske value =Sample titre — Blank titrexN of NaOHx 10

Edible Oils and Fats

229

R.M. and Polenske values are affected by low barometric pressures which occur at high altitudes. Under such conditions, oes the seallitizs as follows: Corrected R.M. value =

(Observed R.M. —10) log 760 + 10 og p

Corrected Polenske value = Observed value (760—45) p—A5

where p = barometric pressure in mm of mercury at the place of determination. Kirschner Value

Proceed as in the determination of the R.M. value, but titrate 100 ml of the. _ filtrate using 0.05 N barium hydroxide instead of 0.1 N NaOH as described earlier. After determination of the R. M. value, add 0.5 g of finely powdered silver sulphate to the solution. Keep in a dark place for 1 hr with intermittent shaking. Filter through a dry Whatman No. 4 filter paper. Add 35 ml of cold, CO2-free distilled water, 10 ml of dilute H2SO,4 and 0.1 g of pumice powder. Connect to the distillation apparatus, and distil 110 ml in 19-21 min. Cool the distillate at 15° C for 10 min, mix and filter through a9 cm Whatman No. 4 filter paper as before. Titrate 100 ml of the filtrate as in R.M. value using 0.05 N barium hydroxide. Carry out a blank determination similarly. Kirschner value = (T,—Tp)

where

(Tr— Ta)] 121 + ¢ [100 dd

‘10,000

7; and T, = sample and blank titre respectively in the Kirschner value

determination. T; and

T, =sample

and blank

. titre respectively

in the R.M. value

determination.

Test for the Presence of Specific Oils

Groundnut Oil Bellier Turbidity Temperature Test (Acetic Acid Method) . This is a test to verify the presence of groundnut oil. Oils containing arachidic - acid give a precipitate when their alcoholic soap solution is treated with dilute - acetic acid or HCI. The precipitate is formed at a particular temperature which is — specific to the oil (Table 10-2). REAGENTS

|

fe

1. Purified alcohol: Reflux 1.2 litres of 95 %-alcohol for30min with 10 g of KOH and 6 g of granulated aluminium foil. Distil and collect ene litre after discarding the first 50 ml. 2.70% alcohol: Dilute the purified alcohol with water to70% (v/v). Check the

230

Analysis of Fruit and Vegetable Products

final strength accurately by determining specific gravity. The specific gravity of

70% alcohol is 0.8898at 15.56° C.

3. 1.5 N Alcoholic potash: Dissolve 8.5 g of KOH in 95% purified alcohol and dilute to 100 ml with the same. Store in a dark bottle. ; 4. Dilute acetic acid (1+2).

PROCEDURE Pipette 1.0 ml of oil to a 100 ml conical flask. Add 5 ml of 1.5 N alcoholic KOH,

fix an air condenser (1.3 metres long) and reflux on a boiling water bath for 10 min with frequent swirling. Cool, neutralize exactly with dilute acetic acid using phenolphthalein as indicator, and add exactly 0.4 ml of acetic acid in excess. Add 50 ml of 70% alcohol and mix. Insert a thermometer (0-60° C with marking to 0.5° C) through a cork to the flask. Ensure that the bulb of the thermometer is immersed in the liquid but does not touch the bottom of the flask. Heat the flask gently over the water bath until the temperature reaches 50° C, and the solution is clear. Allow the flask to cool in the air with frequent shaking until the temperature falls gradually to 40° C. In the case of groundnut oil, turbidity appears between 39 and 41° C. Cool the flask with constant shaking by occasional immersion in chilled water (15° C) so that the distinct turbidity appears which is the turbidity temperature. Further cooling results in deposition of the precipitate which acts as a confirmatory test. Dissolve the precipitate by gently heating the contents to 50° C over the water bath. Again cool as described a‘ove, and note the turbidity temperature. The values should agree within + 0.5° C Sesame Oil

(Modified Villavecchia Test)

The test described below and adopted by AOAC is similar to Baudouin test except for difference in concentration and volumes used.

Add 2 ml of distilled furfural to 100 ml of alcohol. To 10 ml of oil or melted fat in a test tube, add 10 ml of conc HCl and 0.1 ml of furfural solution. Mix for 15 secand

allow the mixture to stand for 10 min. Appearance of crimson colour indicates the presence of sesame oil. Add 10 ml of water, shake and observe the colour. If the colour disappears, sesame oil is absent. Furfural gives violet colour with HCl. Hence, use very dilute solution as specified.

Cottonseed Oil (Halphen Test) To 1% (w/v) solution of sulphur in carbon disulphide, add an equal volume of

amyl alcohol. Mix 5 ml of melted oil or fat with 5 ml of sulphur solution and heat in a water bath (70-80°C)

for ayfew minutes until the carbon disulphide has

evaporated and no foaming is observed in the sample. Heat the tube in an oil bath at 112-115° Cc for 1 to 2 hr. A red colour at the end of the heating period indicates the presence of cottonseed oil.

Edible Oils and Fats

234-

Linseed Oil (Hexabromide Test) To 1 ml of the oil ina dry stoppered test tube,-add 5 ml of chloroform and about 1 ml of bromine dropwise, till the mixture is deep red in colour. Cool the test tubein an ice water bath. Add about 1.5 ml of rectified spirit while shaking the mixture until the precipitate which is first formed just dissolves. Then add 10 ml of ether. Mix and place the tube ‘in the ice water bath tor 30 min. Appearance: ot 4 precipitate indicates the presence of linseed oil. Argemone Oil

Prepare ferric chloride solution by dissolvirig 10 g of crystalline ferric chloride in 10 ml of conc HCI and 90 ml of water. Filter, if the solution is hazy. ( To 5 ml of the oil or melted fat in a test tube, add 2 ml of conc HCI, mix, and heat the mixture for 5 min on a water bath shaking occasionally. Transfer the acid layer: into another test tube, and add 1 ml of ferric chloride solution. Mix the reagent and the acid layerby rotating the tube between the palms of the hands. Do not mix by shaking. Heat the solution in a boiling water bath for 10 min. Argemone oil gives straight or needle shaped reddish brown crystals at the junction of the acid and oil layers. When the argemone oil content is less than 1%, crystals formed are not clearly visibleto the naked eye. Remove the floating particles by means of a capillary tube and examine under a microscope.

Presence of Mineral Oil (Holde Test)

The method is sensitive when the mineral oil content is 05% or more. To 1.0 ml of oil or melted fat in a conical flask, add 25 ml of 0.5 N alcoholic KOH solution. Boil under reflux for about 5 min or until saponification is complete. Add 25 ml of hot distilled water. In the presence of mineral oil, distinct turbidity appears.

For quantitative estimation, follow the method for the determination of unsaponifiable matter.

Fatty Acid Analysis Total Fat Extraction

The fat could be extracted as described under the determination of crude fat or ether extractives (see Chapter 1) using petroleum ether of boiling range 40+60° C but the method is less precise than the chloroform-methanol extraction procedure

described below. The method is applicable to all food products when further characterization of fat is required. PRINCIPLE

np

The fat-is extracted trom the sample using a mixture of chloroform and methanol

\

232.

~—Analysis of Fruit and Vegetable Products

at room temperature. The extract is diluted with the calculated amount of water when the two phases separate. The lower chloroform layer which contains the fat is separated, washed with dilute NaCl solution to remove the extracted proteinaceous material, and dried using anhydrous sodium sulphate. The chloroform extract is then evaporated to dryness in a tared dish and the fat residue weighed. REAGENTS

1. 20%. Magnesium chloride aqueous solution (w/v). 2. 0.1% Aqueous NaC] solution. PROCEDURE

Weigh 5 g of the sample into a boiling tube (20cm long). Add a few glass beads, 5 ml of chloroform, 10 ml of.‘methanol and 0.05 ml of magnesium chloride solution: Mix thoroughly: using a_whirl mixer for 2 min. Add another 5 ml of chloroform and mix again for 2 min. © _Add sufficient volume of water so that the total water content, including the __moisture content of the sample, is 9 ml. For example, if the moisture content of the sample is 60%, 5 g of the sample.taken would contain 3 ml of water. Hence, add 6 mil of water to the tube. Mix again for 30 seconds. Filter the extract through a sintered funnel of porosity No. 1 under mild suction into a boiling tube. Do not filter through the filter paper as phospholipids are absorbed by the paper. Wash the tube in which the sample was taken and the funnel thrice with 2.5 ml portions of chloroform. Swirl the contents, and then centrifuge at 1500 rpm for 5 min. With a fine bore pipette attached to a suction -line, remove the top aqueous layer as much as possible without disturbing the chloroform layer. To the chloroform extract, add 10 ml of 0.1% of NaCl solution, and mix by gentle inversion for 6 times. Do not shake vigorously as an emulsion will form. Salt solution is used to remove the protein that might have been extracted. Centrifuge again for 5 min. Remove the top aqueous layer as before. To the chloroform layer, add 1-2 g of anhydrous powdered sodium sulphate and shake vigorously to dry the chloroform,and filter using sintered crucible of No. 4 porosity into a dry receiver. Wash the tube and the funnel thrice with 2.5 ml portions of chloroform. . Transfer the dried chloroform extract to a tared dish using sma#l partions of ‘chloroform for washing the residue. Evaporate the chloroform on a water bath and dry the dish containing the fat residue in an oven at 100° C for 5 min. Cool in a desiccator and weigh. CALCULATION

Wt of residue Reference.

near

We of sample

xX 100

Folch, J., M. Lees '& G.H'S. Stanley, J. Biol. Chem., 226, 497 (1957).

Edible Oils and Fats

233

Examination of Fatty Acid Composition (Gas-Liquid Chromatographic Method) PRINCIPLE

The sample of fat is methylated, and the methyl esters, after purification, are separated by gas-liquid chromatography. In case of foods, fat separated using chloroform-methanol extraction procedure is preferred.

Preparation of Methyl Esters of Fatty Acids The fats may be converted into methyl esters by transesterifying the triglycerides with methanol under the influence of HCl, sodium methylate or boron trifluoride, by treating the fatty acids isolated from thetriglycerides with methanol and HCl, or with diazomethane. The latter method is generally made use of when lower fatty acids (e.g., coconut oil fatty acids) are present. Two procedures are described below: PROCEDURE A Prepare ammonium chloride-methanol-H2SO, reagent by dissolving 2 g of ammonium chloride in 60 ml of inethanol to which has been added 3 ml of conc H2SO,. Take 200 to 500 mg of fat, and reflux with 0.5 N KOH for 3-5 min. When the solution is hot, add 15 ml of ammonium chloride-methanol-H2SO, mixture, and reflux: for 15 min. Swirl to mix, and reflux again for 3 min. Cool, add light

petroleum ether and shake. Separate the ether layer and evaporate the ether under vacuum or nitrogen. Dissolve the residue in 3 to 10 ml of petroleum ether for determining the fatty acids by gas chromatography. PROCEDURE B Prepare transmethylation mixture by mixing 150 ml of methanol and 70 ml of toluene. Cool under a tap and add carefully, with stirring, 7.5 ml conc. H2SO,4 (sp. gr. 1.84).

Weigh 10-12 mg of fat into a 50 ml round bottom flask. Add 10 ml of transmethylation mixture. Heat on a water bath under reflux for 90 min. Cool, and add 10 mi of petroleum ether (B.P. 40-60° C) and 10 ml of water-Stoppecthe flask, shake well, allow the layers to separate, and pipette out the aqueous layer using a fine pipette. Repeat the washing procedure with a second 10 mlof water. Add 2-3 g of anhydrous sodium sulphate to remove the moisture from the ether solution. ‘Decant the clear ether layer and evaporate to dryness. Dissolve the residue in about 0.3 ml of petroleum ether for gas chromatography.

-Gas Chromatographic Determination of Methyl Esters of Fatty Acids

The operating conditions used for examining methy] esters of fatty acids by gas chromatography are given in Table 10-5. With carrier gas flowing through

Analysis of Fruit and Vegetable Products 234

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Edible Oils and Fats

235

apparatus, raise the temperature to operating conditions. Record the baseline to check for the stability of the instrument. If the flame ionization detector is used, operate at medium rather than maximum sensitivity. If the column is new, condition by holding at approximately 10° C above the Operating temperature with carrier gas flowing for 24 hr or until stable. Disconnect the column from the detector during conditioning. With proper gas flow rate and temperature, methy] linolenate, other Cis esters, and esters of shorter chain length are eluted in 30 min or less. When esters of fatty’ acids of greater chain length are present, increase the gas flow and/or the temperature so that the retention time of the last component is reduced.

With flame ionization detector, dilute methyl esters with isooctane, hexane or other suitable solvent so that 0.5-4 yl contain 0.01-0.1 yl of methyl esters, Take 0.5-4 ul in a micro syringe, pierce the septum éfSample inlet port, and quickly discharge the sample. Withdraw the needle and note’gHie recorder chart small peak due to air or solvent which marks the sample introduction point. Adjust the sample size so that the major peak is not attenuated more than 8 x or preferably less. To keep the peaks on chart paper, change the setting of the attenuator as necessary. Mark attenuator setting on chart. After all peaks have been traced, and when the pen has returned to the base line, remove the chart for calculation. IDENTIFICATION

Identification

of the fatty acids C09 to Cig usually presents no problem.

However, the number of possiblé isomers of the Cgo fatty acids makes assignment more difficult. The following methods are used. Identify peaks by relative position on chart. With polyester liquid phase on chromosorb W, esters appear in the order of increasing number of C atoms and of increasing unsaturation for the same number of C atoms. Cjg is ahead of Cig, and Cig methyl esters appear in the order: stearate (18:0); oleate (18:1), linolate (18:2); and linolenate (18:3). The Cgo saturated ester (arachidic 20:0) usually

_appears after 18:3 ester but may_be reversed on some columns, or position may change with column use. Determine the identity of compounds by analysing known mixtures.

er

A second method of identification: is by plotting loginretention time against carbon chain length. Using this technique, the saturated fatty acids are given whole carbon numbers (e.g. palmitic, Ci¢.0, has a carbon number of 16.0). From a mixture

of known fatty acid methyl! esters, the carbon numbers can be determined for a series of standards and related to unknown peaks from a sample. Gas chromatography is combined with mass spectrophotometry where further

confirmation 1s required.

236

Analysis of Fruit and Vegetable Products

conveniently made from peak height and peak width at 0.607 of peak height. Make appropriate corrections if the attenuator has been changed during the analysis. If the response of the flame ionization detector to fatty acid methyl esters is constant, then peak area is proportional to the weight of fatty acid. Let the peak area of the fatty acids A,B,C, etc., be a,b,c. Then per cent fatty acid

content of A is equal to

_* _» 100. atbtc

Similarly fatty acid.contents of B,C

etc., may be calculated. The chromatogram of fatty acid mixture displays a number of peaks. The resolution being high, the peaks of saturated and unsaturated fatty acids do not coincide and a single analysis gives the full picture of fatty acids. Table 10-1 gives the fatty acid composition of a number of fats. To'identify and establish, whether the fat under investigation is a pure fat or a mixture, it is sufficient to know the

qualitative fatty acid composition. This can be done by spectroscopic or chromatographic methods. Gas chromatography is to be preferred. As can be seen from Table 10-2, many of the vegetable oils are indistinguishable

from one another on the basis of identity characteristics like saponification values

and iodine values. A fatty acid chromatogram immediately reveals a complete spectrum of the fatty acids present. GLC of fatty acids and esters has been reviewed by Ackman’ and readers may refer to it for further information on the subject. References

1. Hartman, L. & R.C.A. Lago, Lab. Pract., 7, 22, 475, 494 (1973). Osborne, D.R. & P. Voogt, The Analysis of Nutrients in Foods, 1978, Academic Press, London and New York. 3. Wessels, J.P.H. et al., J. Sci. Food Agric., 24, 451, 1973. > . Official Methods of Analysis, 12th edn., (1975). Association of Analytical Chemists, P.O. Box 546, i.

Benjamin Franklin Station, Washington DC 20044. . Ackman, R.G., in Methods in Enzymology, Vol. 14, Lipids (Ed.) J.M. Lowenstein, p. 329 (1965).

ws

Tests for Stability of Fats

Several tests have been developed to indicate oxidative rancidity in fats. Some of these tests are purely qualitative, while others give a quantitative indication of the degree of rancidity. The incipient stages of rancidity can be detected by these tests before the spoilage can be detected organoleptically. The fat from the solvent should be extracted as given under determination of crude fat or ether extractive (see page 8). Qualitative detection by Kreis test and Thiobarbituric acid (TBA) test, and quantitative estimation by determining the peroxide value are given below. Kreis Test

The test depends upon the formation of a red colour when an oxidised fat is treated with conc.HCl and a solution of phloroglucinol! in ether. The

Edible Oils and Fats

237

compound in rancid fats responsible for the colour reaction is epihydrin aldehyde. All oxidised fats respond to the Kreis test and the intensity of the colour produced is roughly proportional to the degree of oxidative rancidity. PROCEDURE

|

Mix 1 ml of melted fat and 1 ml of conc HCI in a test tube. Add 1 ml of a 1% solution of phloroglucinol in diethyl ether and mix thoroughly with the fat-acid mixture. A pink colour formation indicates that the fat is slightly oxidised while a red colour indicates that the fat is definitely oxidised.

TBA Test The oxidation of fats produces compounds that will react with 2thiobarbituric acid to give red-coloured products. Essentially, the method ' involves dissolving the fat sample in an organic solvent such as benzene, chloroform, or carbon tetrachloride, and extracting the reactive material with an acetic acid-thiobarbituri¢ acid-water solution. The aqueous extract on heating will develop a red colour in the presence of oxidised fats. REAGENTS 1. TBA reagent: Dissolve 0.67 g of thiobarbituric acid in distilled water _ on a steam bath. Transfer the solution toa 100 ml volumetric flask, cool and make up to volume. Mix an equal volume of this TBA solution with an equal volume of glacial acetic acid. 2. Carbon tetrachloride—CP grade PROCEDURE

Dissolve an aliquot of melted fat sample in 10 ml of carbon tetrachloride. Add 10 ml of TBA reagent. Shake thoroughly for 4 min. Transfer the contents to a separating funnel and withdraw the aqueous layer into a test tube. Immerse the tube in a boiling water bath for 30 min., cool, and note the

colour. Pink to red colour is formed depending upon the extent of oxidation.

Peroxide Value

The peroxide value of an oil or fat is the amount of peroxides present and expressed as milli-equivalents of peroxide per 1,000 g of sample. The sample is dissolved

in solvent,

treated with potassium

iodide, and ‘the iodine

liberated by the peroxides present in rancid fat or oil is titrated with sodium thiosulphate solution often the number of millimoles of peroxide oxygen is reported, and the result is then half that of peroxide value. In this case, the

term ‘“‘Lea-value” is frequently used.

REAGENTS

1. Solvent: Mix 2 volumes of glacial acetic acid and one volume of | chloroform.

' 238

Analysis of Fruit and Vegetable Products

2. Saturated potassium ‘iodidé solution: Dissolve 4 parts of pure potassium iodide in 3 parts of distilled water: Keep the solution in a brown bottle. 3. 0.1. N Sodium thiosulphate solution 4. 0.5% Starch indicator PROCEDURE Weigh an aliquot of the extracted fat of the sample into a conical flask.

Add 25 ml of the solvent and displace the air above the liquid with COp. Add 1 ml of the potassium iodide solution, stopper the flask, and allow it to

stand for 1 min (with shaking). Now add 35 ml of water liberated iodine with 0.1 N sodium thiosulphate solution, indicator. Shake vigorously at the end to remove the last from the chloroform layer. Carry out a blank determination (The thiosulphate consumption should be negligible.)

and titrate the using starch as traces of iodine simultaneously.

CALCULATION

Calculate the peroxide value using the following expression: Peroxide value (milliequivalents or millimoles) Ete

1,000

ee

Sample Blank) _ Normality of sodium titre ~ titre |~ thiosulphate solution Guts O0 Ie

ae

Wt of fat taken

Active Oxygen Method The active oxgen method (AOM) measures the time in hours required for a sample of fat or oil to attain a predetermined peroxide value (P.V.) (100 m.e.)

under specified conditions of test. The principle of determination is that air is passed through the fat maintained at high temperature until the P.V. has reached a certain level. The P.V. is so chosen that it corresponds approximately with the point at which rancidity is organoleptically perceptible. For those fats to which the P.V. does not apply, the end-point should be determined organoleptically. The time required for the fat sample to attain the predetermined P.V. is taken as a measure of resistance to'the development of rancidity. There is a certain relationship between P.V., shelf life, rancidity etc., which has not been possible to establish exactly due to wide variations in storage conditions. Any correlation is ‘based purely on personal experience. The method is empirical and conditions should be rigorously maintained. Do not wash apparatus using cleaning mixture. Use glass distilled water preferably to prevent contamination with copper and/or other metals. Clean the tubes and other Parts coming in contact with the oil using acetone or petroleum ether immediately after determination. If need be, use detergent solution.

Edible Oils and Fats

239

APPARATUS °

1. Air purification train: Pass compressed air through a stainless steel needle valve to three gas washing jars in a series; one bottle containing distilled water and two bottles containing 2% potassium dichromate in 1% H2SQO,. To the last jar,

connect a 5-bulb water-cooled vertical condenser. Connect the condenser to a wide mouthed bottle having glass wool. Connect the outlet end of the bottle (a) to 2 more gas washing jars in series containing distilled water, and (b) to the manifold which takes the sample tubes through a flowmeter. 2. Sample tubes: Use Pyrex or Corning tubes 2.5 x 20cm calibrated or etched at 20 ml level. Fit a neoprene stopper having two holes and fit an air inlet and an ‘outlet tube. to the holes. PROCEDURE

Pour 20 ml of oil or melted fat (note: to melt the fat, do not exceed a temperature of more than 10° C of the melting point) into the sample tube. Insert the aeration tube and so adjust the air inlet tube that it is 5cm below the surface of the oil. Place the tube in a boiling water bath for 5 min and then transfer to a heater which maintains a temperature of 97.8° C. Connect the aeration tube to the capillary manifold, adjust the air rate and note the time. The AOM stability value is the time required for the sample to attain a peroxide value (P.V.) of 100 m.e. and is the average of two samples. At a time slightly less_

than the time required for the development of this value (which by experience could be judged by the odour of the outlet air from the sample tube),determine the P.V. using 1 g of the sample. If the P.V. is between 75 and 175 m.e.,make another determination immediately. If the value is more than 175 m.e.,discard the material and make a new determination. If the first determination gives a value less than 75, make a determination of P.V.

using 5 g.of sample at a time when the P.V. would be about 75. Make a second

determination. exactly one

hour thereafter,

and find the P.V. The

two

determinations so made should give values between 75 and 225 m.e. Plot the P.V. against time on a coordinate graph paper, connect the two points, and find the time: required to give a value of 100 m.e. Repeat the procedure on the duplicate sample, and report the AOM stability value as the average of the two values. With respect to organoleptic behaviour, fats can be grouped as stable and unstable fats. The stable fats generally contain oleic acid and a small amount of linoleic acid as unsaturated fatty acids. The unstable fats contain linolenic acid and higher unsaturated fatty acids in addition to oleic acid and linoleic acid. In the stable fats,-the P.V. increases gradually without being noticeable organoleptically. Animal and vegetable fats behave differently. With animal fats,

the P.V. increases gradually up to 20 (induction period). Thereafter there,is a sharp increase in the P.V. Generally, rancidity occurs in animal fats when the P.V. reaches 20. In vegetable. fats, no such sharp increase is observed in P.V. and distinct induction period cannot be found out. The P.V. gradually increases up to a maximum after which there is decrease. It is generally observed that rancid odour and taste become appreciable when the P.V. has reached 1,00.

240

= Analysis of Fruit and Vegetable Products

The organoleptically unstable fats behave quite differently. A good fat may give a strongly positive Kries test while an unpalatable fat may have a zero P.V. Inthe determination of P.V. of such fats, peroxide formation as well as decomposition occurs. The value would be higher initially and adapt to a normal level thereafter. Such oils, when refined satisfactorily, would be organoleptically acceptable upto a free fatty acid content of about 0.1%. Free fatty acids may be formed in coconut oil and palm kernel oil on storage which may be checked by determining the acid value. Ketone or Perfume Rancidity Butter fat, coconut oil,and palm kernel oil develop a distinctive odour and taste

which are quite different from those of oxidative rancidity. The taste and odour are due to ketones rather than aldehydes and esters which develop in oxidative rancidity, and is due to the action of moulds, mainly Penicillium and Aspergillus on the fat in the presence of moisture and nitrogenous materials. Its detection is based on the development of red colour when salicylaldehyde reacts with the ketones in the presence of H2SO.. PROCEDURE

Blank: Take 160 ml of water in a 200 ml glass stoppered distilling flask fitted with a short condenser. Distil 25-30 ml into a 50 ml glass stoppered cylinder. Add 0.4 ml of pure salicylaldehyde, shake vigorously, centrifuge and allow to settle. Pour off the supernatant until about 4 ml of the residue remains. To the residue, add 2 ml of conc H2SO, from the sides of the tube. Shake the mixture vigorously

and allow to stand. The salicylaldehyde which separates as the upper layer should show only a pale yellow, or at the most, a,faint pink colour. Do not use apparatus having rubber or cork parts. Use purified salicylaldehyde. Sample: Add 5-10°g-of the oil or melted fat to the water remaining in the distilling flask, and distil. Collect 25 ml of the distillate. Test the distillate with salicylaldehyde and H2SO, as in the blank. The aldehyde layer will be coloured pink to deep red in the presence of ketones. If the colour is only faint, immerse in boiling water for 15 min when the colour intensifies. References l.

Official and Tentative Methods, ‘3rd edn., American Oil Chemists’ Society, 508 South Sixth Street, Champaign, Illinois 61820, Vol. 1, Cd 12-57 (1975). 2. Van Der Vet, A.P., in Quality Control in the Food Industry, (Ed.) Herschdoerfer, S.M., Academic Press, London and New York, Vol. 2, p. 355 (1968).

3. Williams, K.S., Oils, Fats and Fatty Foods, 4th edn., Churchill Ltd., London, p. 55, 1966.

Oxidation Level of Fats

When an oil is used for frying, a progressive deterioration of the oi! takes place which has to be rejected at some stage. The main cause of deterioration is the build-

Edible Oils and Fats

241

up of oxidized material. Physical characteristics of deterioration are colour, increased viscosity, tendency to foam, lowering of smoke point etc. Criteria used for determination of oxidation level are peroxide, carbonyls, conjugated double bonds and free fatty acids. The progressive oxidation of oils in frying may be followed by measuring the oxidized glycerides using thin layer chromatography (TLC). PROCEDURE

Prepare TLC plates of size20X20 cm coated with 0.25 mm layer of Silica Gel G. Air dry the plates and activate by heating at 130° C for 30 min. Prepare a 10% solution of sample in chloroform. Spot 10 yl. Develop the plates using 99 parts of benzene and 1 part of diethyl ether to a length or 15 cok Allow the solvent to evaporate in the air. Spray with 50% v/v chromic acid (prepared by adding potassium chromate to conc H2SO, until colour becomes deep red, and then diluting with an equal volume of water). Heat the plates in a hot air oven at 180° C for 15 min. Compare the spots visually. Quantitative determination may be made using a scanning densitometer.

Unoxidized triglycerides have an Ry value of about 0.4 while the oxidized glycerides are held close to the base line. The oxidized fat spot will also contain partial glycerides and certain non-saponifiable components, but the amount of these is small, and affects the results only at low concentration levels. Changes in oil during frying are as follows: Oxidation level (%)

Oil

Before use

Groundnut Palm

Unsuitable for further use

6 9

30-35 34

Soyabean

5

36

Sunflower

5

32-38

Reference

1. Freeman, I.P., Chem.& Ind., p. 623, 1974.

CHAPTER

11

Flavouring Materials SPICES AND HERBS Spices and herbs consist of leaves, buds, flowers, fruits, barks or roots. The

contribution they make to the taste of the food is mostly due to the presence of volatile oils. The aroma must be characteristic of the spice with no portion of flavouring principle removed. It should be free from artificial colouring matter, adulterants or impurities. The PFA specifications for spices and herbs are given in Table 11-1 and the routine and specific methods of analysis are described in the subsequent pages. Curry powder, although a condiment, convenience of description.

is included under this heading for

SAMPLING Spices are packed either in bags or rigid containers. The consignment received may be from one lot or a mixed lot (consisting of different varieties, grades or years of production). Group together containers having products of similar characteristics. Draw samples as follows: Lot size N

Number of containers to be selected n

1-5 6 - 49 50 - 100

All 5 10% of the containers

101 and above

Square root of the number of containers rounded to the nearest whole number.

Make a random sampling. If the sample has come in bags, use sack type spears or triers. To mix and divide the samples, use shovels and dividing apparatus. With the appropriate instrument, draw samples from different parts of each container selected. This forms the primary sample. Mix all the primary samples to form the bulk sample. Divide the bulk sample into 3 parts, (i) for analysis, (ii) for reference and (iii) for seller.

EXTRANEOUS

MATTER

Mix the material thoroughly. Weigh 100-200 g of the material depending upon the nature of the material, hand pick the extraneous matter from the material,

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Wink’s weight equilibrium method,® and the graphical interpolation method.”* The graphical interpolation method is much quicker than the weight equilibrium method and can be employed for the quick and

approximate determination of the ERH. The latter method, however, gives more precise data and also detailed information regarding the storage behaviour of the product in relation to danger, critical points and safe margin, especially when products undergu visible changes in colour, texture, etc. Hence the procedure for determining ERH by the weight equilibrium method is given in detail.

Wink’s Weight Equilibrium Method The procedure involves samples of the product under test to be brought to actual weight equilibrium when exposed to different relative humidities. APPARATUS

The apparatus consists of a crystallizing dish (12.5 x 6.5 cm) as the main body of the test unit. Grind the top edge of the dish on a flat glass unit plate smeared with carborundum paste to remove irregularities. Apply silicone

Flexible Packaging Materials

481

grease to the ground edge to effect a good seal between the dish and the glass plate. Place the saturated salt solution in the crystallizing dish in which the sample held in a Petri dish is kept (Fig. 16.5). The Petri dish.(9.3 X 1.8 cm) is normally supported by a bracket made from 0.6 cm aluminium sheet. The bracket should be 8.1 cm long and 1.25 cm wide and bent to fit the dish snugly. Fasten this bracket to the threaded end of 0.32 cm diameter brass rod by means of two 2-56 brass nuts. The overall length of the rod, 8.1 cm, is

arbitrary and depends on the depth of the test unit and the height of the platform upon which the unit rests when placed in the balance (Fig. 16.6). Bend the top end of the rod into a flat hook for convenient suspension from the pan hook of a balance or from the counterpoise used with a magnetic damping device. Drill a brass cone slip on to the rod and solder in place with the small end down and at a distance of 2.5 cm from the bottom end of the

rod. Cut this cone from a 0.6 cm brass rod. The lower half of the cone should have an angle of approximately 30°. ei

SATURATED

Fig.

SALT SOLUTION

16.5

Fig. 16.6

Cover the top of the crystallizing dish with a square plate glass (13.75 X 13.75 cm) and cut a hole 0.47 cm in diameter which should be 0.6 cm off-centre on a line perpendicular to one edge of the plate and passing through

its centre. This off-centering is necessary because of the limitations in the space in analytical balance (Fig. 16.6). There should be no chipping in the glass plate where the hole is drilled. In place of the above arrangement small desiccators may be used. Petri dish may be supported either on glass tripod or porcelain plate. This arrangement, however, has the disadvantage that during the interval of weighing, the sample atmosphere is changed and requires slightly larger quantity. of salt solution. An arrangement as shown in Fig. 16.7 may also be used’. Use of single pan semi-automatic analytical balance would considerably reduce the time of weighing. .

482

Analysis of Fruit and Vegetable Products — -Glass plate Grease seal

Fig. 16.7

The ERH

Humidity

values for a number

control unit

of saturated salt solutions and H,SO,

solutions of varying normalities are given in Tables 16-11 and 16-12, respectively. Wink® made use of saturated salt solutions while Landrock and Proctor” used H,SO, solutions.

TABLE

16-11: Equilibrium Relative Humidities for Saturated Salt Solutions® Relative humidity %

Chemical

Formula 22.8° C

307°C

37.8° C

(73° F)

(86° F)

(100° F)

NH,H,PO,

92-9

92.0

911

K,CrO,

86.5

86.3

85.6

(NH,).SO,

80.1

79.6

79.1

Sodium chloride

NaCl

isis

75.2

75.1

Sodium acetate

NaC,H,0,

74.8

71.4

67.7

Sodium

NaNO,

64.8

63.3

61.8

Sodium bromide

NaBr

58.5

56.3

53.7

Sodium dichromate

Na,Cr,0,

54.1

52.0

50.0

Magnesium nitrate

Mg(NO)),

53.5

51.4

49.0

Potassium nitrite

KNO,

48.6

47.2

45.9

Calcium nitrate

Ca(NQO3),

51.8

46.6

38.9

Potassium thiocyanate

KCNS

46.6

43.7

41.1

Potassium carbonate

K,CO;

43.9

43.5

43.4

Chromium

Ammonium

phosphate

Potassium chromate

Ammonium

sulphate

nitrite

trioxide

_

:

CrO,

39.2

40.0

40.2

Magnesium chloride

MgCl,

32.9

32.4

31.9

Potassium acetate

KC,H;0,

22.9

22.0

20.4

Lithium chloride

LiCl

11.1

t.2

EE

11.1 ee

Flexible Packaging Materials

TABLE

483

16-12: Specific Gravities and Normalities of Sulphuric Acid Solutions” Providing Specific Relative Humidities at 25°C Relative humidity (%)

:

Specific gravity

Normality

0

1.835

5

1.608

22.0

10

1.552

19.5

15

1.512

18.0

20

1.478

16.8

25

1.450

15.8

30

1.425

14.8

35

1.400

13.9

40

1.377

13.1

45

1.355

12.3

50

1.335

11.5

55

1.314

10.8

60

1.293

10.0

65

1.271

9.2

70

1.248

8.3

75

1.224

7.4

80

1.197

6.3

85

1.165

5:2

90

1.127

3.9

95

1.076

2.3

100

1.000

0.0

PROCEDURE

Procedure for determining ERH of mango custard powder by the weight

equilibrium method is described below.? Pour approximately 40 ml (or more if desiccator is used) of saturated salt solution into each crystallizing dish. Add salt in excess to ensure a saturated condition

of the

solution.

In

solutions,

where

moisture

«is removed

(desorption isotherm), have mounds of the excess salt exposed above the level of the solution. Note the tare weight. Weigh exactly 5-10 g lots of the powder and spread uniformly on Petri dishes. Note the moisture content of the sample by drying . in a vacuum oven at 70° C. Expose the dish to different relative humidities ranging from 0 to 98% inside crystallizing dish or desiccators containing H,SO, solutions of different strengths at 25°C, or saturated solutions of

484

= Analysis of Fruit and Vegetable Products

different salts having definite relative humidities at 25° and 37° C. Determine the gain or loss in weight of the sample at the end of 1, 2, 3, 4, 5 and 6 hr and,

thereafter, at intervals of 24 hr up to 360 hr with a view to determine the moisture equilibrium of the powder at 25° C. In the case of 37° C, weigh the dishes at intervals of a fortnight up to 75/120 days to observe the changes in moisture uptake and also any adverse changes in the powder, such as caking, discolouration, mould growth, etc., and thus get an idea of the critical point and storage characteristics of the product. Note the weight of the powder when there is no further loss or gain. Calculate the equilibrium moisture content at each relative humidity studied using the expression: X = A — B, where X is dry matter content in the sample taken for determination of ERH, A is weight of sample taken, and B is moisture content.

og 100, Equilibrium moisture content (%) = : = where S is weight of sample after equilibration. (Initial weight before equilibration should be same as that of A.) Draw the moisture equilibrium curves (Fig. 16.8) and mark on this curve: i. the initial point (J)—the moisture content and the ERH of the product as prepared;

Content Moisture Equilibrium (%)

0

10

20

30

40

5S

6

70

8

9

Relative Humidity (%)

Fig. 16.8: Humidity moisture equilibrium curves for mango custard powder: 7 by graphical interpolation method; 2 and 3 by weight equilibrium method; 2 at 25° C and 3 at 37°C. / is the initial point, D is the danger point, and C is the critical point.

Flexible Packaging Materials

485

ii. the critical point (C)—stage at which the product just becomes lumpy; and iii. the danger point (D)—a point which is of 5% lower RH than the critical point.

The portion of the curve between D and C is generally defined as the safety

range (SR).”* The portion of the curve between the points J and D may be defined as the safety margin (SM) as a helpful guide in the packaging of the material. The package adopted should not permit the product to reach the danger point so that there may be a safe margin of. error. The ERH data for mango custard powder are given in Table 16-13. The initial moisture content of the product is 2.0% (13.0% RH). When the moisture content rises to 4.4% (32% RH) critical point is reached, and beyond 4.4% caking, discolouration, etc. are observed. At the danger point, the product has 4.2% moisture and 27% RH. Moisture content increases along with an increase in ERH. The increase in moisture content is steep beyond 50% RH. Hence, this product falls into the category of hygroscopic substances. TABLE At room

temperature (25° C)

Graphical interpolation method Equilibrium moisture content

16-13: ERH of Mango Custard Powder (0.94% Moisture)? At 37°C

Weight equilibrium method .

Equilibrium ERH moisture content

ERH

Weight equilibrium method

Equilibrium moisture ERH content

Remarks

%o

Yo

%

%

%

1.87

12.5

0.24

0.0

0.50

0.0

2.89 4.37 7.88

15.0 27.0 38.5

1.11 2.10 4.13

10.0 20.0 30.000

1.32 3.71 4.36

10.0 20.4 31.9

-do-do-do-

8.56

41.5

640 9.40

40.0 50.0

4.75 7.31

40.2 53.7

Colour faded, caking observed Slightly brownish, caking observed

11.59

53.5

12.90

60.0

9.60

61.8

Colour faded, cake formation

14.60

61.5

17.60

70.0

18.04

75.1

80.0

23.47

79.1

Brown and caky, mould appeared after 30 days Brown and caky, mould appeared after 20 days

90.0

24.56

91.1

25.2

NG

¢

35.4

Free flowing, colour not affected

Brown and caky, mould appeared after 8 days

(Reprinted with permission of the Institute of Food Technologists, USA ©)

486

Analysis of Fruit and Vegetable Products

Graphical Interpolation Method’ The graphical method is based upon the principle that the rate of gain or loss in the moisture of a product, at any given initial moisture content and atmospheric relative humidity, increases with increasing difference between the atmospheric and the product vapour pressures. In this method, the duration of exposure is held constant in a true equilibrium (gain or loss of weight method). APPARATUS

1. Crystallizing dishes, prepared as before, with glass covers (Fig. 16.7) or desiccators. 2. Petri dishes. 3. Analytical balance. REAGENTS

H,SO, solutions: Prepare 750-1,000 ml of H,SO, solutions of 10, 20, 30,

40, 50, 60, 70, 80 and 90% relative humidity by referring to Table 16-12. PROCEDURE

Preparation of sample: Prior to the determination of ERH, the sample has to be conditioned to the entire range of moisture content relevant to it (e.g., in the case of dehydrated potato,® the moisture content vary from 3 to 16%). This should be done as given below: i. Use one sample of 50-60 g as it comes out of the production line or from a package. ii. Place another 180 to 200g in a vacuum oven and dry at 40-45°C. Remove one portion (~60 g) after 1 hr, a second after 2 hr and the last after 3 hr. Store the samples at 25° C, immediately after drying in moisture proof bottles with rubber lined caps. These samples will have lower moisture contents than the sample described in i. iii. Place 5 or 6 other portions in a large desiccator containing either water or a solution of relatively high humidity (90%) at 37° C. Stir at intervals to ensure uniformity. Remove each at varying intervals. Immediately transfer into a-bottle or tin as before and store at 25°C. Determine the moisture content of each of the above samples by drying in a vacuum oven at 70°C for 6 hr. Fruit juice powders require longer (16 hr) hearing. Determination of ERH: Note the tare weight of the Petri dishes. Number of dishes required for each sample is equal to the number of different relative humidities at which the tests are carried out. Place 5+ 0.01 g of the sample into each of the Petri dishes, note the weight, and place the dishes in humidity

Flexible Packaging Materials

487

control units (crystallizing dish or desiccator). Place the units in a balance room at 25°C and note the gain or loss in weight in mg of the samples in dishes at intervals of 1 hr. In a similar way, note the gain or loss in weight of

other samples which have been conditioned in different moisture contents.

Equilibrium curves: Plot a graph as shown in Fig. 16.9. Note the loss or gain in weight (mg) per hour at different relative humidities. When sufficient number of points have been drawn above and below zero base line for a sample of given moisture content, draw a smooth curve. Similarly, draw curves for samples with different moisture contents.

weight/hr Mg in Gain

weight/hr, in Loss Mg

10 20

30

40 50 60 70

80

90 100

% Relative Humidity

Fig. 16.9: Graphical interpolation isotherms for mango custard powder (I hr test period at 25° C). (Reprinted with permission of the Institute of Food Technologists, USA ©)

Each graphical interpolation curve constitutes an isotherm. (Isotherm, in general, refers to any curve based on data obtained at a constant temperature.) The zero base line represents no change in weight. The ERH is, therefore, given by the relative humidity at the point at which an isotherm cuts the zero base line. From the ERH values so obtained and the moisture content of the samples, humidity moisture equilibrium curves can be drawn (see Fig. 16.8).

488

Analysis of Fruit and Vegetable Products

Procedure for Selection of Packaging Material'’® 1? Having found the ERH, the next question to be considered is which packaging material will suit best. The following worked out example is given to illustrate the selection of best packaging material for a particular product. Let us suppose that it is required to pack the mango custard powder in 250 g units to withstand 300 days of storage at 25°C and 65% relative humidity. The product when packed is supposed to contain a maximum of 2% moisture and will deteriorate rapidly when the moisture content rises above 4.2%. The bulk density of the material (0.6 g/ml) is such that a suitable bag or pouch to contain 250 g would have a surface area of approximately 250 sq. em. The approximate maximum water vapour transmission of the material of which the package is made may be calculated by assuming that the sealing is perfect and the packaging material is unaffected by creasing. The calculations involved are given below:'°-? Permissible uptake of moisture by the product by weight

—

was

=

Oo,

= 4.2 — 2.0 =2,2%

.'. Permissible moisture uptake

., 2.2

for 250 g of the product

250

i.

es

1 gram mole (i.e. 18.0 g) of water vapour occupies at NTP 22.4 litres

.’. 5.5 g of water vapour occupies =

22,400 18 15.5

6844 ml

The water vapour pressure outside the package may be taken as 65% of saturation vapour pressure at 25° C. The equilibrium humidity is 13% relative humidity at 2% moisture content and 27% relative humidity at 4.2% moisture content. Thus the average for the range may be assumed to be:

(13 + 27)/2 = 20%.

Hence, the average vapour pressure differential (D) between the inside and outside of the wrapper during the period of storage may be assumed to be equivalent to (65 — 20) = 45% of the saturation vapour pressure at 25° C. The saturation vapour pressure at 25°C is 23.78 mm or 2.378 cm (Table 16-14). Therefore, D is 45% of 2.378 cm or 1.07 cm.

The permeability of a film (gas or water vapour transmission of the film) is

defined as the quantity of gas (or water vapour) passing through a piece of

film having unit area, in unit time, with unit differential across the faces. It

may be calculated by using the expression'™ 12 yA q ee

At(p, — p2)

where, P is water vapour or gas transmission of the film; q, quantity of water vapour or gas in cc; t, time of storage in sec; p;, water vapour pressure outside

Flexible Packaging Materials TABLE

16-14: Pressure of Saturated Water Vapour in mm

489

of Mercury

_oO—... SEES a

Temperature °C

Pressure

Temperature °C

Pressure

10

SD

22

19.81

11

9.83

23

21.04

OA

22.35

rligst

12

10.50

13

11.22

25

23.78

14

11.97

26

25.18

15

12.77

27

26.71

16

13.62

28

28.32

17

14.51

29

30.01

18

15.46

30

31.79

19

16.45

35

42.14

20

17.51

40

55.29

21

18.63

45

71.84

the package; p2, water vapour pressure inside the package; p; — po, water vapour pressure differential between outside and inside of the wrapper during the period of storage of the package, expressed in cm of Hg; and A is the effective area of the package in sq cm. Unit of P = cc/cm?/sec/cm of Hg at a given temperature.

The water vapour transmission of the packaging material in the example cited must_not,

therefore, exceed the value given below: P = 6844/(250 x 300 x 24 x 6060 x 1.07)

= 99 X 107 8cc/cm?/sec/em of Hg at 25° C From Table 16-15, it can be seen that high density (0.954) polyethylene of 1 mil (0.001 inch) thickness or polypropylene of 2 mil thickness would give the required protection. Saran film of any thickness would give a very large safety margin. If, in addition, protection from permeability of oxygen is required, use of Saran or laminate of high density polyethylene with aluminium or regenerated cellulose is recommended.

PACKAGING

MATERIAL

FOR

SPECIFIC

PURPOSES

The processor is more interested in the ultimate performance of the package in terms of protection; ease of filling and closing; handling; storage; and display. Properties of different packaging materials on a 0-10 graded basis in most of the qualities likely to be encountered are given in Table 16-16. In addition to the performance, cost is ani important criterion. What is needed is a package that will perform the task required at the minimum cost.

490

Analysis of Fruit and Vegetable Products TABLE

16-15: Water Vapour and Gas

These figures which show the comparative transmissions of different thickness of various expressed in mils Water vapour

Material

1 mil

2 mil

3 mil

Polyethylene (density 0.922)

420

210

140

Polyethylene (density 0.954)

60

30

20

Polypropylene

160

80

53

Polyester (Mylar A)

510

260

170

Saran 517

5.5

Dee

1.8

Nylon 6

2800

1400

930

Nylon 11 (Ralsin)

2,300-

1200-

11,000

5500

7703700

Pliofilm N,

430

220

140

Pliofilm N,

590

290

200

Pliofilm P,

1100

550

370

Pliofilm FM,

5100

2600

1700

Pliofilm 75 BF*

1900

ihe

Fd

630

320

210

4700

2400

1600

PVC

Polystyrene Cellulose acetate (plasticized)

Regenerated cellulose (RH 0%) Regenerated cellulose (RH 100%)

Regenerated cellulose (moisture proof)"

29,000-

15,000—

47,000

24,000

970016,000

“4

cz

94,000

47,000

31,000

as

5000

5000

5000

*Only one thickness (0.75 ml) available. RH—relative humidity. (Reprinted with permission of the British Food Manufacturing Industries Research Association)

Flexible Packaging Materials

491

Transmissions < 10? of Various Materials!?

: (Units: cc (N.T.P.)/cm?/sec/em Hg at 25° C) films have been derived mainly from the permeabilities’? Thicknesses are (thousandth of an inch). Carbon 1 mil

Oxygen

dioxide

Nitrogen

2 mil

3 mil

1 mil

2 mil

28

18

15

78

2.>

U7

4.3

3

1.5

0.5

0.3

1.7 55 1.2

3 mil

2 mil

3 mil

0.4

0.3

0.008

0.005

0.001

0.0007

0.015

0.01

0.04

9.03

3.0

2.0

0.08

0.05

0.5

0.3

0.4-0.5

0.27-0.3

Analysis of Fruit and Vegetable Products 492

ee

se

Speieod urseg—KX sanbedo—O

SS

ee

‘iuaiedsues]—y, ‘pasoysuy—y

Se

-e BS 9 = Oh

FO ES OL et 6

92 38 6s Se mie

8) 8 (Ol Sa --C 29s 9 "¢

Ol scree POUR Ol, 8] SOT 0 ca -F9nrs “9

9 0-0" Ge SS 8) 0.0 Gee OL Pade acl. "LFS Swsi OSD «ON ©€ ©

a.

ia~]

&

iar)

E@

8

a

@)

Bx

3
760 nm

If the light striking the retina of the eye does not contain all of the wavelengths of the visible spectrum, or if their intensities differ considerably, the sensation of colour results. The other wavelengths are either reflected or transmitted according to the nature of the’object and are perceived by the eye as the colour of the object. Because light of different wavelengths is refracted (velocity reduced) to different degrees on passing through a transparent medium, it is possible to separate the different wavelengths of white light by the use of prism and produce a coloured spectrum. Another way in which portions of the spectrum may be removed is by interference. Certain wavelengths of white light may be removed by absorption which is by far the most common. cause of colour in the absorbing material. The colour may be observed as the light transmitted through a solution of the substance in a transparent medium, or as the light reflected from the surface of a substance. The visual colour is complementary to the colour absorbed, that is, it is the colour sensation produced by all of the wavelengths minus the wave-

lengths absorbed. Table 17-1 gives the observed colours when relatively narrow bands of the visible spectrum are absorbed. If the absorption bands are broader or if more than one absorption band is present, the visible colour is altered. In this chapter the measurement of colour based on absorption, and reflection of colour is discussed.

Analysis of Fruit and Vegetable Products

498 ie

TABLE

eee

Se

17-1: Relation Between Absorption and Visual Colour te ee

Se

Colour absorbed

Wavelengths absorbed (nm) —

ee

;

SS

Visual colour

400-430

Violet

Yellow-green

435-480

Blue

Yellow

480-490

Green—blue

Orange

490-500

Blue-green

Red

500-560

Green

Purple

560-580

Yellow-green

Violet

580-595

Yellow

Blue

595-605

Orange

Green-blue

605-750

Red

Blue-green

Theory of Spectrophotometry and Colorimetry When light (monochromatic or heterogeneous) falls upon a medium, a portion is absorbed within the medium, and the remainder is transmitted. The measurement of colour by spectrophotometer or colorimeter is based on Lambert’s law and Beer’s law of absorption of light. Transmittance (T) is the ratio of the radiant energy transmitted by sample (P) to the energy in-

cident upon the sample (P,). Both radiant energies should be obtained at same wavelength and with same spectral slit width.

T= P/ Po Lambert’s law states that when a beam of monochromatic light is passed through a solution of constant concentration, the absorbence (A) is directly proportional to thickness of solution (d). That is,

or

Ax Db

A = a,b, where a, = constant absorbency index

ot . -A,= 4b =logig Io/f where, I, = Intensity of light, and I = Intensity of transmitted light

According to Beer’s law, when a monochromatic light passes through a solution of constant length, the absorbence (A) is directly proportional to the concentration of solution (C). That is, Ao

C

or A. = 4,C = log, Ip/I Combining Lambert’s and Beer’s expressions, we have

Ax be

|

or

A=abC

= logyy Ig/I

or)

A > aC. = logy h/t

|

Colour Measurement

499

Based on Beer’s and Lambert’s laws, the various expressions used in spectrophotometry and colorimetry and their definitions are given below. Transmission (T), also termed transmittance, is the ratio of the transmitted light to that of the incident light. Transmittance (T,) is the ratio of the transmittance of a cell containing the coloured solution to that of an identical cell containing the solvent or a blank solution. Optical density (OD) also called the extinction (E) or the absorbency (A). of a medium is the logarithmic ratio of the intensity of the incidént light to that of the emergent light. (The per cent transmittancy may be converted into OD by using the expression 2 — log T,.) Extinction coefficient (or absorbence index) is the OD per unit path length. Specific extinction coefficient (absorptivity or absorbence) is the optical density per unit path length and unit concentration. When the molecular weight of a substance is not definitely known, this term is customarily used and, usually, the unit of concentration is written as a superscript,

and the unit of length as a subscript. Thus Ei% 440 nm=50 means that for the substance in question, at wavelength of 440 nm, a solution

of length 1 cm (i.e. width of the cuvette used for measuring the colour of the sample) and concentration of 1% (1 g per 100 ml), log I)/I is equal to 50.

:

Molecular extinction coefficient. («) (or molar absorptivity or molar absorbency index) is the specific extinction coefficient for a concentration of 1 gram mole (molecular. weight expressed in grams) per litce (—molar solution) and a path length of 1 cm. Colour in foods is usually due to the presence of natural pigments like anthocyanins, carotenoids or chlorophyll. In the course of chemical analysis of various constituents, colour is dsually developed by the addition of an appropriate chromogenic reagent. The variation in the colour of a system with the change in concentration of some of the components forms the basis of colorimetric analysis. Colorimetry is based on the determination of the concentration of a substance by measuring the relative absorption of light with respect to a known concentration of the substance. In visual colorimetry, natural

or artificial white light is generally used as a light source and determinations are usually made with a colorimeteror colour comparator. When a photoelectric cell replaces the eye (thus largely eliminating the errors due to the personal characteristics of each observer), the instrument is termed photoelectric colorimeter. This is usually employed when light contained within a comparatively narrow range of wavelengths is furnished by passing white light through filters. Hence, the instrument is also termed as filter photometer. A spectrophotometer, on the other hand, consists of a spectrometer and:a photometer combined together. A spectrometer produces coloured light of any selected colour or wavelength, and when it forms the part of a spectro-

500

Analysis of Fruit and Vegetable Products

‘photometer, it is called a monochromator and is generally calibrated in wavelengths (nm, hitherto designated as my). A photometer measures the intensity of the monochromatic beam produced by the monochromator. In spectrophotometric analysis, light of definite wavelength (not exceeding 1-10A in bandwidth) extending to the ultraviolet region of the spectrum constitutes the source of light. The range of electromagnetic radiation extends considerably beyond the visible region. Gamma rays and X-rays have very short wavelengths, while ultraviolet, visible, infrared and radio waves have. progressively longer wavelengths. For colorimetric and spectrophotometric measurements, the visible region and the adjacent ultraviolet region (< 400 nm) are of major importance. The terminology used and units of measurement are given below. Wavelength (A) = Distance between the peaks of waves in cm, unless otherwise specified. Wave number (»’) == Number of waves per cm

Frequency (y)

= Number of waves per second

The relationship between these three is: 1/Wavelength = Wave number = Frequency/Velocity of light. Units of Measurement

1 Angstrom unit (A) = 10-19 metre = 10-8 cm 1 Millimicron (nm or mu) = 104A = 1077 cm

1 Micron

(p)

=10°A = 10-* cm

Velocity of light (¢) Wave number (v’) Frequency (v)

= 2.99796 x 10! cm per second = 1/X waves per cm = ¢/A = 3 x 10!°/X waves per second

1 Freshnel unit (/f)

= 10-?/A waves per second Selection. of Filters

Optical filters are used in colorimeters (absorptiometers) for isolating any desired spectral region. They consist of coloured glass or coloured gelatin coated on glass and possess the property of selectively transmitting light from a specified region of the spectrum, the other regions being absorbed. Choice of filter colour for particular colour of the solution is given below. Colour of solution

Colour of filter

Purple

Green

Otange-red

Blue-green

Yellow

Blue

Violet-red Blue

Purple Red

Colour Measurement

501

The filters are rinwufactured by Kodak, Ilford, Corning, etc. ‘The transmis-

sion ranges for the various filters as prescribed by the manufacturers are given in Table 17-2. . TABLE Number

17-2: Transmission Range for. various Ilford Filters

Colour of filter

_

Transmission region

Peak wave-

(nm)

length. =

{am)

601

Spectrum violet

380-470

405 ’

602

Spectrum blue

440-490

425

603

Spectrum blue-green

470-520

47°

604

Spectrum green

500-540

490

605

Spectrum yellow-green

530-570

520

606

Spectrum yellow

560-610

586

607 .

Spectrum otrange

570 with absorption in-

600

creasing

608 609

Spectrum red Spectrum deep red

.

620 into infrared ~ 650 into infrared

660 690

In addition to the above, Ilford markets a series of bright spectrum filters,

Nos. 621-626, which are considerably brighter (i.e. have a higher transmission) than the standard spectrum filters, but with a slightly wider transmission range. The transmission regions and peak wavelengths of these filters are

given in Table

17-3.

TABLE Ilford bright spectrum filter 621

|

17-3: Transmission Range of Bright Spectrum Filters Colour of filter

Transmission tegion (nm)

Peak transmission _ (nm)

Bright spectrum violet

340-§15

450

622

Bright spectrum blue

375-530

470

623

Bright spectrum blue-green

460-545

490

624

Bright spectrum green

499-575

520

625

Bright spectrum yellow-green

510-590

540

626

Bright spectrum yellow

545-620

‘$70

Interference filters (transmission type) have somewhat narrower transmitted bands than coloured filters and are available commercially. Interference filters are essentially composed. of two highly reflecting, bu. partially transmitting, films of metal (usually silver separated by a spacer film of transparent material). The amount of separation of the metal films governs the wavelength position

of the pass band, and hence the colour of the light that the filter will transmit.

502.

= Analysis of Fruit and Vegetable Products

This is the result of an optical interference effect which produces a high transmission of light when the optical separation of the metal films is effectively a half wavelength or a multiple of a half wavelength. Light which is not transmitted is for the most part reflected. The wavelength region covered is from 330 to1,200 nm. The nominal peak transmittance is 459% and the half-width is less than 20 nm for filters of interest in colorimetry. The half-width is defined as the spectral width of the pass band in millimicrons (or nm) at the level where the transmittance is one-half the peak transmittance. The ideal way of selecting a filter for use with a coloured solution is to construct first, by means of a suitable spectrophotometer, the absorption curve for the visible spectrum. Comparison of this curve with the spectral tratismission curves of the set of filters supplied by the manufacturers enables a suitable choice to be made. Alternatively, OD (or transmittance)/concentration calibration curves may be constructed with a photoelectric colorimeter, using each of the filters in turn. Asa general rule, the best filter to use ina particular determination is that which gives! the maximum absorption or minimum transmission for a given concentration of the absorbing substance. Less satisfactory methods include the use of a filter that gives the smallest transmission for a given concentration and depth of cell, and the use of a filter whose colour is as close as possible to the complementary colour of the solution (Table 17-1). Estimation of vitamins, minerals, colouring matter, etc., based on colorimetric and/or spectrophotometric methods in the analysis of fruit and vege-

table products have already been dealt with in the foregoing chapters and are also included-in the subsequent ones. While these methods are suitable for a solution of the sample, the colour of food products is measured by reflectance which is discussed in the following paragraphs.

Measurement and Specification of Colour Colour is the first quality attribute a consumer perceives in a container of food. It is often regarded as an index of general quality of the pack and so may influence the consumer’s judgment of other attributes such as flavour. Colour of fruits and vegetables, raw or processed, is dependent upon numerous factors. Change of colour is generally accompanied by flavour changes. Colour refers to the sensation arising from the activity of the retina of the human eye and its attached nervous mechanism. Light reflected or transmitted by the food is received by the retina and sensations are conveyed to the brain which give rise to the concept of colour. The colour perceived depends on the spectral composition of the light source, the chemical and physical character of the object or colourant and the spectral sensitivity characteristics of the eye viewing the object. Measurement of colour implies expressing the above concept in terms of numerical dimensions. Two approaches are possible-—a complete quantitative

specification or the use of a numerical index which defines 2 colour adequately

Colour Measurement

503

for specific purposes and enables comparison. Complete specification of colour requires measurement

of three recognizable atccibutes of cofour: .

1. Hue: the kind of colour, red, blue or green. 2. Saturation: the depth or strength of hue or the extent to which the pure

hue is admixed with white. 3. Lightness: which may be understood as the extent to which the hue is diluted with black. It is associated with brightness aspect of colour and usually depends on the relative luminous flux transmitted or reflected by the colourant. A glossary of terms used in colour measurement and specification is given

in Table 17-8. In the measurentent of colour, it is necessary to relate hue, saturation and

lightness of colour to the properties of light which stimulates the retina. The

amounts of light in different parts of the visible spectrum reflected or transmitted by a coloured body can be measured by a spéctrophotometer. A spectral curve showing intensities of light at different wavelengths gives a complete specification of colour in physical terms. ; Coloured objects, such as paint, chips, ceramic tiles, etc., sometimes used as colour standards, may fade or change with age. In order to avoid these difficulties and place the measurement and specification of a colour on a

scientific basis, the International Commission on Illumination (CIE: Commission Internationale de l’Eclairage, 1931) adopted a set of standards which has made it possible to define the colour in absolute terms. The

CIE

System!

“It is well-known that any colour can be matched exactly by a suitable

mixture of only three colours selected from the red or amber (X), green (Y) and blue (Z) parts of the spectrum (Fig. 17.1). The three selected 250 200 150 100 50

350 400 ©

500

600

700

Wavelength ——»

Fig. 17.1 CIE primarics used in matching colour.

504

— Analysis of Fruit and Vegetable Products

colours are called ‘‘primaries,” and the relative amounts

of theni required

to match a given colour are called the “tristimulus” values of the colour.

If light beams of these three colours are directed on a single point, their combination would give the impression of white light. If they occupy the corners of an equilateral triangle (Fig. 17.2) and shine inwards over all parts Equal red values along this line

Equal green values along this line.

Red

Equal blue values along this line

(Courtesy

Fig. 17.2 Colour triz::* . G.J. Chamberlin, Tintometer Sales Ltd.)

of the area of the triangle, the intensity of a particular colour will be 100% at the particular corner from which it emanates, becoming uniformly weaker as the light progresses farther away from the corner. By the time it reaches any point on the opposite side, its intensity is zero. At the centre of the triangle (O), all three lights having travelled an equal distance are present in equal amounts. As shown in the figure, the three coloured lights are indicated by the words red, green and blue. Thus, at the point blue, the light is 100 % blue and at the point Y on the red-green line there is no blue, and at any place along the red-green line, the amount of blue will always be zero. The same applies to the other two lights from the remaining corners. If the power of the red, green and blue lights is suitably chosen, the effect of the combined colours at the point O in the middle of the triangle will be white light. Nearly every colour can be matched at some point in the triangle a suitable combination of the three colours.. The position of any point in triangle can be defined mathematically by means of coordinates and this ables values to be quoted which may be converted into actual colour or

by the envice

versa. Addition of red and.green lights in the appropriate proportions gives a pure yellow and that of red and blue gives violet. At points atong the side of the triangle, between the red and green lights, will fall all those shades of colour which are obtained by mixing these two lights in varying proportions, nantely, orange, yellow and yellow-green. These are examples of the fully saturated hues, because the third primary (namely blue) plays no part in the mixture. On the side between the green and blue lights will be found all those hues

Colour Measurement

505

which can be obtained by a mixture of those two primaries, and along the third side of the triangle will appear those colours resulting from a mixture of blueand red in varying proportions. Colours along this third side do not appear in

the spectrum and are called the non-spectral purples.

If the hues along the sides of the triangle are now compared with similar hues in the actual spectrum, the former will be found to be less rich or saturated than the spectral colours. For example, ifthe red-green which appears midway along the line joining red and green is compared with the nearest colour in the spectrum, the spectral colour will appear much richer. The spectral

red-green must be diluted by mixing with a certain amount of blue light to exactly match the red-green of the triangle. This is similar to imagining the spectral colour to be outside the triangle and being brought inwards by the addition of the blue light so as to match the red-green of the triangle. Adding © blue to the sample is exactly the same, mathematically, -as subtracting blue from the mixture, i.e. ‘adding a negative amount of blue to the mixture’, Hence, matching of any spectral colour with the chosen primaries is possible only at the actual red, green and blue corners of the triangle. All other spectral colours fall outside and have to be admixed with the third primary in order to dilute them and bring them sufficiently towards the white centre to find a place upon the sides of the triangle. If all the points outside the triangle where the spectral colours would fall are joined, a curve as shown in Fig. 17.3 is produced. This curve does not extend beyond the red-blue line because these hues do not exist in the spectrum. Locus of Spectral Colours

Ae eeecira

Fig. 17.3 Diagram showing spectral colours falling outside the chosen primaries (red, green and blue) at the corners of the triangle. (Courtesy: G.J. Chamberlin, Tintometer Sales Ltd.)

Although, within the actual triangle, it is possible to find all the hues that can be appreciated by the eye, it is still not possible to obtain the saturation necessary to match all the rich spectral colours since they fall outside the range of the three primaries which have been chosen. Yet these three primaries are

the most intense that can possibly be obtained because they are the spectral colours.

In the CIE system, this difficulty of having certain colours falling outside the colour triangle has been overcome by adopting three reference primaries which are theoretically assumed to have greater saturation than spectral colours, and consequently fall right outside the spectrum locus. Thus the use of negative values is avoided. Since these three theoretical primaries cannot

506

Analysis of Fruit and Vegetable Products

be produced, nor appreciated by the eye, they are referred to as ‘stimuli’ and not as primary colours. These three imaginary red, green and blue ‘unreal colours’ form the reference stimuli and are designated by the letters X, Y and Z, respectively. Fig. 17.4 shows how they are chosen to enclose all the spectral colours. It is not

necessary to make use of negative values. In actual practice, however, it is necessary to use real colours and, therefore, these three reference stimuli are defined in terms of the thrée real spectral lights mentioned previously by a mathematical formula. When a match has been made with real colours, the

result can be converted by means of this formula to be expressed in terms of X, Y and Z.

The triangle constructed joining the points X, Y'and Z is not equilateral, but, having defined these stimuli, it is simple to plot these points as the cor-

ners of an equilateral triangle and redraw the spectrum locus and the position of the real primaries, red, green and blue, inside it as shown in Fig. 17.5. The Y

IMWZ\IVLAZ WANA

VSI VLVIN EPS IVY VBETLLALA

x

Fig. 17.4 Diagram showing the reference stimuli (imaginary unreal colours-X, Y and Z) enclosing all the spectral colours. (Courtesy: G.J. Chamberlin, Tintometer Sales Ltd.)

Zz

WIP TITAN 8

_ Fig. 17.5 An equilateral CIE triangle. | (Courtesy: G.J. Chamberlin, Tintometer Sales Ltd.)

values of the three stimuli, X, Y and Z, can now be used for calculations just

like those for the real primary colours. By a further transformation of triangles (Fig. 17.6) it is possible to arrange the three points (X, Y and Z) to be at the corners of a right-angled triangle and then only two of the three primaries (X and Y) can be plotted on ordinary graph paper. When the tristimulus values, X, Y and Z, are made to add up to unity, the values are known as ‘chromaticity coordinates’ and are denoted by the small letters x, y and x. These are the relative proportions of the three primaries

Colour Measurement

507

Fig. 17.6 Right-angled CIE triangle. (Courtesy: G.J. Chamberlin, Tintometer Sales Ltd.)

necessary to match the colour and they may be plotted in the same way as

indicated for X, Y and Z. Any given colour to be described in CIE terms can now be located in the spectral locus by the relative distances along the x and y coordinates, representing respectively the values of X and Y. When the total distance along the x coordinate between Z and X in the above

diagram is indicated by units sub-divided into equal fractional parts and the same applies to the y coordinate between the points Z and Y, the diagram becomes the CIE Chromaticity Chart (Fig. 17.7).

aH 700

alee

i

e

urple

date \L Sow | Na"| wader(tol Tt lel |nolo eA aplaedy obit| sabes lie)» 9000

100

Ss4:

380,

io “300°

.300

.400

,

+500

Fig. 17.7 Chromaticity diagram.

.600

.700

800

Analysis of Fruit and Vegetable Products

508

According to the CIE system,? an equation correlating any real colour (©)

as seen by an observer in average daylight and the three standard primaries can be expressed as:

C(C) =X (X) + V(Y) + 24 where an amount of C of the colour (C) is matched by a mixture of the amounts X, Y and Z of the three standard primaries (X), (Y) and (Z). Therefore, X, Y | 7 and Z are the tristimulus values of the colour(C). of amount unit a to equal made To conveniently specify the colour, C is colour, called the trichromatic unit and this can be written as:

1C =x (X) + 9(Y) + 7 @) where,

x =

x

Paty os

°

=

ne

Y XeYsZ an d

xX =

men

ie

X+¥+Z

x, y and z are called chromaticity coordinates (or trichromatic coefficients) and represent the amounts of primaries amber or red (X), green (Y) and blue (Z) required to match one trichromatic unit of the colour. Only two of these, usually x and y, are required to be given since the third can be deduced from

the equation

:

= 1 Gib)

In addition to the measurement of coordinates, it is necessary to measure

the total amount of light in the colour which is defined. by the percentage reflection or transmission. This is done by assuming that two of the reference stimuli, X and Z, have zero luminous efficiency, which means that all of the light energy represented by a colour is regarded as coming from Y. The amount

Y of the primary stimulus (Y) is then a direct measure of the lightness of the colour and is called luminance factor (or the transmission or reflection.

factor). If Y values are plotted perpendicularly

to

the

chromaticity

plane, the irregular colour solid or colour space is created within which any real colour

can

be fixed as a

unique point with the CIE coordinates x, y, and Y. To determine the visual dimensions of a particular colour,

;

“erie

Ve me

a

45° _ the chromaticity diagram (Fig. 17.8).

x Fig. 17.8 Determination of dominant wave-

length and purity of colour.

mark

the illuminant (white light), S, on

Draw ‘a line from this point to the point of intersection of the x and y chromaticity coordinates of the colour (C) and extrapolate to

the edge of the figure (D), which is the locus of the wavelengths of the

Colour Measurement

509

spectral colours. Reds are located at the right of the chromaticity diagram,

greens at the top and blues at the bottom left.

.

For colours, such as C; falling into the triangular region of the chromaticity

diagram (Fig. 17.9) formed by the corners S, 380 nm and 770 nm (i.e. the

region of the purple or nonspectral colours) only the complementary wave-

Fig. 17.9 Determination of complementary wavelength and purity.

length can be determined because a dominant wavelength does not exist for such colours. Find the complementary wavelength by intersecting the straight line through C, and S with the spectrum locus; in the present case it is 540 nm. To distinguish the complementary wavelength from the dominant wavelength, attach either a negative sign to the former or place a C after it (as, for

example — 540 nm or 540 (nm).° Colour dimensions may then be represented, with reference to Fig. 17.8,

as given below. 1. Hue of the colour C is given in terms of the dominant

wavelength at

the point D. 2. Saturation of colour C is measured in terms of purity which is the ratio of the distance SC to the distance SD. 3. Lightness of colour C is given by its Y coordinate perpendicular to the chromaticity plane and is represented as Y %.

- Hue and saturation together with lightness (Y %) give a better conceptof colour. For example, two colours having same x and _y values but with a diffe-

rent Y could only differ in brightness. Two colours with the same Y plotted along the same line would differ only in purity. Colours of different dominant wavelengths would lie on different. lines when plotted from the illuminant.

The CIE system has certain disadvantages. For example, equal differences in perceptibility between colours are represented by lines of unequal length

510

Analysis of Fruit and Vegetable Products

in different parts of the colour space.

Further, it is very difficult to form a

mental picture of a colour from its CIE coordinates. In the above system of describing what a colour looks like, certain differences may arise due to an observer’s individual reaction to colour. Therefore,

the CIE system has defined a “‘standard observer” which was arrived at by asking a number of observers to match a sufficient number of points on the

colour triangle by means of the three agreed real colours and then taking an average of their readings. It has been agreed internationally to adopt three types of illumination given below as standards.1 I/uminant A: Corresponds to the light from a gas-filled tungsten lamp ope-

rated at a colour temperature of 2,856° K. Iluminant B : Corresponds to the more yellow type of average daylight which —

is commonly found in Europe, and consists of the standard illuminant 4 in conjunction with a colour filter consisting of two solutions B, and B,, each 1 cm in thickness and contained in a double cell made of colourless optical glass. ; Iiluminant C : Corresponds to the light from the sky rather than sunlight and has been compared with the north sky. It consists of the standard illuminant 4 used in conjunction with a colour filter consisting of two solutions C, and C, in a cell as in the case of illuminant B. Dg; : A new standard illuminant operating at a colour temperature of approximately 6,500° K and is intended to reproduce neutral daylight more exactly and includes some near ultraviolet down to 300 nm. At present no actual lamp has been recommended which fulfils this requirement.4 The chemical composition of the solutions B,, B,, C,, and C, are given below.

Copper sulphate (CuSO,.5H,O) Mannite (C,H,)OH, Pyridine (C;H;N) Water (distilled) to make

Cobalt ammonium sulphate [CoSO,.(NH,),SO,.6H,O]

Copper sulphate (CuSO,.5H,O) Sulphuric acid (sp gr 1.835) Water (distilled) to make

COLOUR

B, 2.452 g 2.452 g 30.0 ml 1,000 ml B,

C, 3.412 g 3.412 g 30.0 ml 1,000 ml Cy

21.71 g

30.580 g

16.11 g

22.520 g

10.0 ml 1,000 ml

10.0 ml 1,000 ml

MATCHING

In the visual matching of the colour of food against standard colours, ob-

servers should be tested for normal colour vision. Ilumination is of utmost

Colour Measurement

511

importance in colour matching. Metameric colours (colours having different spectral curves appearing identical to the human eye) may match under one

and not under another illuminant. Colour dictionaries, disc colorimeters and Lovibond tintometers are used for colour matching of foods.

Colour Dictionaries The dictionary of Maerz and Paul5 is most commonly used. The dictionary consists of 56 charts. Seven main groups of hues are presented in order of theit spectra. For each group, there are 8 plates, the first printed on white and the successive ones on deepening shades of grey until the colours approach black. The Maerz and Paul colours have been defined in CIE terms and may be converted to Munsell values.? In place-of colour dictionary, colour reproduced, on secondary standards such as painted test panels, rings, discs or plastic models may be used.® The disadvantages of colour dictionary are given below. 1. Difficult to match when surfaces are different of a glossy wet surface against a matt printed 2. Definite jump in colour between neighbouring makes matching difficult. 3. The printed paper standards may deteriorate

(e.g. to match the colour chart). patches of the chart which

through exposure.

Matching procedure : Use a mask of neutral grey having two openings. The size of each opening should be equal to the size of the individual colour patch in the sheet. Place one opening over the sample and the other over different patches on the chart until a match is achieved, and note the colour.

Dise Colorimetry Disc colorimetry is an additive system based on the use of coloured discs.

The discs have radial slits so that a number of them may be slipped together with varying portions of each showing. The discs are spun on a spindle at about 2,700 rpm so that the colours merge into a single hue without flickering. The test sample is placed adjacent to the spinning disc under controlled illumination, and both are viewed simultaneously.’ If the sample is not homogeneous

in colour, it may also be spun.

Disc colorimeters

both disc and sample are viewed through scanning prisms rotated

speed or having an optical system

in which

at high

which brings the sample and the disc

together are also available. In conjunction with disc colorimetry, Munsell system of colour classification has most frequently been used. In the Munsell colour space (Fig. 17.10), colours are specified according to the three attributes—hue, value and saturation. There are 10 hues—R red; YR yellow-red; Y yellow; GY green-yellow; G green; BG blue-green; B blue; PB purple-blue; P purple; and RP red-purple. Each hue is subdivided into 10 shades. Value (lightness) is evaluated on

a scale that ranges from 0 at black to 10 at white, The chroma is expressed

512.

Analysis of Fruit and Vegetable Products

Black

Fig. 17.10

Munsell colour dimensions.

on an arbitrary scale of intensity of saturation ranging from 0 to 18. On each scale, the divisions represent approximately equal value steps. When the colour of a sample is matched against a Munsell disc, the results may be recorded in terms of the areas of the discs exposed (percentages of the total area) or as a ratio of the two chromatic discs, or these values may be

transferred to x, y, x values of the CIE system by calculations,® or by the use of chromaticity diagrams on which Munsell notations are superimposed. The discs are printed with either glossy or a matt finish to facilitate matching with samples of varied surface characteristics. Matching of colour with Munsell discs is a tedious procedure and differences between individual observers may be considerable. EXAMPLE

The US standards for grades of canned tomato juice specify the Munsell system for colour grading. Grade-A sample must reach the following standard or contain more ‘red’ than that produced by spinning the Munsell colour discs in the following combinations: Disc No.

Colour of the disc

Area of the disc exposed

Colour

(%) I

Red

65

2

Yellow-red

21

3* 4*

Black Grey

14 14

5R 2.6.13 glossy finish (Hue 5 red with a value of 2.6 and a chroma of 13) 2.5 YR 5/12 glossy finish (Hue 2.5 yellowred with a value of 5 and a chroma of 12). Ni glossy finish N4 matt finish

*Either disc 3. or disc 4 with 14% exposed or 7% of the area of disc 3 and 7% of the area of disc 4 exposed, whichever most nearly matches the reflectance of the product, may be

Colour Measurement

513

The comparison as made under a diffused light source of approximately: 250 foot candles intensity and having a spectral. quality approximately that of daylight under a moderately overcast sky and a colour temperature of 7,500° Kelvin + 200. In the Macbeth-Munsell Disc Colorimeter, the light source is directly over the disc and product observation is made at an angle of 45° from a distance of about 24 inch from the product. Tintometers

The Lovibond tintometer is a subtractive colorimeter based on direct observation of an illuminated sample through a viewing tube. The instrument is provided with sets of red, yellow and blue glass slides to be used as permanent standards. The slides form an evenly graded series from very light tints (0) to deep colours (20), numbered according to their depth of cclour. The colour intensities in each series of slides are additive so that the slide of

value 10 is equivalent to two or more slides having a total value of 10. The three series are so related that when three slides of equal value are combined and viewed against white, they appear grey or neutral in colour. By a system of prism and mirrors, the sample is made to occupy half the field of view, ‘while the other halfieceives reflected light from a standard white surface which passes through ‘coloured glass slides mounted im racks. When the colour of the sample is matched, it is specified by the values of red, yellow, and blue slides required :for comparison as: 14.0 R + 6.0 Y + 1.0-B.

The Lovibond unit colour is an arbitrary one, but has become generally accepted throughout the world and has the ailvantage of extreme simplicity. The drawhbsick:of tintometer és the extremely small area which appears in the “‘half-field” under the viewing eyepiece, and the eye-fatigue which occurs relatively ‘quickly when making colour matches. However, a good reproducibility occurs between operators who do ‘not suffer obvious vision defects. The results x 0.6- 210ae Wace Sa ans aes eRe ee 2 (asty(Sey" 0.5 2 1/0-5

1300 x 0.6

G Mi

-

i

1

1/0-5+2

- 210}

2

( 2 V(es+3)(o3 +2)

ae EEii ec 24

2/

a

7\

MPR

2AL

APKiln

AP

2(_—2 wee 1300 x 1300

Int

A ai

1300

_ 1847.92

~ —Rie

1

(16)

al n

1300 x 0.6

Kiln (=A

\?

2x 210

(0.6)2—

ost)

-210)

4

Teak ost!

_ 972 ~ Kiln

(17)

Q=01+Q2 1847.92

Kime

972

+ Fin = 9-22 m/sec

1847. Kin eR

12817.83 = 1.28 x 104

Measurement of Viscosity, Consistency and Texture

543

Where the two curves intersect, find the corresponding value of 7 to be 0.65 and

log K’™ to be 3.1. Hence,

log KY°® = 3,1 1/0.65 log K = log K = K = =

3.1 3.1 X 0.65 = 2.015 antilog of 2.015 103.5142 dynes (sec)°™/cm?

Determination Using a Tube Viscometer Consistency Parameters

Kand n Neglecting the Yield Stress ‘PROCEDURE 1. Plot flow rate (Q).vs pressure drop per unit length (AP/AL) values given in Table 18-2 on a log-log paper (Fig. 18.9). 2. From the graph, find the slope, 1/n, to be 5.25/1.85 = 2.84. Hence, n = 1/2.84

0.352. 3. To solve K, use the following equation for flow rate Q. If

Q=

(=a)

Gres +3 ) (Re ane

10 8

6 4

2

; i]

=,

'

y (7) =

t 1

'

~

1

E 0-8

a

we 0-6

ae

A! papel loc re

1/n =2-8378 0°4

n= 0-3524

0-2

0-1

1

eld da dt

20 S@ 6 “6°10 20 A PJAL(dynes/cm3 in thousands)

Fig. 18.9 Flow rate vs pressure drop per unit length. Determination of K and n using tube viscometer neglecting Q.

8)

544

Analysis of Fruit and Vegetable Products

Rewriting the equation (18)

x=

,

4(S47) (aera a)

(19)

For conditions, where Q = 0.22 ml and AP/AL = 1300 dynes/cm?.

na,

11300

$098? fo 22/Teox

=e = 570\ 2 )

(sossa73) 0-6

(1/0-352)+3

)

= 12061229.41 K=310.9

Comparison of Power Law Constant Using Tube Viscometer K ube. ‘ dynes sec”/cm? Considering the yield stress 103.51 Neglecting the yield stress 310.9

n

C dynes/cm?

0.65 0.352

210 —-

Reference 1. Charm, S.E., Adv. Food Res., 11. 355 (1962)

Determination of Consistency Constants using Brookfield Synchrolectric Rotary Viscometer In the tube viscometer, the whole fluid is subjected to high shear rate, and the measured value is more representative of the true consistency!. The high shear rate is an important advantage of tukc viscometer over rotational viscometers. With the Brookfield viscometer, the maximum shear rate is normally less than 100 (1/sec), while the coaxial viscometers can develop shear rates upto 100 (1/sec). In the rotational viscometers, high shear rate may cause a separation of

suspended solids in a puree resulting in erroneous value of consistency, usually : lower.

Determination of consistency constants using Brookfield viscometer, model LVT, is described below. Brookfield viscometer has been selected because of its

wide use in the industry, and as it can handle many fluids that cannot be treated with other viscometers. DETERMINATION CALIBRATION OF THE SPINDLE (BOB) FOR THE END EFFECT

During measurement, the drag on the ends of the cylinder has to be corrected. Two procedures may be used to calibrate the bob.

Measurement of Viscosity, Consistency and Texture

545

Procepure A?”

The basic equation is: ntl - TDL

(20)

where, 7 = shear stress (dynes/cm7) A = torque (dynes cm) D = diameter of the bob (cm) L = equivalent length of the bob which is larger thanthe value actually found.

To solve equation 20, proceed as follows: (i) Using a Newtonian fluid of known viscosity, note the dial readings at different rpm. (it) Calculate the torque corresponding to the dial reading using the following equation:

Dial Torque for full scale gre Rr _ reading in dynes cm A : : Full scale reading on the dial Brookfield Viscometer Model No.

LVF, RVF, HAF, HBF,

LVT RVF-100, RVT HAT HBT

(21)

Torque for full scale dyne cm

673.7 7,187.0 14,374.0 57,496.0

(iii) Calculate the shear rate yy using the equation

y=4a0N

(22)

where, N = revolutions per second. (iv) Calculate the shear stress 7 using equation 2. (v) Solve for L using equation 20 for the 7 calculated above. Equation 20 gives the shear stress corresponding to the torque obtained for every rotation speed applied to a fluid of known viscosity. The viscosity is calculated from equation 3 which becomes 7 = u(‘y)100 when 4 is in centipoises

and r is in dynes/cm?. PROCEDURE B4

At any one speed, vary the depth of the immersion of the spindle in the fluid, note the scale reading, and calculate the torque corresponding to the scale reading using equation 21. The depth of immersion can be varied by placing the sample “container on glass plates or tiles, and removing one plate after another after

noting the scale reading.

546

Analysis of Fruit and Vegetable Products

EXAMPLE

The actual length of spindle number 4 of Brookfield LVT model viscometer is 3.09 cm. To calculate the end effect using mango pulp, the spindle was dipped to varying heights in the pulp, and the scale reading noted at 30 rpm. The results are

summarized in Table 18-4. Plot torque vs depth of immersion (Fig. 18.10). Extrapolate toa value of zero for TABLE 18-4: Effect of Varying Depth of Immersion of Spindle in Mango Pulp on Scale Reading

Depth of

Scale

Torque

immersion

reading

dyne cm

cm 3.09

20.0

134.74

2.51

18.5

124.6

2.09 1.72

15.5 13.5

104.4 90.95

140

@

ro)

(dynes-cm) Torque

ho

1

2

3

h

Depth ot immersion (cm) Fig. 18.10: Determination of end effect from the data found by immersing the spindle to various depths.

Measurement of Viscosity, Consistency and Texture

547

torque and find the intercept (4) on the negative portion of the X axisto be 0:85 cm. The corrected length of the spindle is found from the expression:

L=h+h, where, L = corrected length / = actual length

(23)

ho= correction

For spindle No. 4 immersed in mango pulp, L = 3.09 + 0.85 = 3.94 cm. Determination of Fundamental Consistency Constants without Considering the Yield Stress The procedure described by Garcia-Borras? is given below: (i) Using the sample, note the dial reading at 3 or more rotational speeds. (ii) Plot dial reading vs rpm on a log-log paper. Determine the slope of the resulting straight line. The slope gives » which is the flow behaviour index. (iii) Find the true rate for the non-Newtonian fluid from the equation: (nena aru

Jing

(24)

By

(iv) Plot ona log-log paper, shear stress calculated from equation 20 vs the shear rate from equation 24. The slope of the line so found is 7. The intercept on the shear stress axis at a shear rate of 1 is the consistency index K. (v) The shear. stress corresponding to any shear rate may be found from the graph or by calculation using the following power law equation?

LAK (yy

(25)

(vi) Viscosity of the fluid at any shear rate is calculated from

Napp in centipoise =

——_,, x.100

(26)

Determination of Consistency Constants Considering the Yield Stress

As in the tube viscometer, determination of the power law constants in the presence of the yield stress is more difficult. Determination of the consistency constants of mango pulp using Brookfield viscometer by the procedure of Charm’® is given below. The power law for a fluid having a yield stress is:

(4)

T= K(y)+C The rate of shear is equivalent to

(27)

= U cs aU

kee

ee):

where, dU/dR = velocity gradient U = linear velocity at R R = distance along the radius from the centre

The shear stress, t, is related to torque (A) by

7(2 mRL)R=A

a)

548

= Analysis of Fruit and Vegetable Products

The shear stress-shear rate relationship for a fluid is a substitution of equation 27 in 4 which becomes:

Ue

wht,

gt aih)

29)

The relationship between torque and rotation in a single cylinder viscometer is given in the equation:

2 t NK

A 1 ‘alRe [ seRZ-° |gi [-x]*

I/n _

(30)

where, R, = radius of the spindle, and

R2 = the distance from the centre at which the velocity of the streamline flow is 0. The limits of R are from R, to Re. This will occur where the yield stress, C, is equal

to the shear stress. The point at which this occurs may be determined by substituting C for 7 in equation 28, and solving for R as given below: be

/

Ro =

A

5 aeee

(31)

To determine C, it is necessary to plot \/A/L vs./ N and extrapolate the curve to N = 0. From this extrapolated value, C may be calculated using the equation:

in which

.32

* ®) rE ot(A/L)o is(yeCs N= 0. the force when (%

:

From equations 4, 27 and 28, the relationship between ¥ and N in the presence of yield stress is as follows: K

tae

\

¢

Greg

A

Yn

“ \ Ie cri)

e

From equations 30 and 33 aN

:

but dN/dR is proportional to the speed of the cylinder or y=BN where B is a constant. Since yy = BN, equation 4 can be rewritten as

T- C= K(BN)"

bue

(35)

ie oer A

Hence,

(—e-¢ A

(57 RLO

I/n

1)

1/fn

) = (K)""

Yn

Sie (BN)

(37)

K

ra ey.

(BN)

(38)

Measurement

of Viscosity, Consistency and Texture

549

The value of 1/” can be determined by plotting log N vs log (ee

- 1).The slope of the resulting line is 1/7.

Having found C and n, K may be found by graphical integration of equation 30. Assume different values of R between R, and Ro, substitute in equation 40, and

calculate. Plot the values found versus corresponding Rona coordinate paper, and determine the area under the curve. The area so found is equal to 2 + N (K/C)""

from which calculate K using the equation: Area K=C (SN |

EXAMPLE

(39)

Determination of the Consistency Constants of Mango pulp using Brookfield Viscometer

1. Connect the spindle to the coupling nut of the instrument, and find the spindle suitable for making measurements. 2. Determine the diameter and the length (upto the mark) of the most suitable spindle using a vernier caliper. 3. Determine the end effect correction using procedure A or B (see page 544) and add to the length of the spindle. This gives the corrected L. 4. Immerse the spindle in the fluid, and level the instrument. 5. Note the dial reading at different speeds. Convert the speed in revolutions per minute (rpm) to revolutions per second (rps) and designate as N.

6. Find the torque (A) corresponding to dial reading using equation 21. 7. Enter the data as shown in Table 18-5 and calculate \/A/L and \/N. TABLE 18-5: Data Found Using Brookfield Viscometer (LVT) for Determination of

Viscosity Constants, K, 2 and C of Mango Pulp Spindle

= Number 4

Diameter

= 0.306 cm

Radius (R)

= 0.153 cm

Length of the spindle upto the mark End effect correction required*

= 3.09 cm = 0.85 cm

Corrected length of the spindle (L)

= 3.94 cm

Torque on full scale

= 673.7 dynes cm

Temperature of the product

Speed (rpm)

=

Speed (rps)

= 25° C

i

3.0

6.0

12.0

30.0

N

0.05

0.1

0.2

0.5

10

10.5

12.5

15.0

19.5

24.0

A

70.67

84.13

100.95

131.23

161.32

A/L

17.94

21.35

25.62

33.32

40.99

V/V A/L

4.23

4.62

5.06

5.71,

6.4

O46.

E6094 2 409%

1.01.72

2.34

Dial reading with pulp Torque (dynes cm)**

Root of rps

Torque/length of spindle Root of A/L

ay

L2nR3C

VN -

0.224

0316

*Correction found using procedure B described earlier. **Torque corresponding to dial reading calculated using equation 21.

0447

0707

60.0

10

550

Analysis of Fruit and Vegetable Products

8. Plot \/A/L vs \/N on a coordinate graph paper and extrapolate the resulting curve to N = 0 as shown in Fig, 18.11. Find the intercept which gives \/(A/L)o to be 3.5. Hence (A/L)o is (3.5)?.

9. Substituting (A/L)o value in equation 32, find

1 C= 3.5)2 |——>--_-—— aE geod verb = 83.3 dynes /cm? 8-0

Tx

5

= 80 a

wv 20 c=

60

7)

» 40 20 0

r

12

24

zo

30

571

210 “=

=

0 12 24 36 48.6972

Time (sec)

o

(Ge

Time(sec)

kre

eee

Pee

Time (sec)

150 x ov 9120

=°o 100 i oS

75

wv

50

S

o

E o

x

e

IS

zz. cS

45

a

ELs0 xo

%»5H

25

0

ow « 25

ee ©

10

20

30

=

40

a

Time (sec)

Fig. 18.24

0

0

12

2

36

48

60

72

Time (sec)

Typical force-time (deformation) results obtained for baked beans with A. Pea Tenderometer;

B. OTMS

plate extrusion; C. wire shear;

D. Shear-compression, and E. back extrusion. (Reprinted from Voisey, P.W., and Larmond, E., J. Texture Studies, 2, 96 (1971), with the permission of Food & Nutrition Press, Inc.)

meaningful results. For example, baked beans tested in a variety of test cells gives

different types of curves (Fig. 18.24). Pea tenderometer cell and the shear compression cell give curves which are almost similar with a single peak showing the onset of shearing. When the shear compression blades are withdrawn, a negative force related to adhesion of the sample to the blades is obtained. In OTMS plate extrusion cell, wire shearing cell and the back extrusion cell, additional compression force is seen even after the onset of shearing. Thus, to obtain a clear

record of onset of shearing or rupturing behaviour, shear-compression cell is best suited. Exploratory studies showed that the OTMS wire extrusion cell detected differences in baked beans caused by variety, temperature, storage period, and removal of the sauce.

A variety of products have been tested in this way with a specific test cell for each product. The test cells selected using the Ottawa Texture Measurement System for different fruits and vegetables are given in Table 18-10. The instrumental methods of measuring the texture are based on applying force under controlled conditions. The success of the procedure in predicting the quality is, however, dependent on the design of the test cell employed for the.purpose.?!

572.

_—Analysis of Fruit and Vegetable Products TABLE 18-10: Test Cell for Measuring Texture of different Fruits and Vegetables”

a

Product

Selected Test Cell a

Peaches Cherries

Raspberries Cranberries Strawberries Fruit puree Apple skin Apples Canned pears Tomato firmness Carrots Peas

Snap beans Instant potato Onion firmness

Cabbage Baked beans Sweet corn pericarp

Wire extrusion Puncture 0.01 in. wire extrusion Wire extrusion Puncture . Back extrusion and Kramer universal cell Puncture Wire extrusion Wire extrusion Fruit crushing cell Warner-Bratzler shear Wire extrusion Kramer shear compression Back extrusion Puncture and compression Puncture Wire extrusion Puncture

Force Deformation Curve

Each product in each test cell produces a characteristic curve.!® Factors which influence the magnitude of the curve are: (i) elasticity, viscoelasticity or the viscosity of the material; (ii) rupture behaviour of the material; (iii) sample test cell; (iv) sample temperature; (v) deformation rate; (vi) sample preparation

method; and (vii) particle size and homogeneity of the sample. The force deformation curve (Fig. 18.25) contains lot of information about the material, the interpretation of which presents great problems. In general, the curve exhibits the following characteristics for the cells used: (i) An initial non-linear portion is seen as the sample is packed into the cell, or as the cell components make uniform contact with the sample surface. This does not provide a great deal of information. (ii) An approximately linear portion as the material is compressed. The slope of this line is related to the apparent elastic properties of the sample, and may indicate firmness which is defined as the force required to attain a given deformation. At the end of the compression phase, the sample is compressed toa density at which the next phase is initiated. (iii) An abrupt change in the slope is seen when the sample begins to rupture. This indicates the stress (bioyield force) required to start shearing of the sample and overcome the forces holding the sample together. (iv) In some cases, the change of slope coincides with the peak of maximum force during deformation. This peak is due to the combined effects of elastic behaviour and rupture of the sample.

Measurement of Viscosity, Consistency and Texture

573

——_————_» Force

Deformation

——____,

Fig. 18.25 Mechanical properties derived from typical force-deformation curves of processed Baldwin apples under slow rates of loading.

ade-Force deformation curve d-rupture point

ed-rupture force ae-rupture deformation area under curve ade-rupture energy slope of straight line through bc-indicates elastic modulus. (Reprinted from Fletcher, $.W., Mechanical damage to the processed fruits and vegetables, in Theory, Determination and Control of Physical Properties of Food Materials by Rha, C. (Editor), with the permission of D. Reidel Publishing Company, Holland)

(v) The behaviour after the rupture point varies with the product and the type of test cell like the following: (a) Force reduces rapidly with further deformation showing that the sample had been compressed to the point of catastrophic failure. This shows the resistance of the sample to shearing. (b) Force decreases less rapidly which may be due to further shearing or resistance of the sample. A small amount of sample adhering to the shearing device or extrusion forces may have a similar effect. (c) An approximately horizontal plateau indicating that the successive layers of the sample being sheared are under conditions of constant stress, and that the compressive forces are in balance with the combined effects of shearing, adhesive and extrusion forces. Erratic fluctuations about this plateau indicate the homogeneity of the sample. The product is continuously ruptured and the mean

height of the plateau is a theoretically reliable measure of cohesiveness of the sample. Throughout the rupturing phase, the sample is probably compressed to a constant density, and resistance to shearing, adhesion and extrusion are constant.

(d) The slope of the curve changes to a less extent than in the compression phase but the force continues to increase. It shows that the sample is further

574

Analysis of Fruit and Vegetable Products

compressed after rupture begins, and the sample density may increase changing resistance to shearing. Adhesive and extrusive forces also change and may be large. In this case, the force at which the change in slope occurs must be used as the index of cohesiveness. The maximum force gives a gross index but depends on the extent to which the sample is deformed in the cell. To obtain meaningful results, the depth of penetration of the moving cell into the stationary cell component must be kept constant.

Texture Measurement Using Different Instruments Although many texture measuring instruments have been designed, no one device furnishes all the needed information. Factors to be considered in the selection of measuring device are: purpose of measurement, characteristic to be measured, accuracy and conditions of measurement, design features, cost, portability, etc.?! Among the factors, the most important one is the purpose—quality control or research. For quality control, one textural parameter might be sufficient, while for research, it may be desirable to obtain a texture profile of the product in terms of a number of parameters. The decision of the parameter to be measured is a crucial one. It can be on the basis of intuition, experience or research. The conditions under which the measurements are performed must be carefully selected. If the goal is correlation with organoleptic data, then both environment, and the rate and type of force application should simulate the conditions of sensory evaluation. With temperature-sensitive materials, the effect of temperature

on __

textural characteristics must be considered. Conditions in the mouth are kinetic and not static both with respect to the environment and the acting forces. For example, the rate of application of force in the mouth changes with the firmness of the food—the harder the product, the slower the speed with which the teeth move down. The instrument must be adjusted accordingly. The accuracy of the measurement is governed by the magnitude of differences in the product, natural variations, and the purpose of the data. If the aim is to correlate with consumer acceptance, the difference should be big enough in objective numbers to be discernible by the consumer. Instrument accuracy and precision depend upon the design which in turn influences the price of the unit. Measurement of texture using different commercially available instruments are given in subsequent paragraphs.

Hand Operated Pressure Testers

The characteristics of the different types of pressure testers are given in Table 18-11. In all these instruments, maximum force required to rupture the sample is measured by pushing a metal probe, usually cylindrical, into the sample to a given. depth. The Magness Taylor Pressure Tester is most commonly used to determine the softening of fruits during maturity. Instruments with 10 Ib or 30 Ib force spring are

Measurement ‘of Viscosity, Consistency and Texture

575

TABLE 18-11: Particulars of Hand-Operated Pressure Testers Type

Force ranges

Magness Taylor

10 Ib or 30 Ib

Tip diameter (in) 5/16, 7/16

pressure tester

D. Ballauf Co., 619, HSt.,

;

Chatillon

500,

pressure tester

Efi-Gi

Supplier

N.W. Washington D.C. 20001

0.026

John Chitillons & Sons

1000 g, 5 lb

0.032, 0.468

Div. Aero-Chatillon Corp.

10 Ib, 20 Ib 40 |b

0.058, 0.063 5/16, 7/16

83-30, Kew Gardens Rd., -Kew Gardens, N.Y. 11415

26 Ib

5/16, 7/16

fruir.tester

Effi-Gi, Carso Garibaldi, 48011, Alfosine, Ravenna, Italy. _

UAC. Fruit firmness tester

10 Ib, 30 Ib (other force

(with stand)

ranges available)

5/16, 7/16

Western Industrial Supply . Inc., 236, Clara St. San Francisco, Cali. 94107

TABLE 18-12: Pressure-test Reading and Degree of Ripeness for Varieties of Apples Pressure test reading in pounds Degree of ripeness

Variety Delicious

Hard Firm Firm-ripe

Ripe

Rome Beauty

17-20

18-23

14-17 11-14

15-18 12-15

8-11

10-12

available. Each of them is provided with one or two punches—5/16 or 7/16

inch in diameter. The large diameter punch is used on softer materials and the smaller one on the firmer materials. Choice of 10 Ib or 30 Ib spring scale depends on the firmness of the test material.

The plunger is held against the surface of the fruit and forced into the fruit with steady pressure to attain the force necessary for breaking the flesh. The fruit is usually peeled to overcome the interference of the skin with the action of the plunger. The force recorded on the pressure tester indicates the BUSS of the fruit (Table 18-12).

In the study of force distance curves of a single punch pressed into foods, Bourne?? found three characteristically different shapes of curve (Fig. 18.26). The type-A curve is typical of freshly harvested apples of most varieties. In this type, there is a rapid rise in force over a short distance as the pressure tip moves into the food. This is because of the whole item deforming under the load, and there is no puncturing. The stage ends when the pressure tip begins to penetrate into the flesh and is represented by a sudden change in the slope called the “yield point”

576

Analysis of Fruit and Vegetable Products

Force(kg)

o=—2

o—2

0--2

Distance (cm) Fig. 18.26 Characteristic force-distance curves obtained in puncture tests. YP-Yield point MT-Reading which would have been obtained from the Magness-Taylor fruit

pressure tester. (Reprinted from Bourne, M.C., Texture measurementsin vegetables, in Theory,

Determination and Control of Physical Properties of Food Materials by Rha, C., (Editor), with the permission of D. Reidel Publishing Company, Holland)

(YP) or the “bioyield point” which marks the beginning of the second stage. The second stage, represented by the rising saw-tooth line, occurs when the pressure tip penetrates the tissue. With type-A commodities, the pressure test depends upon the depth of penetration of the metal tip into the fruit, anditis a higher value than the bioyield point. Hence, the pressure tester must be pushed with increased force to make the pressure tip penetrate to the required depth. After the yield point is passed, each increment in pressure causes a corresponding increment in penetration. Unless the pressure is increased, there is no further penetration. The steeply sloping second stage of the type-A force-distance curve traces this behaviour. With this type of commodities, the pressure test could be stopped exactly when the penetration reaches the required depth as indicated by the line inscribed on the pressure tip. A

AN

The type-B curve is characteristic of apples stored for a considerable\period. In this type, the first stage is same as type-A. The second stage after the yield point, however, appears as a plateau which is approximately constant over a considerable

distance of penetration of the tip. In this case, the pressure. ntest is almost independent of the depth of penetration of the metal tip, and the readling obtained is approximately equal to the yield point.Hence, in such products, the force on the pressure tester must be increased until the yield point is reached, at which time, the

Pressure tip continues to slide into the tissue without further increase in the

Measurement of Viscosity, Consistency and Texture

Potato

577

Squash

Cucumber

Carrot

Force

O20

2

0.

2

00

2

Ot?

Distance (cm) Fig. 18.27 Characteristic force-distance curves for puncture tests on vegetables. Each of them is type C curve.

_pressure. With this type of test, it is possible to stop the pressure tip with some

difficulty when it reaches the marked line. The type-C curve is characteristic of vegetables like potato, carrot, beet, etc. (Fig. 18.27). The first stage is the same as type-A, but in the second stage, the force decreases sharply after the yield point. In such commodities, the yield point and the pressure test are identical; the force on the pressure tester must be increased until the yield point is reached, at which time, the pressure tip plunges into the flesh very rapidly and is stopped by the splash collar. It is almost impossible to stop the penetration at the marked line. Bourne?} recommends that the hand pressure testers are good instruments to measure the texture of vegetables although many workers consider the test to be unsatisfactory for vegetables. The instrument is simple, inexpensive and portable. The tips of the hand-operated pressure testers may be fixed to force-deformation recording machines like Instron to get more information than just the maximum force required to break the sample. The results are comparable |(Table 18-13).

Texture meters for Peas and Beans

The texturometer for peas consists of a grid of blades rotated at a constant speed through a second grid so that the force on the latter is counter-balanced by a. pendulum. As the peas are.cut by the blades, the maximum force is indicated by the angular movement of the pendulum displayed by a pointer moving over a graduated scale. The instrument has been officially recognised for determining the

578

Analysis of Fruit and Vegetable Products TABLE 18-13: Comparison of Punture Test using Effe-Gi hand Puncture Tester and Instron”

Commodity

Irish potatoes Summer squash Beets

Effe-Gi Tester Mean force

Instron ‘Mean force

kg 10.76

kg 10.86

9.78

f

12.23

9.50 12.59

(5/16 inch diameter punch, mean of 25 punches)

maturity of the peas for canning. A value of 90-95 indicates fancy quality peas while the value over 100 indicates a lower grade standard. Peas testing 100-180 may be expected to yield a substandard end-product. Maturometer, Kramer Shear Press and the Ottawa Texture Measuring System have also been made use of for determining the maturity of peas. A texturometer designed by Voisey and Hansen?‘ in which the shear blade is similar to that of the Warner-Bratzler shear apparatus?> has been made use of for determining the maturity of French (green) beans.”¢ Using a single blade, the force required to shear the beans placed edgewise was found to be generally more than that required in the flat position in tender beans, and the trend was reversed in mature beans. A value of 9 kg force and above indicates over-mature beans,while below 9 kg the beans are tender. Kramer Shear Press

This is the first machine developed to measure the texture of foods. The machine consists of a hydraulic press in which the ram drives the moving component of the texture testing cell into the stationary component which is supported on a frame. In the commercial version, manufactured by Food Technology Corporation of USA, the ram speed ranges from 0 to 12 inch per min,

and the ram force, greater than 5,000 lb. Two proving rings with dial gauges indicate the force. The ranges are 0-300 lb and 0-3,000 lb. The machine also has electronic force recording charts. General Foods Texturometer

This instrument is based on the principles made use of earlier in the Denturometer developed in the fifties by Proctor and his group in MIT, USA. The machine consists of deformation mechanism, force indicating system and texture cells.2” The deformation mechanism consists of an electric motor which drives an articulator up and down at 12 to 24 strokes per min. The articulator is a lever pivoted near the centre with the plunger deforming the sample at one end, and the motor drive mechanism connected to the other end. The plunger motion is sinusoidal. The mechanism can be adjusted to vary the extent of deformation of

Measurement of Viscosity, Consistency and Texture

579

the sample. The sample is supported under the plunger bya platform mounted on a beam transducer. The strain gauges on the beam detect the forces applied. Flat bottom brass or acrylic plungers with diameters ranging from 17-50 mm, and a plunger with three serrations are also supplied. A flat-plate is provided to support

solid samples and a shallow dish for liquid or semi-liquid products. The instrument is standardised by positioning the articulator arm for a clearance of 0.125 inch (75% compression) between plunger and sample stage at a point of maximum drive. A standard sponge rubber sample 0.5 inch in height is maintained for standardisation. The standard should be referred to at intervals

during a day’s run of samples. All samples, wherever possible, should be made to a height of 0.5 inch and an area at least equal to that of the plunger base. If other sample heights are used, corrections have to be made. Making use of this instrument, it is possible to measure the texture profile of the sample. Figure 18.28 represents a typical curve obtained from the recommended standard test involving two successive cycles (or bites) on the

sample in the

texturometer, peak A, being the first bite and peak A2 the second chew. From this \ curve. the following parameters can be made out.?’ \ Cohesiveness = 2 Ai

Springiness =C-B

C=Time constant for clay

Fig. 18.28 Typical texturometer curve

(i) Hardness: It is the peak force during the first compression and is measured as the height of the force peak on the first compression cycle. Since the height of the sample is 0.5 inch and the plunger-travel is fixed to reach within 0.125 inch of the plate, 75% compression is involved. All hardness values are normalized to a onevolt output by the following method.

Hlardocte:t

Height of first peak Volts input

580

Analysis of Fruit and Vegetable Products

(ii) Cohesiveness: It is measured as a ratio of the area under the second peak and the area under the first peak (A2/A1). Since the parameter is measured asa ratio of two

areas, the input voltage is not critical. However,

the voltage used for

measurement should be sufficient enough to keep the peak heights within the confines of the recorder paper. The value is a direct function of the work needed to overcome the internal bonds of the material. If the food sample exhibits adhesiveness, coat the material with talcum powder before recording cohesiveness profile to overcome the distortion created by the negative adhesion peak.

(iii) Springiness or Elasticity: It is measured as the difference between the distance B measured from the initial sample contact to the contact on the second chew, and the distance C, the same measurement made on a completely inelastic

standard material such as clay. With the instrument, the value of C is 68.5. Preferable sample height for this measurement is 1.0 inch. Springiness = C—B Currently, springiness is defined as the height that the food recovers during the

time that elapses between the end of the first bite and the start of the second bite. (iv) Adhesiveness: It is measured as the negative force area beneath the base line (As) representing the work necessary to pull away the compressing plunger from the sample. Measure using samples 0.5 inch high and input voltage of 5. (v) Fracturability (or brittleness): It is easily characterised by the multipeak

shape of the first bite, and is measured as the height of the first significant break in the peak. Conditions for measurement are:sample height of 0.5 inch and input voltage of 1. (vi) Gumminess: Hardness X cohesiveness. Multiply the results by 100 to eliminate decimals. (vii) Chewiness: Hardness X cohesiveness X elasticity.

Measurements made on the texturometer are reported in arbitrary instrument units which can be converted into CGS units by the use of standard references. The texture parameters described by the General Foods Group give excellent correlations with the sensory evaluation of foods. An additional textural parameter called “crushability index” has been introduced. This is obtained by subtracting twice the area of decompression of the first bite from the total positive area of the first bite, and calculating the ratio D/A,. D represents crushing work and A, represents elastic work during compression. The crushability index (D/A,) has a correlation with the tenderness in some foods.

The dimensions for the texture profile parameters described by the General Foods Group are given in Table 18-14. The determination of texture of strawberry ts given below to illustrate the use of the Texturometer.?® The instrument was fitted with a flat 17 mm diameter brass plunger. The speed of chewing was set at a rate of 12 thews per min, and the distance between the supporting platform and the plunger in its most downward position was fixed at 1.2 mm. Six to 15 individual determinations were made per lot and the result averaged. Fresh and frozen strawberries were evaluated as halves

Measurement ofViscosity, Consistency and Texture

581

TABLE 18-14 Dimensional Analysis of the General Foods Group

Texture Profile**

Mechanical

Measured

Dimensions of

parameter

variable

measured variable*

Hardness Cohesiveness

Force Ratio

ml/¢? Dimensionless

Elasticity

Distance

Adhesiveness

Work

: mi /¢

Brittleness

Force

ml/$2

Chewiness Gumminess

Work Force

mE /¢2 ml/¢2

Viscosity”

Flow

B/t

(fluids only)

* m = mass; / = length; ¢ =.time » Viscosity isi frequently measured as volume flow per unit time, the measured variables having dimension /?/2.The property of viscosity has the dimensions, m/ lt.

and placed on the platform with the cut surface down. Freeze dehydrated berries were evaluated as slices. All the tested samples were of the same height initially. If necessary, slices were built up to the required height. Firmness and crispness are expressed as grams force. Cohesiveness is a. dimensionless parameter representing a ratio of the recorded area for the second and the. first chew. Adhesiveness is a function of work, and is expressed as grains force multiplied by centimetre chart. The degree of juiciness was determined by placing one piece of Whatman No. 41 filter’ paper under the sample and another piece of the filter paper above the sample and determining the total wetted area after three consecutive instrument “chews”. A typical texturometer curve for a strawberry is shown in Fig. 18.29, and the objective characterisation.of underripe, ripe and overripe fresh strawberries in Table 18-15, and fresh, frozen and freeze dried strawberries in Table 18-16.

TABLE. 18-15: Objective Texture Characterisation of Fresh Strawberries 28 (New Jersey Sparkle)

Underripe a

Firmpess (g) Cohesiveness

Crispness (g) Adhesiveness (g. cm)

(% showing) Juiciness (cm?)

F

;

1470

0.14 4.1

Overripe

1380 |

1180 .

-0.16

1550 (55%) - 58.0

Ripe

1160 ;

8.0

(100%) , 645

0.15

1000 3.2

(83%) 67.1

582.

Analysis of Fruit and Vegetable Products F

Force—> Ad

Ai

A2 Time—

Fig. 18.29 A typical texturometer curve for a strawberry C-Crispness F-Firmness Ad-Adhesiveness A,-First chew

Ao-Second chew ,

Ao/Ai-Cohesiveness.

(Reprinted from Szezesniak, A.S. and Smith, B., J. Texture Studies, 1,65 (1969), with the permission of Food & Nutrition Press, Inc.)

TABLE

18-16 Objective Texture Characterisation of

Fresh and Processed Ripe Strawberries?® "Fresh (sparkle)

Firmness (g)

Cohesiveness

Crispness (g)

Adhesiveness* (g cm) Juiciness (cm?)

1380

0.16

1160

8.0 64.5

Frozen in

Freeze dried

syrup (Fancy

rehydrated

North West)

(Shasta)

290

290

0.44

170

6.2 75.0

0.35

0

10.6 43.0

* All samples showed the adhesiveness peak.

Instron Testing Machine This machine was initially used for testing engineering materials and was first reported for measuring the texture of foods in 1966.!2 The machine consists of two parts: (i) the drive mechanism, which drives a moving crosshead in a vertical

direction by means of twin lead screws at selected speeds in the range of 0.05 to 50 cm/min,; and (ii) the load sensing and recording system which consists of electric bonded-wire strain gauges whose output is fed to a strip-chart recorder. A sensitivity selector switch and five different load cells enable full scale deflection of the recorder pen over the load range of 2 g to 5,000 kg. The recorder chart and the crosshead are synchronously driven from the same power supply, and hence the time axis of the chart is a measure of the movementof the crosshead.

Measurement of Viscosity, Consistency and Texture

583

A rectangular space of approximately 28 X 20cm is available for working parts, and there is a stroke length of 70 cm. The working parts (fixtures) include (i) flat

compression plates, cylindrical compression box, (ii) assortment of needles and punches, (iii) a single star-shaped needle from a commercial cherry-pitter, etc. A careful choice of fixtures permits the measurement of tensile, compressive, flexural or shear streams within the sample. The recorder chart can either run _ according to time (force-time) or at five different ratios to the crosshead speed (force deformation).

Instron measures force-distance-time functions, and is most useful. for measuring tension, compression and bending required for establishing fundamental material properties. The Instron is extensively used to measure the

texture of fruits, vegetables, processed foods etc. A few examples are given below: Simple Compression Figure 18.30 shows the force-distance curve during the compression of a whole apple between the flat surfaces of Instron. Deformation of the apple starts with the application of a small degree of compression which results in a steep increase in force upto the point A. No physical damage is observed in the apple upto the point A. However, the distinct break in the curve at the point A indicates some damage to the apple though not seen physically. Cutting the apple into halves at this point shows an internal fracture in the flesh of the apple shearing away from the body of the fruit in conical form. Point B shows the splitting of the apple skin and rupture of the whole apple. Further application of compression breaks the whole apple in the form of opened petals of a flower bud. The CD portion of the

E

|

i) 1)

B

A

=

fe) w

00

,

:

Compression —>

Fig. 18.30’ Force-distance plot for compression of a whole apple in the Instron. (Reprinted from Bourne, M.C., Interpretation of force curves from instrument

texture measurements, in Rheology and Textu:e in Food Quality by DeMann J.M. P.W. Voisey, V.F. Rasper and D.W. Stanley (Ed.), p. 263, with the permission of the AVI Publishing Co., Box: 831, Westport, CT 06881)

584

. Analysis of Fruit and Vegetable Products

curve represents this phase. Continued compression results in complete crushing of the apple which spreads out thinly on the flat surface of Instron. The increase int force from D to E is the pulverizing force required to disintegrate the apple into small particles. Tnis force decreases to zero as the crosshead is reversed at point E. Extrusion Shear

A simple test for measuring the texture of some foods is to place the food ina strong container open at the top, and to compress the food with a loose fitting plunger until the food flows between the plunger and the walls of the strong container. This is described as the ‘back extrusion’? or the ‘extrusion test’ (Fig 18.31).

Force —>

0 A

0 Fig. 18.31

Distance

Typical force-distance curve obtained with the extrusion test on fruits and

vegetables.

A to B—Force necessary for progressive deformation and compression of peas and vegetables.

B—Force at which the peas are packed solidly and the liquid pressed out from the peas. B to C—Transition from packing to extrusion when more juice is pressed out. C—Rupture point at which the liquid flows through anular space or the force necessary to begin the process of extrusion. C to D—Plateau showing the force needed to continue extrusion. D-—Termination point of extrusion.

Firmness of Tomato

Holt}! measured the firmness of the different parts of a tomato by attaching a plunger with a 0.04 inch diameter tip tapering to a 0.02 inch diameter to the crosshead shaft of the instrument,and driving it into the tomato at a crosshead speed of 1 cm/min. Distinct peaks were observed on the force distance curve

Measurement

250

200

of Viscosity, Consistency and Texture

-

585

xocarp

ye

2)

= t'S.0 9

~ 100

O° w

Mesocarp peseulas

stran

Pou ocarp » Locular juice

Distance Fig. 18.32 Instron force-distance curve for a ripe tomato.

(Reprinted from Holt, C.B., J. Texture Studies, 1,491 (1970), with the permission of Food & Nutrition Press Inc., Westport, (T 06881)

(Fig.18.32) which are related to the firmness of the pericarp which consists of the skin (exocarp), the flesh (mesocarp), and the inner skin (endocarp). The main peak characterises the passage of the plunger through the exocarp; a plateau is found in the mesocarp tegion, and a smaller peak in the endocarp region. Finally, a plateau is obtained as the plunger enters the locular juice. The instrument can be used to follow the changes in firmness during ripening, to detect ripening abnormalities, to measure differences between varieties, and to detect variations in firmness over the surface of individual tomatoes.

Texture Profile Analysis **8 The Instron machine may be used to make a texture profile analysis as in the case of the General Foods Texturometer. The texture profile analysis of pears has been made by cutting a 2 cm diameter cylinder of tissue from each pear at right angles to the core axis with a cork borer, and trimming to a height of 1 cm. Each piece is punctured with a 7/16” diameter Magness Taylor pressure tip mounted in the machine. The cross head speed is set to 5 cm/min and chart speed to 50cm/min (exactly 10 times). Since both chart and drive motor are synchronously driven, the areas under the Instron curves are both force-time and force-distance

integrals. A typical force-distance curve of the first and second bites is sh~wn in

Fig. 18.33 which is the mirror image of, the tracing. The ‘pip’ is a:. .ndication when every time the crosshead commenced a downward stroke. Some of the parameters which can be derived from the curves are as follows: Elasticity: Drop a perpendicular from the peak of the second bite and measure the distance along the baseline from this point back to the point where the compressing plate contacted the pear.

Analysis of Fruit and Vegetable Products

586

Upstroke Gounatrgne ardness 1

Hordness 2

Fracturability

Adhesive force

Time

——>

Fig. 18.33: A generalised TPA curve from the Instron Universal Testing Machine.

(Reprinted from Bourney, M.C., Food Technol., 32 (7), 62 (1978) /permission of Institute of Food Technologists (C))

with the

Cohesiveness: Drop a perpendicular from the peak of the first bite. Note the area to left of ‘the perpendiculars in the first and the second bites and find the ratio

(A2/A,) which is dimensionless.

e area to the left of the perpendicular measures the work done by the machine on the food during compression, and to the right refers to the work during decompression which is excluded. As a further improvement, the area under the decompression is subtracted from the compression area to give the net work done during the two compression cycles. Gumminess: Hardness X cohesiveness (Hardness X A,/Ae2) Chewiness: Gumminess X elasticity (Hardness X A,/A2 X springiness) Adhesiveness: Area below the base line (A3)

Ottawa Texture Measuring System

This instrument consists of a press having an aluminium framework with the controls and motor at the top. The motor drives a screw that moves the crosshead from the top. The speed of the motor ranges from 2 to 29 cm per min, and the speed may be noted, if need be, by attaching a digital speed indicator. The crosshead stops to positions to + 0.1 mm for single speed drives, and to + 0.3 mm for variable speeds. The stationary texture test cell component fits into the slides in the base of the press, and a chuck on the crosshead holds. the moving part. The nominal load capacity of the press is 1,500 kg.

Measurement of Viscosity, Consistency and Texture

587

A single speed quality control version of the instrument is also available. Force is detected by a compact strain-gauge force transducer and has a capacity of 500 kg ranging from 0-2 kg to 0-500 kg recorded to the accuracy of + 0.25%. The system has a variety of test cells to suit the requirements of different products. Succulometer

Succulence or juiciness is associated with the moisture content. Kramer and Smith*4 developed the succulometer to measure the maturity of sweet corn. A cylindrical cell is fitted with 100 g of corn kernel. A plunger which is 0.127 mm smaller than the inside diameter of the cylinder is then placed in the sample. The pressure is applied gradually by a hydraulic press until the pressure applied

reaches 500 lb/inch’, and is held at that pressure for 3 min. The expressed juice j is collected in a measuring cylinder, and the volume is noted which gives an indication of the succulence or maturity of the sweet corn. The succulometer is also made use of to determine the residual storage life of apples and the water holding capacity of reconstituted dehydrated products.*® Apple is peeled, cored and shredded. Fifty grams of the material is used for noting the yield of the juice in the same way as in the case of corn. Freshly harvested apple yields 15-20 ml juice which gradually drops! to 10 ml during storage, and then abruptly to 5 ml. At the time when this abru rupt drop in juice yield is observed, apples show first signs of surface wrinkling. The dehydrated fruits and vegetables do not usually reconstitute to the original fresh weight. Furthermore, the absorbed water may not be held firmly so that the reconstituted products have more of a watery appearance rather than the juicy appearance. If the water expressed using the succulometer is more, the.

reconstituted product is less likely to be juicy.

Accuracy of Objective Methods In spite of the multiplicity of the objective methods for measuring rheological properties of foods, at times, their use in evaluation of complex natural products may be limited. The properties of Newtonian liquids could be measured

accurately. Non-Newtonian fluids and semi-solid foods are difficult to measure accurately.

The parameters to bemeasured should be defined precisely with respect to both ‘sensory characteristics and textural properties. The results of sensory and textural data or their logarithms should be subjected to statistical analysis by the use of correlation regression. It is only after elaborate studies that an objective textural

procedure could be considered which would match the sensory textural characteristics with sufficient accuracy.Readers may refer to the suggested books?*3® on the subject. Problems in Texture Measurement”

Texture is a combinationofdifferent physical properties which are more ecsy to sense than measure as the instruments measure either one or a tew of them. In

588

Analysis of Fruit and Vegetable Products

some materials like the maturity changes in French beans or the ripening changes in pears, the changes occur in unison. Similarly, the changes in peaches, either free or clingstone, irradiated. carrots.and peaches, the textural changes take place in unison. In such fruits and vegetables, it is comparatively easy to develop an instrumental method which correlates well with the sensory characteristics. In contrast, textural changes of apples during maturation and storage do not occur in unison. In sweet corn kernels, most of the changes during maturity occur in pericarp which forms only 5% of the weight of the kernels and becomes increasingly tough. Although pericarp content could be estimated,” it does not pres the texture. There is no suitable instrument to measure the toughness of

the pericarp although succulometer which measures the volume of thejjuice expressed from 100 g of sweet corn subjected to 3,000 Ib of pressure for 3 min is one of the methods for measuring the maturity of sweet corn. e Vegetables like French beans, broad beans, lady’s fingers, asparagus, celery, etc.,|

become fibrous as they mature. In the raw state, they are easy to cut and shear but the cooked product becomes soft, and the fibrousness becomes prominent during

eating. It is difficult to measure these changes and the problem remains unsolved. Physical and Chemical Indices of Maturity A number of physical and chemical methods have been made use of as objective methods for determining maturity (see. Chapter 7) and texture. All of these indirect methods assume that there is a definite relationship between texture and specific characteristic tested, and that there are no modifying effects exerted by other parameters. The indirect parameter would be a suitable index only when physico-chemical changes occur in unison during maturity or storage. Some of the parameters which have proved to Le of practical value are described below. Moisture

Total solids or moisture are a useful index in determining the tenderness of the vegetables at the time of maturity. Sweet corn having a moisture content of about 80% is too watery; 75% yields a product suitable for being graded as US Fancy or Grade A; and 65% renders the corn so starchy which results in the product being gtaded as substandard. Different methods of determining moisture are given in Chapter 1. Alcohol Insoluble Solids

Insoluble solids are more a measure of texture than an index of maturity. From the prepared sample, soluble solids are leached off and the residue is dried in an’ oven which gives a measure of the insoluble solids content. Alcohol insoluble solids (AIS) content has been adopted as the official method y in USA for determining the quality of canned peas and sweet corn. The residual. dried material contains mostly starches, cellulose, fibre, pectins, and proteins. A

lower alcohol insoluble solids content indicates less maturity and better suitability

Measurement of Viscosity, Consistency and Texture

589

for purposes of canning and freezing. In grade-C cream style corn, the weight of AIS of the washed, drained material should not exceed 27%. In grade-C canned whole corn kernels. the limiting line for AIS is 27% of the drained weight. In

grade-C peas, the AIS content of early type and sweet type should not exceed

23.5% and 21% respectively.“’ In the case of frozen peas, the AIS content of smooth. skin variety and sweet green wrinkled: variety should not exceed 23% and 19% respectively. The procedure for the determination of AIS in canned and frozen products is given in Chapters 26 and 27. Fibre Content Vegetables like asparagus, French (green) beans, wax beans, okra, etc. become

more fibrous with maturity. The fibre contributes to the toughness of the product. The crude fibre includes cellulose and lignin content of the plant tissue. The AOAC method of determining the crude fibre content (see Chapter 1) involves digesting

the prepared sample first with acid and then with alkali, thereafter washing, straining and drying. Wax or (French) green beans containing more than 0.15% crude fibre are considered substandard. Kramer et al.‘? made use of a more simplified method of blending with water, straining, washing the residue, and drying. The procedure involves blending 100 g of deseeded pods with 200 ml of water in a blender for 5 min, transferring the blended material to a previously weighed (A) 30 mesh monel metal screen (4 inch diameter with side walls 1 inch

high) and washing with water until the fibrous material is clear. The screen and the fibrous material are dried for 2 hr at 100 + 2° Cina hot air oven, cooled ina

desiccator and weighed (B). The difference in weight (B—A)is reported in terms of percentage. Since the seed material is finely comminuted in the blender, the whole ‘ beans may be cOmminuted. If that is done, only values below 0.07% may be

considered as safely within the standard level for fibrousness, whereas 0.09% indicates the limit of fibrousness when only the podwalls are used. The method was originally developed for use with canned products, and the values obtained closely related to. the data obtained by the AOAC procedure. However, when applied to raw green beans, other tissues are not clearly separated. Hence, the

matetial must be first_cooked by boiling in water for about 20 min or under pressure for about 5 min. After cooking treatment, the fibre can be isolated by

blending and screening.” To determine fibrous matter in frozen French (green) beans, AOAC

have

adopted a procedure involving maceration with cold alkali, washing and drying“ (see Chapter 27 for. procedure).

Sugar-Acid Ratio taste appeal, and more so in It is an important criterion from the standpoint of juices. For quality control purposes, sugar-acid ratio is the ratio of sugar value as determined using a refractometer to the grams‘ of acid content determined by titration. The ratio is placed at 40:1 for apple products and 10:1 for orange juices.

590

Analysis of Fruit and Vegetable Products

Density

The brine floatation method has been made use of for tenderness-maturity grading of canned peas (Table 18-17)’. Shelled peas are blanched, floated in the brine and separated into floaters and sinkers. The method is rapid and simple, and provides not only a measure of average quality but also uniformity. In addition to peas, the method has been applied for maturity grading of lima beans (Table18-

17)* and water chestnut.” yi

TABLE

18-17: United States Standards for Grades of Canned Peas

(Maturity and Tenderness)‘ Sweet Peas (Wrinkled varieties)

Alaska or Smooth Seeded Peas (Early peas) -

Grade A (Fancy grade)

Not more than 12% by count will sink in a 11% salt solution

Not more than 20% by count of peas will sink in a 11% salt

and not more than 2% will sink in a 13% salt solution

solution and not more than 2% will sink in a 13%% salt solution

Grade B (Extra standard)

Not more than 15% by count will sink in a 13% salt solution and not more than 4% will sink in a 15% salt solution

Not sink and in a

more than 30% by count will in a salt solution of 134% not more than 8% will sink 15% salt solution

Grade C (Standard)

Not more than 10% by count will sink in a salt solution of 15%

Not more than 10% by count of peas will sink in a salt solution of 16%

Brine Densities used for Peas and Lima Beans (using a canner’s salometer scale)‘

Peas

30° to 35 to 38 to and

Lima beans

36° S for No. 1 Fancy 42° S for No. 2 Choice 46° S for No. 3 Standard larger

First separation: 43 — 47°S Second separation: 47 — 60°S Third separation: 60 — 70°S

One of the primary requisites of well-canned potato is that it should not disintegrate or slough during processing “’ Immature potatoes with a specific

gravity of less than 1.075 can generally be canned without disintegration or sloughing during processing even without the use of calcium salts; sloughing can usually be prevented in specific gravity lots of 1.075 to 1.095 with added calcium When the specific gravity exceeds 1.100, excessive sloughing occurs even with added calcium salts.

Measurement of Viscosity, Consistency and Texture

591

Colour Measurement

In some fruits and vegetables, there is a close correlation between colour and* texture, and hence colour may be used as an index of texture. Residual green pigments in peaches and, apricots have proved to be most satisfactory for measuring their ripeness.“* Residual chlorophyll content serves as an index of measuring the ripeness of tomato in which the red colour is due to lycopene.” Even with varieties having lycopene content as low as 3.2 mg/100 g,.tomato

ketchup which would meet the colour requirements of US gradesA and C could be provided

prepared

the chlorophyll

content

is not

more

than 0.027

and

0.12 mg/100 g respectively. Procedure for determining chlorophyll is given on page 91.

Weight to Length Ratio In French (green) beans, factors which contribute to the sensory tenderness,

appearance and flavour arehee as associated, and change in conjunction with each other as the bean matures.” Hence, a single objective test which has a good simple

correlation with the sensory quality factors would correlate highly with the others also. In the Burpee stringless variety of French beans, based on ten physico-chemical characteristics studied during growth, weight-to-length (W/L) ratio has been found to be closely correlated to the sensory characteristics. The beans are very tender upto a W/L ratio of 0.4 g/cm, tender from 0.41 to 0.5 g/cm, which is optimal mature stage, mature from 0.51 to 0.6 g/cm, and over-mature thereafter. Miscellaneous

New

developments

and techniques for measurement of texture include

resonance and optical properties of peaches as related to flesh firmness, mechanical resonance within red delicious apples and its relation to fruit texture, and developments in quality control which make use of optical properties, electrical properties, x-rays, gamma-rays, microwave, etc..are compiled in the publication of the American Society of Agricultural Engineers. ke

References 1. Brandt, M.A., E.Z. Skinner, & J.A. Coleman, J. Food Sci., 28, 404 (1963).

2.,Szczesniak, _A.S., M.A. Brandt & H.H. Friedman, J. Food Sci., 28, 397 (1963). 3. General Foods Corporation, Sensory Texture Analysis Manual 1970. General Foods Corporation, White Plains, New York.

4. Sherman, P.,J.,Food Sci., 34, 458 (1969). 5.. Szczesniak, A.S. & M.C. Bourne, J, Texture Studies, 1, 52 (1969).

6. Szczesniak, A.S. & BJ. Smith, J. Texture Studies, 1, 65 (1969). 7.. Szczesniak, A.S., J. Food Sti., 28, 385 (1963). 8. Caffyn, J.E. & M. Maron, Dairyman, 64, 345 (1947).

592.

Analysis of Fruit and Vegetable Products

9. Caldwell, R.C., J. Dasry Res., 38, 188 (1959).

10. Szczesniak, A.S. & E. Farkas, J. Food Sci., 27, 381 (1962). 11. Tiemstra, PJ., Food Technol., 18, 921 (1964). 12. Bourne, M.C., J.A. Moyer & D.B. Hand, Food Technol., 20, 522 (1966).

13. Ferry, J.D., Viscoelastic Properties of Polymers, John Wiley & Sons, New York (1961), p. 105. 14. Scott-Brair, G.W., Adv. Food Res., 8, 1 (1958).

:

15. Szczesniak, A.S., J. Food Scé., 28, 410 (1963). 16. Bourne, M.C., J. Food Scsé., 31, 1011 (1966), erratum, 32, 154 (1967). 17. Szczesniak, A.S., Food Technol., 20, 1292 (1966).

18. Finney, Jr. E.E., Agric. Eng., 50, 462 (1969). 19. Bourne, M.C., in Rheology and Texture in Food Quality,

DeMann, J.M., P.W. Voisey, V.F. Rasper,

& D.W Stanley (Eds), 1976, p. 244. The AVI Publishing Co. Inc., Westport, Connecticut, USA 20. Voisey, P.W., J. Texture Studies, 2, 129 (1971). 21. Szczesniak, A.S., Food Technol., 20, 1292 (1966).

22. Bourne, M.C., Food Technol., 19, 413 (1965).

23. Bourne, M.C., in Theory, Determination and Control of Physical Properties of Food Materials, Rha, C. (Ed.) D. Reide! Publishing Co., Dordrecht, Holland, 1975,. p. 131. 24. Voisey, P.W. & H. Hansen, Food Technol., 21, 355 (1967).

25. Bratzler, LJ., Rept. Animal Husbandry Section, Animal Industry, U.S.D.A., and Kansas Agri. Expr. Sta., 1933. 26: Ramaswamy, H'S., S. Ranganna & V.S. Govindarajan, J. Food Quality, 3, 11 (1980). 27. 28. 29. 30.

Friedman, H.H., J.E. Whitney & A.S. Szczesniak, J. Food Sci., 28, 390 (1963). Szczesniak, A.S. & BJ. Smith, J. Texture Studies, 1, 65 (1969). Bourne, M.C. & J.C. Moyer, Food Technol., 22, 1013 (1968). Kramer, A. & J.V. Hawbecker, Food Technol., 20, 209 (1966).

31. Hole, CB, J. Texture Studies, 1, 491 (1970). 32. Bourne, M.C., J. Food Scé., 33, 223 (1968). 33. Bourne, M.C., Food Technol., 32 (7), 62 (1978). 34. Kramer,-A. & H.R. Smith, Food Packer, 28, 56 (1946). 35. Kramer, A.& B.A. Twigg, Quality Control for the Food Industry, 3rd edn., Vol. 2,1973. The AVI

Publishing Co. Inc., Westport, Connecticut, USA. 36. Kramer, A. & AS. Szczesniak, Texture Measurement of Foods, 1973. D. Reidel Publishing Co.,

Dordrecht. Holland. 37. Rha, C., Theory, Determsnazson and Control of Physical Properties of Food Materials, 1975. D. Reidel Publishing Co., Dordrecht, Holland. 38. DeMann,J.M., P.W. Voisey, V.F. Rasper&D.W. Stanjey, Rheology and Texture in Food Quality,

(1976), The AVI Publishing Co._Inc., Westport, Connecticut, USA 39. Kramer, A., Ra. Guyer & L.E. Ide, Proc. Am. Soc. Hort. Sci., 54, 342 (1949). 40. Kramer, A. & J. Cooler, Proc. Am. Soc. Hort. Scs., 70, 379 (1957).

41. The Almanac of the Canning, Freezing and Preserving Industries, Edward E..

Judge & Sons Inc., 79, Bond Street, West Minister, Marviand. 21157, USA, 1979. 42. Kramer, A., R.B. Guyer & LE. Ide, Food Packer, 30 (9), 41 (1949). 43. Kramer, A., (1979) (Personal Communication).

44. Association of Analytical Chemists, Official Methods of Analysis_(12th edn.) (1975), 32.052. 32.054, AOAC,

P.O. Box 540; Benjamin Franklin Station, Washington, D.C., 20044.

—

45. Joslyn, M.A.& A. Timmons in Food Processing Operations..Joslyn, M.A. & J.L Heid (Eds), The AVI Publishing Co. Inc., ‘Westport, Connecticut, Vol. 3, p. 55 (1964) , 46. Rodriguez, R., P.C. Agarwal, & N.K. Saha, J. Food Sci. Technol., 1, 28 (1964). 47. Talburt, W.F. & O. Smith, Potato Processing, 3rd edn., 1975, p. 581, The AVI Publishin Westport, Connecticut, USA.

g Co. Inc.,

Measurement of Viscosity, Consistency. and Texture

593

48. Kramer, A. & H.R. Smith, Food Technol., 1, 527 (1947). 49. Pairat, N. & S. Ranganna, Indian Food Packer, XXX1(2), 42: (1977).

50. Fox, A. & A. Kramer, Food Technol., 20(12), 88 (1966). 51. Gaffney, J.J., Quality Detection in Foods, ASAE Publication, 1-76 (1976), American Society of

Agricultural Engineers, 2950 Niles Road, St. Joseph, Michigan 49085, USA.

CHAPTER 19

Sensory Evaluation

Quatity is the ultimate criterion of the desirability of any food product to the consumer. Overall quality depends on quantity, nutritional and other

hidden attributes, and sensory quality. Quantity is an attribute of quality inasmuch as the quantity of juice in a beverage, net and drained weight in a can, quantity of fruit in jam, etc. are of consequence to the consumer. Nutritional and other hidden attributes : The absence ot nutritional qualities

and ptesence of harmful or toxic ingredients are parameters which ate vital to the consumer. However, as the consumer is “unable to judge them easily, he is protected by stringent government controls in the shape of food laws. Sensory quality : This parameter is of great importance to both the processor and consumer—to the processor, since it attracts consumers; to the consumer,

since it satisfies his aesthetic and gustatory sense. Sensory quality is a combination of different senses of perception coming into play in choosing and eating a food. Appearance,which can be judged by the eye, ¢.g., colour, size, shape, uniformity and absence of defects, is of first importance in food selection. Objective instrumental methods are available for measuring most of these attributes (Chapter 17) and they could be correlated to consumer preferences.

Kinesthetics, the next important attribute, principally concerns texture and consistency (Chapter 18). Instrumental methods based on measuring deformation, compressior. and shear have been developed and _ kinesthetic factors have been correlated to consumer preference. Flavour embraces the senses of taste, smell and feeling. As far as human beings are concerned, it is generally agreed that the sense of taste is limited to sweet, sotir, salty and bitter. The dimensions of these tastes could be estimated chemically, but their optima in relation to consumer preference, especially when-they occur in combination in a complex food are not fully understood. Feeling (asteingency, bite, etc.) is an attribute which is of significance to flavour especially in spices, wine, coffee, etc. Odour, a vastly complex sensation, is the most important factor in flavour. In recent times, there have been many attempts to relate gas chromatographic peaks to flavour acceptability but the picture is terribly confusing with literally hundreds of peaks having to be correlated to consumer acceptance and preference.

é

Sensory Evaluation

595

Another dimension of food quality, the affective characteristics, is not the

Ptoperty of the food, but the subject’s reaction to the sensory qualities of foods.

This reaction is highly conditioned by a variety of psychological and social factors and, in the final analysis, plays a vital role in the acceptance and preference of foods. Appropriateness of the different quality characteristics is an important factor in this dimension, Evaluation of Sensory Qualities

The sensory qualities, particularly the flavour attributes; are essentially to be measured subjectively. From early times,this judging has been the preserve of experts, who had trained themselves to remember and distinguish small differences in odour and taste of specific products like tea, coffee, wine, etc.

With the development of sensory evaluation techniques on scientific lines, the experts are being replaced by panels whose sensitivity and consistency have been established by training and repeated tests. The panel members analyze food products through properly planned experiments and their judgements. are quantified by appropriate statistical analysis for determining the significance of variation of average scores and the contribution of the individual quality characteristics to the overall quality. The trained panel is generally constituted of a small number of people,who,in a rigorously controlled set-up in the laboratory look after quality control of in-line and final product, process development, and, to a limited extent, preliminary acceptance testing. For market testing, where the natural reaction based on emotion (the affective characteris-

tics) to the selected food products. is required to be ascertained, an untrained panel, constituted of a large number of men selected tobe representative of the population to be surveyed, is used and this is known as consumer survey. Such surveys, by their nature, are time consuming and costly, and are restricted to products selected through a series of laboratory panel tests and presented ‘generally in their final marketable forms. The data from these surveys are analysed ‘statistically to discover the significance of preference and rejection. When people are used as the measuring instruments, the importance of planning and control of each experiment cannot be over-emphasized. It is recommended that the sensory tests are well integrated with the overall plan

of development of the product and the sensory test plan is completed in detail before the samples are prepared and packed. This includes the definition of the specific objectives or questionstequired to be answered by-the tests, the selec-

tion of appropriate method, the numbers ot evaluations and the statistical design to decide the quantity uf sample to 0€ prepared, and the statistical treatment of data. Preliminary experimentation is required to decide the optimum state, quantity and presentation of samples so that homogeneity, appropriateness and randomization are achieved to take care of different types of bias that could affect the judgements

of personncl in the test panels. The necessary

conditions for sensory tests are obtained by attention to the following requisites.

596

Analysis of Fruit and Vegetable Products

Laboratory Set-Up and Equipment The set-up may be simple or elaborate; the important considerations being that independent judgement in an atmosphere of relaxed concentration and free from any distraction should be possible. The general decor should be amenable to easy maintenance of cleanliness and of pleasing natural shades of colour. A typical layout should consist of three separate areas : (i) the reception and briefing room providing ‘people control’; (ii) the preparation room equipped for preparing and serving all kinds of foods, and with. extra-large storage space and plenty of serving utensils; and (iii) the panel booth area located in between or adjacent to the other two rooms. The whole area, or at least the panel booth area, is preferably airconditioned. The booths should be identical, uniformly illuminated, and provided with

drinking

water, a glass,

clean towels and a basin or receptacle for convenient examination. Provision may be made to have coloured lights whenever needed to mask small differences in the colour of samples. Stainless steel, glass and China dishes and cups, and plain serving trays are the most convenient as utensils. The utensils suggested are : ruby-red glasses (3 oz size); custard type China glass dishes (6 oz size); cups (3 oz size); glass shifters (8-12 oz size); small stainless steel forks and spoons; watch glasses as covers; and glass-stoppered bottles for odour tests (4 oz size). Electric rather than gas equipment is preferred for cooking.

Panel Selection and Training The requirements for panel membership are : (i) good health; (ii) average sensitivity; (iii) high degree of personal integrity; (iv) intellectual curiosity and interest in sensory evaluation work; (v) ability to concentrate and learn; and (vi) availability and willingness to spend time in evaluation and submission to periodic tests for acuity and consistency. Candidates possessing these qualities must be indexed with details of age, sex, specific likes and dislikes, availability, etc. The candidates must then be put through basic qualifying tests of odour and taste recognition and thresholds, and their performance recorded in the index cards. Laboratory panels must then be carefully trained tor specific products or purposes. These tests aim at finding differences in specific quality characteristics between different stimuli and also direction, and/or

intensity of the diff-

erence. Periodically the panel is given refresher training and tests. Mlavour profile and texture profile panels should be given a still higher degree of training for detailed qualitative and quantitative analysis of complex natural and processed foods. These trained panels are usually constituted of small numbers (5-10) and are principally used in research and development.

Sensory Evaluation

597

Discriminative, communicative or semi-trained panels: These panels ate consti- |

tuted of technical people and their families, who are normally familiar with the qualities of different types of food. They are capable, with few preliminary test runs, of following instructions for tests given, discriminating differences and communicating their reactions. Such panels of 25-30 are used to find the acceptability or preference of final experimental products prior to large scale consumer trials. Consumer panels: Such panels are made up of untrained people chosen at random to represent a cross-section of the population for which the product is intended. The greater the number, the greater the dependability of the result; a group of not less than 100 is considered the minimum.

Design of experiments : Experimental error and bias can be minimized through use of techniques of randomizing. A statistical design is used in order to mea-

sure variables separately and together, and to establish the significance of results. The experiment should be designed on the basis of the accuracy needed and the amount of sample available.

Samples : The number of samples, control and experimental, required for the entire test or series of tests should be carefully worked out and obtained prior to the start of the evaluation. The number of samples used in any one session is dependent upon the sensory nature of the test product and the evaluation method used. Proper sampling of the control and test samples should be done, and samples for presentation must be from homogeneous lots. Samples should be prepared in exactly the same manner for each lot and for each examination. Appropriateness of temperature, time and sometimes the quantity have to be taken into account in the presentation of samples.

Samples are presented with three or five digit code markings to obscure the identity of the samples. The order of presentation should also be randomized within each test session.

Judging Quality

Judging should be done in individual booths. This assures independent judgement and no communication between panel members should be allowed except for consultation with the panel leader on any point of doubt. The best time of day for sensory testing is the hour which is clearly an hour after

any normal meal,e g 10.00 a.m. to 12 noonand 3to 5 p.m. that panel members

It is preferable

do not smoke or chew pan or supari for at least half an

hour prior to the test.

Judgements should be done quickly, but not hurriedly. Odour observations

by sniffing should be done before tasting. While panel members should be allowed retesting, prolonged testing is not conducive to reliable judgements. Tasters may either swallow or spit out the samples after testing. Rinsing bet-

598

Analysis of Fruit and Vegetable Products

Method

Type

Panelists

Numi

No. of samples per test

A. - Difference ~(Qualitative)

1. Paired comparison 2. Duo-Trio

3. Triangle (Triad)

Trained Untrained Trained

Trained

B. Rating (Quantitative differences) "1, Ranking Trained Semi-trained

5-12

3

(2 identical and) 1 different 3 (2 identical and) 1 different

2-7

Untrained

2. Single sample (Monadic)

Trained Untrained

6-25 72-80

I

3. Two sample difference 4. Multiple sample

Trained

difference 5. Hedonic

6. Numerical scoring

D.

5-12

2

5-12 10-25 72-80

and quality

C.

5-12 72-80

Trained

Semi-trained Semi-trained

Untrained Trained

7. Composite

Trained

Sensitivity 1. Threshold

Untrained

2 Dilution

Trained

Descriptive Flavour Profile Trained

6-25 6-25

4 pairs of unknown and control sample 3-6

10-25

Including control and depending on

10-25

number of quality factors evaluated 5-10 Larger number only if mild

72-80 5-12

flavoured or rated texture 1-4

for colour or

1-6 5-10 Larger number only if mild flavoured or rated only for texture

5-12

1-4

-

5-10

12-24

5-10

3-6

1-5

(specially in the technique) OT A AEOL PS UOUER R E

EO

AS

Sensory Evaluation

599

Sensory Evaluation Methods

Statistical analysis of data

Purpose

A. Binomial distribution Table 19-4

Comparing

samples for specific characteristics

X?-test Table 19-10 2. Binomial distribution Table 19-4

3.

Binomial! distribution Table 19-4

1. Rank analysis Table 19-5

Detecting difference when carryover, after-taste may be present. Also for training and testing panels.

Detecting differences when intersample effects (after. taste etc.) are minimum. Also for training and testing panels. '

Determining order according to one specific charac-

teristic or determining preference used in product and process improvement; selection of best sample. '

2.

Analysis of variance Tables 19-6,

Pilot consumer analysis. Consumer preference analysis. Detecting difference from normal product, off flav-

19-7, 19-8 and 19-9.

our,

. Analysis of variance Tables 19-6, 19-7, 19-8 and 19-9. t -Test, Table 19-11 . Analysis of variance Tables 19-6, 19-7, 19-8 and 19-9. Duncan's

off taste

and

direction

when

aftertaste

and

carryover are present. Evaluation of new product. Marketing and consumer analysis. Difference between samples, quantitatively and directionally. Comparing samples with more than one variable in the same session (reduced reliability.)

Multiple Range Test Table 19-9 Dunnett Test, Table 19-12. . Analysis of variance Tables 19-6,

19-7, 19-8 and 19-9.

. Analysis of variance Tables 19-6, 19-7, 19-8 and 19-9.

. Analysis of variance Tables 19-6, 19-7, 19-8 and 19-9. 1. Threshold value 2.

Dilution number

Pilot consumer analysis for screening by preference

Consumer analysis for preference. Screening for quality development, new product quality, maintenance, ingredients or product grading and selection of trained panel. Comparing scveral products of samc type and grading. Selecting panel members for evaluating ingredients, packaging matcrial; maintaining quality. Odour and flavour evaluation of foods, ingredients; product devclopment ; quality control. specially useful for spices.

Sample characteristics expressed in. common terms. Intensity expressed on agrecd scale. Used in new product development, product improvement and storage studics.

600

Analysis of Fruit and Vegetable Products

ween samples or use of materials such as bread a strong flavour is recommended. In any one followed should be adhered to by all and for all Evaluation card: The questionnaire or score carefully for each test. The card should be clearly

or puffed rice to remove evaluation, the procedure samples. card should be prepared

typed or printed. It should be simple and use unambiguous terms and directions in the desired sequence of action as a guide to the evaluation.

Test Methods

The different test methods based on both laboratory analysis with trained panelists and consumer analysis with untrained panelists are described in Table 19-1. The methods are grouped according to their techniques of evaluation and further classified by design of the experiments. The table also gives the current recommendations from different organizations on the type and number of panelists, number of samples, statistical method for analysis of data, and indication of the purpose for which the methods have been used.

The selection of a test method will depend on the defined objective of the test. The method should be appropriate to the problem and situation, efficient and practical, and should be as simple as possible. Sensory problems are so individual that standardization of sensory test methods becomes difficult. Therefore, a guide to the application of sensory test methods to food industry problems is given in Table 19-2. Specimen evaluation cards tor different methods are given in the following pages. Each method is illustrated with an example and the results are statistically analysed. Reference may be made to Chapter 33 for the statistical methods used in this chapter.

Difference Tests

More often comparisons of two similar products are required to be made to establish whether or not two materials are perceptibly different in some respect. Paired comparison, duo-trio, and triangle tests are made use of for this purpose.

A, Paired Comparison Test These methods are used tor difference, intensity, or preference tests depending

upon the objective of the test.

Two samples, one the standard or control and -the other an experimental

one, are presented. Trained or untrained panelists are asked to indicate : (1)

if the samples are different and/or (ii) the sample that hasthe greater or lesser degree of intensity of a specified sensory characteristic. A positive answer is

Sensory Evaluation

601

TABLE 19-2 : Application of Sensory Tests to Food Industry Problems Type of-problem and objective

Appropriate tests (see Table 14-1)

I.

New product development

2.

Product improvement : by formulation

3.

Process improvement : to measure the‘effect of

Ay,

B,,

B,,

B,,

Ay,

B,, B,, B;,

C,, D

A,

B,,

B,,

C,,

A,

B,,

B,

Ay,

Ag,

As,

B;,

C,,

D

D

process change on quality of product Cost reduction : te produce equal product quality at lower cost by changes in raw material or ptocess simplification.

;

Selection of new source of supply: to

find pro-

B,

duct as good as standard Quality maintenance during production and

A,

Ag,

Storage stability

Bg,

B,,

Product grading and rating :to grade products

Bg,

B,

As,

By,

Bg,

B,

marketing D

and ingredients correctly and consistently Selection of best sample: to select the best tor the intended 10.

Ay, By, Be

use

Market testing of a new or imptoved product:

Aj,

B.,

B;

to determine customer acceptance II.

Consumer preference

Ay,

B,,

B,

12.

Selection of trained panelists : to select the

A,

As,

Bg,

C,

testing group best able to make the specific evaluation

required. one-half. compared ted at ane

required

The chance probability of placing the sample in a certain order is If more than two treatments are being considered, each treatment is with every other in the series. The number of sample pairs presen-

session is limited hy the degree of fatigue induced. by sensory testing.

In consumer analysis, the same test is used but the judgement asked for is for overall quality.

Analysis of Fruit and Vegetable Products

602 Specimen

FExvalyation

Card

PAIRED Nantes?

Ae

COMPARISON

TEST

ees

Date :

You are given one or several pairs of samples.

Evaluate the two samples in the pair for....... * Is there any difference between the two samples in the pair ? Code no. of pairs

Yes

No

Signature Nore: The less preferred sample need not be of poor quality and may still fall in the acceptable category and this should be decided by a separate test.

*Mention the specified sensory characteristics to be studied, e.g. sweetness, texture, flavour, or overall quality and use separate cards for each characteristic.

Paired difference testing (without direction) is used to determine if the -difference between two samples can be discriminated. If the judges find a difference, they are asked to describe the difference based on which a series of pairs of samples are presented ina randomized design (Table A). TABLE A: Randomized Design for Presentation of sample for Paired Comparison Difference Testing

Judge number Pair

number

1

1 2 3 4

AB BA BB AA

2

AA AB Om) AVN BB

3

4

BB AA AB BA

BA BB AA AB

For paired intensity testing (with direction), two distinct samples are presented,

and the panelists are asked which sample has greater or lesser intensity of

particular characteristic like sweetness, toughness, flavour, etc. In testing for the flavour intensity, a pretest is made to determine the concentration at which the

Sensory Evaluation

603

flavour becomes perceptible and then presented along with control sample which contains no added flavour. Panelists are asked to judge.whether the samples in a pair differ, and if so, to identify which of the two contains the more intense characteristic flavour. Pretesting helps to verify the flavour level, orients the panel to test problem, and enables screening of the performance of judges. This test is called a one-tailed test and the chance of probability is 50 to 50. The pretest is followed by the actual test. The sample containing the correct concentration established in the pretest, now called control, is compared with a sample containing the same concentration of flavour under investigation. Table B gives the design for sample presentation to the panelists. The number of judges should be more. TABLE B: Design of Sample Presentation for Paired

Comparison Intensity Testing Judge number Pair number

1 2 3 4

=

1

2

3

4

AB BA BA AB

BA AB BA AB

BA BA AB AB

AB AB AB BA

The results of paired comparison tests are analysed using Table 19-4 (one-tail test). As discussed above, when one sample is known to be more intense than the

other or when testing is done to determine whether the samples are the same or different, the test is considered a one-tailed test, and the values given for the one-

tailed test in the table should be compared. Most paired comparison tests are twotailed which means that the panelists can choose either of the two samples, e.g., preferred sample. In such cases, the values given under two-tailed test in the table should be made use of for comparison. Note that a one-tailed test has lower limits than a two-tailed test. EXAMPLE 1

In the pretest, unflavoured orange squash is prepared and divided into two portions. To one portion, a detectable quantity of orange oil from source A is added and to the second portion, no oil is added and used as control (C). The two squashes

are diluted (1+3) with water. To each judge, 2 randomly arranged pairs are presented, and they are asked to pick the sample having the stronger flavour. The results of analysis by 15 judges are given in Table C. Fifteen judges and 2 replicates result in 30 judgements. From Table 19-4 for

one-tailed test, note that 24 correct answers are required for significance at (0.001X100)

99.9%

confidence

level. The

results

(25 correct

judgements)

604

= Analysis of Fruit and Vegetable Products TABLE C: Results of Paired Comparison Pretest of Flavoured Orange Squash vs Control

Judge

1 2

Sample chosen as having 5 strong flavour

Correctness of judgement

I Pair

II Pair

A A

A Cc

++2 meen |

3

C

A

a+ |

4 D 6

A A A

A A A

++2 ++2 ++2

7

A

A

Peesy.

8 9 10

A A C

A A 6

++2 ++2 --0

11 12

A A

A A

eran? ++2

13 14

C A

A A

-+1 ee 2

15

A

A

eyo?

Total25

indicate that the concentration of orange oil used is adequate for judging. If we desire to select a group of sensitive panelists, repeat the test for over 10 times and analyse the panelists’ data similarly to select those who significantly could identify the more intense sample. If most of the panelists fail to identify correctly the level at which the test was carried out, increase the intensity, and repeat the test. The actual test is intended to determine which of the two, brand A or brand B,is

best suited. Squash is prepared in the same way, and to two parts, the two oils are added in the same concentration found best in the pretest. The samples are presented to the 12 judges in 2 pairs on 2 occasions and asked to pick the sample having the characteristic flavour. The results are shown in Table D. This test is a two-tailed case as it is intended to select either brand A or brand B.

From Table 19-4 for a two tailed test, a sample should be picked up 36 out of 48 times to be considered as having stronger flavour. Brand A is accordingly best suited. In paired comparison test, if the null hypothesis is considered, that is, there is no

difference between the samples, the probability, p, of the taster identifying by chance, a particular sample in each of the several trials is half. The results can be

analysed by applying x” test.

yee LX = Xa) =P n

Sensory Evaluation

605

TABLE D: Results of Paired Comparison of Two Brands of Orange Oil

Response for 4 pairs Brand preferred

Judge Number 1 2 3 4 5 6 v|

A B A B A A A

A A A B A A A

A A A B A A A

A A A B B A A

8 9

A A

B A

A A

A A

10 11 12

A A B

A B A

B A A

A A A Total A = 38 B= 10

where X and X2 represent the score for the two samples, x is the total number of trials and x? is based on one degree of freedom. Substituting the values found in the pretest wherein X, = 25 and X2 = 5 and n = 30.

gun Arisin (25+5)e 197 esegai Dn

x=

12.03

By comparing with the tabulated value of 7.83 (Table 19-10), the observed value is significant at 0.5% level.

Substituting the values found in the actual test in the expression ?

2 _gia! ((38-10)-1)" _ 48

15.2

The tabulated values of x? at 5, 1 and 0.5% levels are 3.84, 6.63 and 7.83

respectively. On the basis of test results, sample A at 0.5 level is significantly preferred to sample B.

A, Duo-Trio

Test

This test employs three samples: two identical and one different. The panel

is first given one of the pair of identical samples as known reference sample R and then the other two successively in random order, and asked to match one of these with the first. A positive answer is required even if it is a guess. The chance probability of placing the samples in a certain order is one-half.

606

Analysis of Fruit and Vegetable Products

Specimen Evaluation Card

DUO-TRIO TEST Name

Dateisr

ertrene eo ice.

voters

The first sample ‘R’ given is the reference sample. Taste it carefully.

~, From the pair of coded samples next given, judge which sample is the same as ‘R’. A positive answer is to be made even if it is a guess. Set no.

Code no. of pairs

Same as ‘R’

I

——

-—_—

ee

II

ay

2 a

ee

Il

Mee

ee

eS

IV

——

—— Signature

The duo-trio test is essentially a paired comparison discrimination method although three samples are used. The probability is that the odd sample will be chosen half the time by chance in a single trial. The significance of the results can, therefore, be determined by summing up the total number of comparisons made,

the total number of correct responses, and referring to the tabulated data (Paired Duo-Trio difference) in Table 19-4. EXAMPLE2

To determine if 25% substitution of mandarin orange juice with sweet orange juice in the manufacture of orange squash could be detected, a duo-trio test was used. The test was performed on two successive days using six panelists. The results are shown in Table E. TABLE E: Duo- Trio Test to test the Difference

in Two Samples of Orange Squash

Panelist

lst day

2nd day

Matching

Matching

1

-

+

2

+

+

3

+

+

4

+

2

5

+

+

6

-

+

i4 4.5te to the reference sample

Matching 8 ccorrectly 1}

5

Sensory Evaluation

607

For 12 panelists in a two-sample test, 10 correct judgements are required for significance even at 5% level. The results show that the substitution cannot be detected. The test is a one-tailed situation as only the coded control should be correctly matched to the known reference sample. The probability of either of the sample being matched to reference sample is 1:1. This type of test does not provide information on the nature of difference even if there be some, and on the intensity of the difference.

A, Triangle Test

This test employs three samples, two identical and one different, presented simultaneously to the panel. The judge is asked to determine which of the three is the odd sample. A positive answer is required even if it is a guess. Since all three samples are unknown, the chance probability of placing the sample in a certain order is one-third. .

Specimen Evaluation Card TRIANGLE TEST

1. Here are three samples for evaluation. Two of the three samples are identical. Determine the odd sample for any difference.

Sample*

Check odd sample == = ==

AAB ABA BAA

' Did you check by guess?

Yes O

Nod

2. Indicate the degree of difference between like and odd samples. INOME ivijsecccteccsecesertcrss Slight iacc5 eases Moderate «0.0... Muchnett..sceeeee 3. Acceptability Odd sample preferred Like sample preferred

YOS icageseennsece ING sgten cies: Ves aan! DINO ieceeccs2-5:

4. Comments, if any.

*In the actual test, use three figure numbers

Like the duo-trio method, the triangle test also utilises 3 samples. In the former, the first sample is always identified as the reference and one of the remaining two is stated to be identical to the reference sample. In the triangle test, all the three samples are presented as unknown. Of the three samples, two are identical and one is different. The panelists are required to identify the odd sample. The triangle test is a one-tailed test used primarily to detect flavour differences. Since the triangle test is based on the ability of the individual to identify the odd sample on the basis of flavour, all samples should be as uniform as possible with

608

Analysis of Fruit and Vegetable Products

respect to other characteristics. If the odd sample has a different colour from the paired samples, the panelist need not even taste to make the correct identification. Correct identification on the basis of chance alone is 1 in 3. Table 19-4 gives the number of correct identifications required from a given size of panelists for the results to be considered statistically significant. The triangle test can also be extended to measure degrees of difference and preference. After identifying which of the three samples is different, the panelist is asked whether this difference is slight, moderate or large, and may also be asked to indicate whether he prefers the odd sample or the paired samples. In evaluating preferences for degrees of difference by the triangle test, only those results are used where the panelists have correctly identified the different sample. The triangle test is primarily intended to establish the difference. Once this has been identified, further questions regarding the degree of difference or degree of preference are frequently an afterthought without any critical tasting. If all the panelists correctly identify, and the panel is virtually unanimous in preferring one sample or the other, the preference ratings are undoubtedly reliable. If the number of correct identifications is just enough to reach the level of significance, and the panel is divided in its preference, a separate preference testing with a larger panel should be attempted.

EXAMPLE 3 To differentiate if a difference existed between orange marmalade containing 40% Seville orange pulp, and the marmalade containing 25% Seville orange pulp TABLE F: Test Design and Results of Triangle Test of Orange Marmalade Panelists, response

First day Panelist

Sampling

number

— sequence

Second day

Odd

Odd

sample chosen®

Degree of difference?

Preferred sample?

Sample Degree of chosen __ difference

Preferred sample

1

ABB

(A)

Moderate

B

(A)

Moderate

B

2

BAB

(A)

Much

3 4

BBA ABB

Moderate

(A) (A)

Much Much

B B

(A)

Moderate

A

5

ABA

(A) B (B)

B A

Moderate

B

A

6

BAA

A

7

AAB

(B)

Moderate

B

(B)

Much

B

8

ABA

A

(B)

Moderate

B

A

Se eee ee eee eee Letters in parenthesis indicate correct identification. bn; ; ) Difference and the preference rating of panelists not correctly identifying the odd sample a1 not considered. A. Marmalade containing 40% Seville orange pulp on fresh weight basis B. Marmalade containing 25% Seville orange pulp and 15% Tenali orange pulp.

Sensory Evaluation

609

mixed with 15% Tenali orange pulp, ten panelists were asked to identify the odd sample on two consecutive days. The test design and results are summarised in Table F. Eleven out of sixteen times, the odd sample was correctly identified. According to tabulated data for triangle difference test (Table 19-4), the difference is significant at 1% level. The conclusion was that a difference existed between the samples.

The degree of difference indicated by the panelists who correctly chose the odd sample was: Moderate = 7 Much =4 The next part of the test was to choose the preferred sample. Of the 11 panelists. who correctly identified the odd sample, 9 preferred marmalade sample containing 25%. Seville orange pulp and 15% Tenali orange pulp. This preference is not significant at 5% level (see Table 19-4 under paired preference). Even though the odd sample was significantly identified, the preference did not attain statistical significance. To establish whether there exists a significant preference, a paired comparison test could probably be carried out with more number of panelists. Referenca

Stahl, W.H., & M.A. Einstein. “Sensory Testing Methods,” Encyclopedia of Industrial Chemical Analysis, 17, 608

(1973), John Wiley and Sons, Inc.

Rating Test

B, Ranking Test This test is used to determine how several samples differ. on the basis of a: single characteristic. A control need not be identified. Panelists are presented

all samples simultaneously (including a standard or control if used) with code numbers

and are asked to rank all samples according to the intensity of the

specified characteristic. In consumer analysis, the panelists are asked to rank the coded samples according to their preference. Specimen

Evaluation

Card RANKING

INA

TEST

:

Saaverxtiveag, 134 +

Date:

:

....

PLOGUCE Se aoc a eye Please tank the samples in numerical otder according to your preference or intensity of aroma/taste characteristic of the product.

Intensity| Preference First

Sample code

Second

— ae.

Third

—

Fourth

—

Comments : (Type of off-flavour, etc.)

610

Analysis of Fruit and Vegetable Products

When more than three samples are required to be compared, the ranking method is particularly suitable. The method can be used for comparing one treatment to others, or for comparing treatments among themselves. The ranks

for these treatments are placed in columns and the replicates in rows. The ranks for each treatment is totalled. By referring to the rank-sum tables (19.5a and b), the significance of the differznce

between

the observed

sums

rank

is

determined. Example 4 is given to illustrate multiple comparison of the samples. EXAMPLE 4

A bottler has developed orange squash and intended to test the product with those of the competitors. He collected random samples of the competitors’ products and submitted them to preference ranking by 10 panelists assigning the ranks 1, 2, 3, 4 and 5 in the order of preference. In the tabulation of the results

(Table G), samples which had been ranked equally were tabulated as average of ranks the samples would have occupied, if there were no ties, or ranked individually.

TABLE G: Preference Ranking of Orange Test 26 Panelist

sample

Market sample A

B

C

D)

5

1

2

1

BD

5

2

1

2

5

3

3 4

1 3

2 1.5

5 5

3 1.5

5

1

3

5

2

6

10

2 3 1 2

1 1 2.5 1

2

5 4 5 4.5

1

3 2 25 3

4 5 4 45

5

3

4

Rank sum

18

16

47

28

41

7 8 9

Refer to Tables 19-5 a and b for 5 treatments and 10 replicates. The tabul. values are as follows: ea

ee

At 5% level (P < 0.05) eer

Upper pair Lower pair

At 1% level (P< 0.01)

reentenneemrnemeremrereeee

20-40 23-37

ee

18-42 20-40

|

Sensory Evaluation

611

For the samples to be considered significantly different, at least one of the rank sums of either thetest sample. or any of the market sample must be lower than the lower limit or Higher than the higher limit in the upper pair. In the present, example, the rdnk sum 16 of market sample A is lower than the rank sum of 18 at 1% level. Similarly, rank sum data higher than the higher tabulated limits are also seen in the With 4 degrees of freedom for numerator and 36 degrees for the denominator

by interpolation

referring to Tables 19-6 and 19-7, the calculated F value must exceed 2.64and 3.91 for 5 and 1% levels

of significance. The F value 35.78 is, therefore, significant at 1% level. Similar examination made with

regard to panelists indicates no difference.

Calculate the shortest significant range (Rp) using Table 19-9 for 5% level in which. the rows depict the degrees of freedom in the error term, and the columns show the number of means. Since there are 5 means in the example, there will be 4

ranges to find. Find p corresponding to 36 degrees of freedom (since 36 is not given use figures between 30 and 40). Multiply the means (p) by the standard error (SEz) to find shortest significant range (Rp).

p (5%) Rp (5%)

2 2.88 0.91

3 3.02 0.96

4 3.11 0.99

5 3.18 1.01

Arrange the sample mean scores given in Table M in the ascending order of magnitude as shown below: E Di -¢ B A Sample No. 4 3 0 2 1

Mean store

28

49

5.051

6,1

8.0

Calculate the range between two means and compare with the shortest range for the relative position. Begin by testing the largest against the smallest, the largest against the second smallest and so on until the range between the two means is less than the significant range for the number of means grouped in the range. Underline the means in this grouping to indicate they are not significantly different.

The means within the grouping need not be tested, since the difference between the two means cannot be significant if both are contained in a group of means which has a non-significant range.

Sensory Evaluation

621

When the largest mean has either been grouped or found to be significantly being different from all other means, test the second largest mean, first against

the smallest mean, then against the second smallest, and so on. Continue until the second smallest mean is tested against the smallest, unless all means have already ‘been grouped. In the example of means given, proceed as follows:

A-E A-D A-C A-B

Rp (5%)

= = = =

8.0-28=5.2>.1.01 (Rs) 8.0 - 4.9 = 3.1>0.99 (Rs) 8.0 - 5.0 = 3.0> 0.96 (Rs) 8.0-6.1=1.9> 0.91 (Re) Between all these pairs of samples, there is significant difference B-E = 6.1.-2.8=3.3> 0.99 B-D = 6.1 -49=1.2> 0.96 B-C = 6.1-5.0=1.1> 0.91

(Ry) (Rs) (Re)

|

Between all these pairs of samples, there is significant difference C-E = 5.0-2.8=2.2 >.0.96 C-D = 5.0-49=010.91 (Re) There is significant difference between D and E

Sample code

4 EE

3 PD

0 se

1 B

2 A

Any two means underscored by the same line are not significantly different. Samples 1 and 2 prepared using spice oils A and B differ significantly from the reference sample 0, and are inferior to other samples. Sample 4 prepared using oleoresin B differs significantly and is superior to others including the reference sample. Sample 3 prepared using oleoresin A does not differ from the reference sample, but is superior to samples prepared using spice oils. Dunnett Test

The Dunnett Test is applied to compare the treatments against a pre-determined control, and the objective is to determine which of the sample means differ

significantly : from the mean of the control. Dunnett has published tables of t‘ values, where ¢ has been adjusted upward (Table 19-12). For selected values of

significance, they show the ¢-value. This value should be used to find the significance of the differences between the reference and the test sample means. The calculations are made using the following expressions:

(a) When the number of observations (N) for all the samples are equal, for example, 10 panelists evaluating all the samples, the expression used is

622

Analysis of Fruit and Vegetable Products

A=tsX

V/p/N

where, A = the amount of the difference between a sample mean and the control required for significance. t = value from Table 19-12. 5 = standard deviation (square root of the average variance) of the

p+1 distributions. p = number of samples excluding the reference sample N = number of observations on which each mean is based

df = (ptl) (N-1)

(b) When the number of observations (N) for all the samples are not equal, the expression used is:

A

a

"

Aly

+

os ta

NS

zi" 2

z

~Z

af = N-(p+1) The application of Dunnett test is illustrated below taking the example of tomato ketchup prepared using spices (sample 0), oleoresin A (sample 3) and

oleoresin B (sample 4). Table O gives the data required for the Dunnett test.

TABLE O: Data for applying Dunnett Test

0

. X?

Panelist

».¢

1 2 3 4 5 6 7 8 9 10

4 6 6 4 5 5 6 5 4 5

16 36 36 16 25 25 36 25 16 25

50

256

Total (x)

3

4

».¢

x2

6 5 4 6 4 5 5 6 4 4

36 25 16 36 16 25 25 36 1616

49

247

xX

xX?

1 4 5 4 3 2 4 2 1 2

1 16 25 16 9 4 16 4 1 4

28

96

x

5.0

4.9

2.8

C.F,= (2X)?/N

250.0

240.1

784

x (X- X=

7

= [X-x)? naa) Nal

Se

al

=) SX

OR

= 256-250

247-240.1

= 6.0

= 6.9

6.0 t ai 0.67

69 — = 0.77

96-78.4 =17.6 17.6 os = 1.96

Sensory Evaluation

623

: 0.67 + 0.77 + 1.96 Average variance, s? = ————-— = 1.13

3

5s = 1.13

= 1.0646

Degree of freedom, df = (2 + 1) (10-1) = 27

t-value from Table 19-12 for 27 degrees of freedom at 5% level. = 2.00

at 1% level = 2.745

A = 2.00 X 1.0646 X 2/10 xf =0.95 at 5% level A = 2.745,X 1.0646 X

2/10 = 1.31|at 1% level

0 ~ 3 =5.0~ 49 = 0.1 Not significant 0~4=5.0~ 28 = 2.2 Significant The results show that tomato ketchup sample 4 differs significantly from the reference sample, and sample 3 is comparable to the reference sample. References 1. Larmond, E., Methods Agriculture, Ottawa.

for Sensory

Evaluation.

Publication

1284, Canada

Department

of

2, Guide for Sensory Evaluation of Foods: Part III. Statistical Analysis of Data, 1S: 6273 (Part III) 1975,

Indian Standards Institution, New Delhi.

B, Hedonic Rating Test Hedonic

rating

relates

to pleasurable

or

unpleasurable experiences. The

hedonic rating test is used to measure the consumer acceptability of food products. From one to four samples are served to the panelist at one session. He is asked to rate the acceptability of the product on a scale, usually of 9 points, ranging from “like extremely” to “dislike extremely.” Scales with

different ranges and other experience phrases could also be used. The results are analysed for preference with data from large untrained panels. Semi-trained panels in smaller numbets are used to screen a number of products for selecting a few for consumer preference studies. When pronounced after-effects are met with, precluding testing of a second sample or when independent judgements are sought for, separate cards are

used for each product. When telative preference is the object of study, cards with multiple columns for the numbet of test samples-atre used. Specimen

Evaluation

Card HEDONIC

RATING

TEST

624

Analysis of Fruit and Vegetable Products

Taste these samples and check how much you like or dislike cach one. Use the appropriate scale to show your attitude by checking at the point that best describes your feel-

ing about the sample. Please give a rcason for this attitude. Remember you are the only ane who can tell what you like. An honest expression of your personal fecling will help us.

Code:

Like extremely

Code:

Code:

=

Like very much

a

Like moderately

=

eo

es

oe

a

Like slightly

Neither like nor dislike

Dislike slightly Dislike moderately

Dislike very much Dislike extremely Reason

ae Se ee

sae

Signature

The test is more useful in determining preferences than in determining differences. Two samples which are quite different in flavour can easily be rated as ‘neither like nor dislike’ by the same panelist. To analyse the results, numerical values are assigned to each point on the scale, 1 point usually being given for ‘like extremely’ and 9 points for ‘dislike extremely’ or vice versa. The scale may be reduced to 7 or 5 points. The scores received by each sample are then averaged and compared with the average score received by other samples in the series. Hedonic rating can also be evaluated by ranking method. For this purpose, the

ratings given by a panelist to each sample are arranged in increasing or decreasing numerical order and ranking assigned. Example 8 is given to illustrate this. EXAMPLE 8

Factory sample of orange marmalade is rated on a9 point hedonic scale against 3 competitive brands to test for relative superiority (Table P).

Considering the mean score, the rating of the quality of the marmalade is in the following order:

Sample A > Factory sample > Sample B > Sample C The rank sum for each sample is shown in Table P. In arranging the rartks, cqual ranks were tabulated as average of the ranks the samples would have occupied if there were no ties.

Sensory Evaluation

625

TABLE P: Conversion of Hedonic Scale to Ranks ——o—o—o—ooo

a

ee

Competitive Brands Panelist

SS 1 2 3 4 5 6 7 8 9 10

Puctoay, sesigle Score ieee 4 3

3 2 2 3 3 2 3 3

28 Mean

Rank ave Py 2; 1 2 2 1 2 2 1 2 2

7

2.8

Sample A

Ea

Score a 3 4 1 1 3 2 2 3 2 2

23

Sample B

Rank

1 2 1 1 2 1 1 2 1 1

3

P55

Score A 5 5 6 4 9 4 5 4 5 4

Sample C

Rank Score Rank eae hs he i oe 3 6 Bape | 3.5 5 3.5 3 8 4 4 3 3 4 5 3 3 5 4 3 6 4 3.5 ° 4 bi) 3 8 4 3 6 4

51

33

5.1

ee

ae

5.6

score (X) TABLE Q: Rearranged Ranking for Factory Sample

and Sample A of Marmalade Panelist

Factory sample

1

2

i,

2 3 4 B) 6 7 8 9 10

1 Zz 2 1 2 2 1 2 2

23 jlo 1 2 1 1 2 1 1

17

Sample A.

13

From Tables 19-5a and 19-5b, for 4 treatments and 10 replications, the tabulated

values are 17-33 at 5% level and 15-35 at 1% level. The tabulated values total rank 19-31 . 17-33 show that for significance, a low of 16 or a high of 34 would be required at 5% level

and a low of 14 or a high of 36 would be required at 1% level. Sample A is significantly superior at 1% level and samples B and C are significantly inferior at 5% and 1% respectively. Factory sample is qualitatively placed between sample A

and samples B and C. If it is desired to know whether the factory sample or market sample A, which were found to be comparable is superior among the two, their ranks are rearranged, omitting the samples B and C (Table Q). By referring to rank total

626

Analysis of Fruit and Vegetable Products

tables, it may be seen that the factory sample and the market sample A are comparable in quality. Calculation of Fiducial limits for Hedonsc Rating

The rank sum method of evaluating Hedonic ratings fails to account for variations in the panelists,scores. For example, in the case of sample B, scores of 4 and 9 by panelists 4 and 5 respectively have been ranked as 4. A more accurate evaluation of Hedonic ratings is the determination of fiducial limits for the reference sample. The fiducial limits represent a range of average scores (above and below average scores for the control) within which an average score for a

particular sample is not significantly different from the control. Average scores above or below the fiducial limits are significantly different from the control. The steps involved in the calculation of the fiducial limits are the following: i) Determination of the standard error of the mean of the reference sample. ii) Calculation of ‘limits’ which is the product of the standard error of the mean,

and the factor ‘#’ found from Table 19-11 depending upon the degrees of freedom, and the level of significance. iii) Determination of the fiducial limits by adding and subtracting the limits from the average score on the control sample. iv) Determining test samples which are superior, inferior or not significant from the control sample, when their Hedonic mean scores fall above, below or

within the fiducial limits. The fiducial limits method may also be used to evaluate the results of paired comparison tests. — Calculation of fiducial limits for the factory (reference) sample are given below (Table R). TABLE

R: Data for Calculation of Fiducial Limits of Facto:y Sample

Scores of factory sample, X

Squares of the scores, x2

4

16

3 3 2 2

9 9 4 4

3 3

9 9

2

4

3 3

9 9

X= 28

X?2 = g2

Sensory Evaluation

627

the agreed quality descriptions and scores, Without this understanding the

iting will not be of any use. veciinen

Evaluation

Card NUMERICAL

SCORING

TEST

628

Analysis of Fruit and Vegetable Products Please rate these samples according to the following descriptions : Score

Quality description

90

Excellent

80

Good

7o

Fair

60

Poor

Sample

Score

Comments Signature

EXAMPLE9

Canned tomato from 3 different manufacturers was rated for texture. The numerical scoring test was used rather than multiple sample difference test as there was no reference sample with which to compare. Ten panelists rated the coded sample according to the evaluation card given above. Table S shows the panel score. TABLE S: Panel Score for Canned Tomato in Juice Panelist

1

2

3

Total

1

6

7

9

22

2 3 4 5 6 7 8 5 10

7 6 5 6 > 6 6 7 5

5 8 6 6 6 7 9 6 6

8 7 9 9 8 9 7 9 8

20 21 20 21 19 22 22 22 19

Total

59

66

83

208

(Norte: Actual scores are divided by 10 for convenience in calculation) \

CALCULATION

The results were analysed by the analysis of variance.

Total numberof judgements

Total score

Correction factor (C.F.)

= 30

¥

= 208

= (208)?/30 = 1442.13

Sensory Evaluation

Sum of squares of samples

>

Sum of squares ofpanelists

22 (22? 2

- 1442.13 = 30.47

10

'

Total sum of squares of scores

ed 662 + 832

629

2 20> ..

i422 a AP) 1442.13

a 1)Aw

= (@2 +724 ...... + 92 +82) - 1442.13 " wi)» ie.)Ni

Between the samples, there is significant difference at 1% level (Table T). TABLE T: Analysis of Variance Sources of variance

Degreés of freedom

Sum of

Mean

squares

square

3-1= 2 10-1 =9

30.47 454

15.24 0.50

Error

29-(9+2) = 18

18.86

1.05

Total

29

53.87

Samples Panelists

F

Tabulated F* 5%

1%

14.51

6.01

3.55

*From Tables 19-6 and 19-7

The samples which are significantly different from the others are determined by the use of Duncan’s Multiple Range (see Example 7) Test. The calculations

involved are as follows:

Sample means Rank means in the descending order Standard error, SE = «/(1.05/10)

1

2

3

59/10

66/10

~~ 83/10

= 5.9

= 6.6

= 83

A 3

B 2

Cc 1

8.3

6.6

5.9

= 0.330

(2)

(3)

p*

4.07

427

Rp**

1.343

1.4091

*From Table 19-8 for 18 degree freedom

**> X SE

A-C=83-59=24>14A41 A- B= 83 -66= 1.7 > 1.34 A is significantly different from B and C B-C=66-59=07

Salty (Stock solution5.845 g of sodium

H

*

Sweet (Stock solution34.23 g of suctosc

.

.

chloride per litre) per litre) side

Crean nislealenes essa =weicaleisiouaetnte cialssteaouint wreace

ml of stock solution to be diluted to 1 litre

I

0.0002

2 3

4

0.0016

ml of stock solution to be diluted to. ‘x ‘litre

2

, pb §

Sour (Stock solution21015 & of cit.

.

.

.

tic acid per litre) feine per litre)

GH.

a

.

Bitter (Stock solution19.41 g of caf-

saxcanroaranerviaissieeinche O*anreeeeaiietaren eine

ml of stock solution to be diluted to 1 litre

2

0.00005

0.0004

4

0.0001

I

0.0008

8

0,0002

2

16

©,0004

4

4

6

16

0.5

ml of stock solution to be diluted to 1 litre

Os I

5

0.0032

32

32

0.0006

6

6

0.0064

64

64

0.0008

8

8

7

0.0128

128

128

0.0010

10

10

8

0.0256

256

256

0.0012

12

12

9

0.0512

2.994 g/litre

17.526 gilitre

0.0014

14

14

10

0.1024

5-988 gilitre

35.052 gi/litre

0.0016

16

16

II

0.2048

11.976 g/litre

70.103 gilitre

0.0032

32

32

12

0.4096

23.953 gilitre

140.206 gilitre

0.0064

64

64

Source : B. Jellinek, J. Nutr.

Dist., I, 219 (1964).

Sensory Evaluation TABLE

633

19.4 : Significance of Duo-Trio Paired Comparison and Triangle Methods

Paired Duo-trio difference |

Paired preference

| Triangle difference _——

Number

Minimum. agreeing ‘judgements to establish of] significant preference

(Two-Tail Test)

judges or

[—

0.01

Probability level

Probability level

Probability level

0.05

blish significant

—_differentiation

|

ation (One-Tail Test)

judge-

ments

Minimum correct judgements to esta-

Minimum correct judge- J ments to establish significant differenti-

0,001

0.05

0.01

0.001

q aus 9.01

0.001

.

aoe

3

sc8

2 3

3

4

4

5

:

5

6

:

6

7

7

8

8

9

5

5

5

6

7

7

5

6

7

8

7

8

6

7

8

8

9

8

9

6

7

8

10

9

10

.

9

10

10

7

8

9

11

10

II

II

9

10

II

7

8

9

12

10

11

12

10

II

12

8

9

10

13

11

12

13

10

12

13

8

9

10

14

12

13

14

II

12

13

9

10

11

15

12

13

14

12

13

14

9

10

12

16

13

14

15

12

14

15

10

iI

12

17

13

15

16

13

14

16

10

I

13

18

14

15

17

13

15

16

10

12

13

19

15

16

17

14

15

17

11

12

14

20

15

17

18

15

16

18

11

13

14

21

16

17

19

15

17

18

12

13

15

22

17

18

19

16

17

19

12

14

15

23

17

19

20

16

18

20

13

14

16

634

Analysis of Fruit and Vegetable Products Table 19-4: (Contd.) Paired preference

Number of

“Minimum

agreeing judgements to establish

judge-

{Two-Tail Test) °

judges or

ments

_—Significant preference

Paired Duo-Trio difference

Triangle

Minimum correct judge-

Minimum

significant

a

establish

correct judgements to estab-

_different-

lish significant

iation (One-Tail Test)

_"_ probability level a

to

ments

difference

differentiation

Probability level

an

0.05

0.01

0.Cor

24

18

19

21

17

19

20

13

14

16

25

18

20

21

18

19

21

13

15

orp

26

19

20

22

18

20

22

14

15

17

27

20

21

23

19

20

22

14

16

18

28

20

22

23

19

2

23

15

16

18

29

21

22

24

20

22

24

15

17

19

30

2i

23

25

20

22

24

15

17

19

31

22

24

25

21

23

25

16

18

19

20

32

23

24

26

22

24

26

16

18

33

23

25

a7

22

24

26

17

19

20

34

24

25

27

23

25

27

17

19

21

35

24

26

28

23

25

27

18

19

21

36

25

27

29

24

26

28

18

20

22

37

25

27

29

24

27

29

18

20

22

38

26

28

30

25

a7

29

19

21

23

39

27

28

31

26

28

30

19

21

23

40

27

29

31

26

28

31

20

22

24

41

28

30

32

27

29

31

20

22

24

42

28

30

32

27

29

32

21

22

25

43

29

31

33

28

30

32

21

23

25

44

29

31

34

28

31

33

21

23

25

45

30

32

34

29

31

34

22

24

26

46

31

33

35

30

32

34

22

24

26

47 48

31 32

30 31 31

32 33 34

36

23 23 23

25 25 25

27 27

32

36 36 37

35 36

49

33 33 34

5O

33

35

37

32

34

37

24

26

28

Source

: E.B. Roessler, G.A. Baker

& M.A.

28

Amerine, Food Res., 21, 117 (1956);

E.B. Roessler, J. Warren, & J. F. Guymon, Food Res., 13, 503 (1948). (Courtesy :Institute of Food Technologists, USA, Copyright ©)

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Sensory Evaluation 635

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Analysis of Fruit and Vegetable Products

Sensory Evaluation TABLE

-19-6 : Variance Ratio—1%

637

Points for Distribution of F

n,—Degrees of freedom for numerator n,—Degrees

of freedom for denominator

The numbers given in this table arc the valucs of F for which the arca to the left equals 0.99 for the indicated numerator and denominator degrees of freedom. ny ai?

12

24

Inf.

I 40$2.20 4999-59 $403. 40 $624.60 5763.60 5859.00 $981.10 6106.30 6234.60 6365 90 99.00 98.50 99. 17 99-25 99. 30 799. 33 99. 37 99 -42 99- 46 30.82 34.12 29. 46 28.71 28. 27% 49 60 27 +05 18.00 21.20 16. 69 15.98 14. 80 14 +37 93 13.27 06 16.26 11.39 89 10. 29 9. 47 -78 10.93 13074 8 -I0 9-15 7 31 12.25 6 84 45 6 .07 9-55 7-85 7.O1 8.65 11.26 6 -03 “59 5 -28 8.02 4 +73 “99 5 -47 olDW CO AM ON Aw 4 +33 55 7-56 5 .06 22 4 4 4 95 3 4 3 4 3 4 3 3 3 3. 3 ae 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 3 2 BRRRUG 3 2 Vunu VwuwMuvwvVnu Ww=) 3 2 2 ee eh 0h Soh 5.° 2 2 AYUYUYYUYYUYYG AHR APPAR oon AA AAA DNNDNA~N I. 95 2 so oO @o Oo NKR ER NWWKWWWwWWHWKWww NNN NN NN RU © Am LR HwYYwWHYKwYYWYWYHYwWwWAA RVC RUUY RA A An © 2

re

aay

pee LAS AAA SEE RAR AAR SM Daw &

Source ; M. Merrington & C.M. Thompson, “ Tables of percentage inverted beta (F) distribution,” Biometrika, 33, 73 (1943). (With the permission of Prof. E.S. Pearson.)

points of the

638

Analysis of Fruit and Vegetable Products

TABLE 19-7:

Variance Ratio—5°/, Points for Distribution of F

n,—Degrees

of freedom

for numerator

n,—Degrees of freedom fot denominator The numbers given in this table are the values of F for which the area to the left equals 0.99 for the indicated numerator and denominator degrees of freedom.

ets

2

3

4

5

6

8

Iz

24

Inf.

N,

I 2

161-45 18e§$la.

199.50 19200

215-70 19s1Gs.

224.60 19625)

230.16 19e30bs

234.00 19233.o

238.90 198379

243-91 T9sd4Iige

249.0§ 254.31 19e45P—19.50

3 4

«10.13 7-71

9°55 6.94

9.28 6.59

9.12 6.39

9.01 6.26

8.94 6.16

8.84 6.04

8.74 5-91

8.64 Se77ux

8.53 5-63

Soo

Sn Gx

5-79

5-41

5:19

—-§ 05

4.95

4-82

4.68

4.53

4.37

6

5:99

5-14

4-76

4.53

4.39

4.28

4.15

4.00

3.84

3.67

7

5-59

4-74

4-35

4.12

3-97

3.87

3-73

3-57

3-41

3.23

8 9

5.32 5.12

4.46 4.26

4.07 3.86

3.84 3.63

3.69 3.48

3.58 3.37

3.44 3.23

3.28 3.07

3a82a5 239050

2.92 2.71

10 II 12 13 14 15 16 17

4.96 4.84 4-75 4.67 4.60 4-45 4.49 4-45

4.10 3.98 3.89 3.81 3.74 3.68 3.63 3.59

Sat 3.59 3.49 3.41 3.34 3.29 3.24 3.20

3.48 3-36 3.26 3.18 Ser 3.06 3.01 2.96

35,33 3.20 3541 3.03 2.96 2.90 2.85 2.81

3e22 3.09 3.00 2.92 2.85 2.79 2.74 2.70

3.07 2.95 2.85 2.77 2.70 2.64 2.59 2.55

2.91 2.79 2.69 2.60 2.53 2.48 2.42 2.38

2a74ox 2.61 2.50 2e42—y 233% 25208 2.24 2.19

2.54 2.40 2.30 2.23 2.53 2.0% 2.01 1.96

18 19 20

4.41 4.38 4-35

3.55 3.52 3.49

3.16 3.13 3.10

2.93 2.90 2.87

2.77 2.74 2.70

2.66 2.63 2.60

2.51 2.48 2.45

2gh Sm 2sllg 2.08 2.05 2,03-

1.92 1.88 1.84

21

4.32

aA

3.07

2.84

2.68

2.57

2.42

22

4.30

3.44

3.05

2.82

2.66

2055

2.40

2.34 2.31 2.28 2.25 2.23

23 24 25 26

4.28 4.26 4.24 4.23

3.42 3.40 3.39 oan

3.03 3.01 2.99 2.98

2.80 2.78 2.76 2.74

2.64 2.62 2.60 2.59

2.53 2.51 2.49 2.47

2.38 2.36 2.34 2.32

2.20 2.18 2.16 2505

2700» 1.98 1.96 1.95

27

aa2t

3.35

2.96

2.73

Bas7.

2 AG,

2.31

2.13

TSOSe

28

4.20

3.34

2.95

2077

2.56

2.44

2.29

chan ws

T29te

.1.76 1.73 1.71 1.69 1.67 1.65

29

4.18

3.33

2.93

2.70

255

2.43

2.28

2.10

1.90

1.64

30

4:17

3.32

2.92

2.69

2.53

2.42

2227,

2.09

1.89

1.62

40

4.08

3.23

2.84

2.61

2.45

2.34

2.18

2.00

60 120 Inf.

4.00 3.92 3.84

1.79

$35 3.07 3.00

1.§1

2.76 2.68 2.60

2.53 2.45 2 atk

2-37 2.29 2.21

2.25 2.18 2.10

2.10 2.02 1.94

1.92 1.83 1.75

I.7O 1.61% I.§2

1.39 1.25 1.00

points

of the

Source :M. Merrington & C.M: besa “Tables of percentage inverted beta (F) distribution”, Biometrika 33, 73 (1943). (Reprinted with the permission of Prof. H.S. Pearson.)

1.81

1.78

639

Sensory Evaluation

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odpny oduey wal 640 Analysis of Fruit and Vegetable Products

\\

) Sénsory Evaluation TABLE

641

19-10: Values of X’ Required for Significance at Various Levels.* Level of Significance

.

Degrees of Freedom

10% ee

eee

e

2.5%

1%

0.5%

|

3.84

5.02

6.63

7.83

4.61 6.25

5.99 781

7.38 9.35

9.21 11.3

10.6 12.8

ye 5 Ore ee Se

7.78 9.24 10.6

9.49 11.1 12.6

11.1 12.8 144

13.3 15.1 16.8

149 16.7 18.5

Ek, eae aaa ne POE ee oi an OR BNF Soniacs « RS.

12.0 13.4 14.7

141 155 169

16.0 17.5 190

18.5 20.1 21.7

20.3 22.0 23.6

“Cae

16.0

18.3

20.5

23.2

252

22SOIR to. os Aa

Se

es ee

17.3

19.7

219

24.7

26.8

0 O. Ayaeantlbedlps

18.5

21.0

233

26.2

28.3

a ee eck. 3 Ate hk: 13. S02... eigen

19.8 211 22.3

22.4 23.7 25.0

247 26.1 275

27.7 29.1 30.6

29.8 313 32.8

. ) Sees 17). O88 So. Oe

23.5 248 26.0

263 27.6 28.9

28.8 30.2 31.5

32.0 33.4 34.8

343 35.7 37.2

mee LS a) re Fh ere

27.2 28.4 29.6

30.1 314 32.7

329 342 35.5

36.2 37.6 38.9

38.6 40.0 414

22. Ac. tie 1, A re Bi Cen eo at

30.8 32.0 33.2

33.9 35.2 36.4

36.8 38.1 39.4

40.3 41.6 43.0

42.8 44.2 45.6

Ae

34.4

377

40.6

443

465

35.6 36.7

38.9 494

41.9 43.2

45.6 47.0

48.3 49.6

37.9 39.1 40.3

413 42.6 43.8

44.5 45.7 47.0

48.3 ae 50.9

51.0 52.3 53.7

‘By.

ES

5%

* ee

On

fo

MEE th PSP. CY 2 /)Nae ee re, © ee ae ot ere

:

* Abridged with permission of the publisher from a table that originally appeared in an article by Thompson, Catherine M., . »metrika, Vol. 32, pp. 188 and 189. ;

642

= Analysis of Fruit and Vegetable Products

TABLE 19-11: Values of ¢ resources for significance of various levels for two-tailed hypothesis Level of Significance

10% 5%

5% 2.5%

2% 1%

1% 0.5%

0.1% 0.05%

Race y ig 3 Fd Moe id 8 S Ca: Se

6.31 2.92 2.35 2.12 2.02

12.71 430 3.18 2.78 257°

31.82 696 454 3.75 336

63.66 9.92 5.84 4.60 4.03

636.62 1.60 12.94 8.61 6.86

GEAR, iocei ee& pe ce ee C2: SIRES. AS HO) ete Ae ie

1.94 1.90 1.86 1.83 1.81

245 2.36 231 2.26 2a

3.14 3.00 2.90 282 "276

3.71 3.50 3.36 3.25 3.17

5.96 5.40 5.04 4.78 4.59

he SPs iF ties oe UY coer Agee i, a ee tt meee ER

1.80 1.78 1.77 1.76 1.75

5 ate 218 2.68 2.16 2.65 214 262 2.13 2.60

3.11 3.06 3.01 2.98 2.95

444 432 4.22 4.14 4.07

7

Degree of Freedom

1.75

He

258

2.92

4.02

NTS arose; He Ses ete Se

os

1.74

21h

257

2.90

3.96

Ch 2 032 + 2 eee 7S

1.73 1.73 1.72

210 209 209

2.55 2.54 253

2.88 2.86 2.84

3.92 488 3.85

Diln ers. So (7 23) a ee Se BAS as du thaabe >? ee

1.72 1.72 1.71 1.71 1.71

208 2.52 207 251 207%, %. 756 2.06 2.49 2.06 2.48

2.83 2.82 2.81 2.80 2.79

3.82 3.79 3.77 3.74 3.72

7 27 7 29) 30:

1.71 1.70 1.70 1.70 1.70

206 205 205 2.04 204

248 2.47 2.46 2.46 2.46

2.78 2.77 2.76 2.76 2.75

3.71 3.69 3.67 3.66 3.65

1 as SET GOL GALS, osace cc SZORSES. So. cc

1.68 1.67 1.66

202. 200 198

..:2.42 2.39 236

2.70 2.66 2.62

3.55 3.46 3.37

\'0)-— «+ BORSA

1.64

1.96

2.33

2.58

3.29

a ee, Sage SIA rea nnn eet VPP. oe

* This table is abridged from Table III of Fisher and Yates, Statistical Tables for Biological, Agricultural and Medical Research, 6th edn., Oliver and Boyd, Edinburgh, 1963, by permission of the’ authors and publishers. > Two-tailed hypothesis. * One-tailed hypothesis.

Sensory Evaluation

643

TABLE 19-12: Table of t for one-sided comparisons between p treatment means and acontrol fora joint confidence coefficient of P=99 (significance levels of 5 and 1 per cent) * iy

Degrees of Freedom

15. Mellie: EhAna

P

P Number of Treatment Means Excluding Control

1

2

3

4

95

L735.»

207

(2.24

2.36

99

260

2091

3.08

3.20

MGs cisenasn 2e02

95 99

1.75 258

2.06 2.88

2.23 3.05

2.34 3.17

eo.

95 99

174 257

2.05 2.86

2.22 3.03

2.33 3.14

Mawes: 3a. boos

95 99

1.73 255%

2.04 12:84

2.21 3.01

2.32 3.12

rag tallies

95 99

Tih 254

Els 283

2.20 2.99

2.31 3.10

20: ssyourecibaci

95 99

1.72 253

2.03 281

2.19 2.97

2.30 3.08

ee

95 99

171 LAG

201 LT

2.17 2.92

2.28 3.03

bpichw.

95 99

170 246

°1.99 2.72

2.15 2.87

2.25 2.97

1 OR

95 99

168 242

197 268

"233 2.82

2.23 2.92

GON

95 99

1671.95 239 264

2.10 2.78

2.21 2.87

0,

95 99

166 2.36

1.93 2.60

2.08 2.73

2.18 2.82

A9OL geisrentbenclYs

95 99

164 233

1.92 2.56

2.06 2.68

2.16 2.77

te0t. 2k

TE Clee.

|

* Adapted from Dunnett, C.W., “A Multiple Comparison Procedure for Comparing Seve-al Technical Means with a Control”, Journal of the American Statistical Association, 50, 1096 (1955).

644

Analysis of Fruit and Vegetable Products

C, Dilution Test Dilution tests are designed to establish the smallest amount of an unknown material, developed as a substitute for a standard product, that can be detected when it is mixed with the standard

product, e.g., margarine in butter, dried

whole milk in fresh milk, synthetic orange flavour ingredients with ratural flavour and so on. The quality of the test material is represented by the dilution number which is the percent of the test material in the mixture of the standard product such that there exists a just identifiable difference in odour and taste between them. The bigger the dilution number the better is the quality of the test material. By a preliminary test, establish the higher and the lower per cent of the test material in the mixture which are likely to be respectively definitely detectable by all and not detectable by all. Define a series of mixtures including those which represent the upper and lower: limits. Usually six are enough, although eight may be used if greater precision is desired. Give a simple paired difference test with this test series against the standard starting with the mixture containing the lower per cent of test substance. Obtain 15 to 20 judgements for each sample of the series. Determine the mixture where the frequency of the difference judgement just

reaches the 5% significance level. The per cent of the test material in that mixture is the dilution number. Questionnaire and analyses are similar to those in the threshold and difference tests. An interesting variation of this test is the dilution flavour profile test which analyses a product by stepwise dilution and records the detectable flavour components in their sequence of appearance and intensity. This is useful in analysing complex processed foods, and helps in studying the flavour secrets of successful products and also the influence of variety, processing, ageing, storage, etc. Selected

References

1,

Amerine, M.A., R.M. Pangborn & E.B. Roessler, Principles of Sensory Evaluation of Food, Academic Press Inc., New York (1965).

2.

Amerine, M.A., E.B. Roessler & F. Filipello, Modern Sensory Methods of Evaluating Wine, Univ. of California, Hilgardia, 28, No. 18 (1959).

ze Basic Principles of, Sensory Evaluation, STP 433, (1968). Ametican Society and Materials, 1916 Race Strcet, Philadelphia, Pa 19103.

4. Bengtsson, K., Wallerstein Lab. Commun., 16, 231 (1953). §-

Bradley, R.A., Biometrics, 9, 22 (1953).

fot Testing

Sensory Evaluation

645

Caul, J.F., Adv. Food Res., 7, 1 (1957).

Committee on Sensory Evaluation of the Institute of Food Technologists, Technol. (Chicago), 18, 1135 (1964).

USA,

Food

Correlation of Subjective-Ob jective Methods in the Study of Odonrs and Taste, STP 440, 1967. American Society for ‘Testing and Materials, 1916 Race Street, Philadelphia, Pa 19103. Ellis, B.A., Guidebook for Sensory Testing, Chicago Co., Inc., Chicago, Hlinois

Technical Center,

Continental Can

(1961).

Gridgeman, N.T., J. Food Sci., 16, 171 (1961). Il.

Jellinck, G., J. Nutri

Dict., 1, 219 (1964).

12.

Kramer, A., & B.A. Twigg, Fundamentals of Quality Control for’the Food Industry, 3r¢ edn., Vol I, AVI Publishing Co., Westport, USA (1970).

pz.

Kramer, A., Food Technol..

14.

Larmond, E., Methods for Sensory Vivaluation of Food, Publn. 1284 (1970).

15.

Manual on Sensory Testing Method, STP 434 (1960). American Society for Testing an

(Chicago), 14, 576 (1960). Canada Department of Agriculture

Materials, 1916 Race Street, Philadelphia, Pai9103. 16.

Quality Control in the Viood Industry,

Vid. S.M. Herschdoerfer,

|Academic

Press Inc.,

Westport, USA (1966). 17.

Read, D.R., Biometrics, 20, 143 (1964).

18.

Scheffe, H., J. Amer. Statist. Ass., 47, 381 (1952).

19.

Snedecor, G.W.

&

Ames, Iowa, USA 20.

W.

G.

Cochran, Statistical

Methods, Yowa State University Press.

(1967).

_Tilgner, D.J., Food Technol. (Chicago), 16 (2), 26 (1962) ; 16 (3), 47 (1962).

CHAPTER 20

General Instructions for

Microbiological Examination Cleaning

of Glassware

GLAsswARE used in bacteriological examination should be chemically clean. New glassware should be cleaned with hot water and soap using a brush, and rinsed thoroughly, first in tap water and then in distilled water. Washing soda, sodium metasilicate, trisodium phosphate, or synthetic detergents may be used instead of soap. Place glass containers with discarded cultures in 3% lysol, or other suitable disinfectant, then boil in soap solution, brush and wash in running tap water, Occasionally wash in ethyl alcohol containing 5%HCl or cleaning solution. Clean pipettes by leaving overnight in the cleaning solution in a tall glass cylinder. Wash in tap water and finally rinse in distilled water. Keep the washed articles inverted and allow to dry in the air. Glassware for use in bacteriological examination should be neutral. New glassware, especially the cheaper

varieties, gives off free alkali. To test for this, fill the bottle nearly full with distilled water at pH 7.0. Add enough 0.4%

phenol red solution to give a

yellow colour. Autoclave for 30 min at 15 psi (120° C) and cool to room tem-

perature. If the tory. A pink or Repeat the test glassware may

colour of the water remains yellow, the glassware is satisfacmagenta colour indicates that alkali is liberated from the glass. again. If no alkali is liberated on the second autoclaving, the be taken for use, otherwise it should be discarded. Sterilization

of Glassware

Any glassware sterilized in a hot air oven should be dry and the oven should not be hot when the articles are placed in it. Steadily raise the temperature to the required level, and maintain for the full time necessary for sterilization. Cool the oven before removing the articles. Pipettes :Place them in copper or stainless steel cylinders with a lid, and sterilize in a hot air oven at 160° C for 1 hr or at 140°C for 2 to 3 hr. Place a pad of cotton at the bottom of the container and on the inside of the cover to protect the tips of the pipettes from damage. If suitable pipette containers are not available, wrap each pipette with kraft paper in a spiral manner and sterilize in the hot air oven. Such wrapped pipettes remain sterilized for a /

General Instructions for Microbiological Examination

647

long time. A cotton plug should be thrust into the mouth of the pipette before sterilization.

Petri dishes : Wrap them in kraft paper in sets of 3 to 5 and sterilize in a hot air oven at 160° C for 1 hr. The dishes may also be sterilized without any wrapping in cylindrical tinned copper boxes. Sampling bottles and dilution bottles : Sterilize them in hot air oven or in autoclave. For bottles with stoppers, insert a strip of paper 3 x 0.5 in. between the stopper and the neck of the bottle to prevent the stopper jamming and the glass cracking on cooling. Ensure that at least half the strip projects outside the bottle so that when the stopper is taken out, the paper can be removed without touching the rim. Sterilize at 15 psig for 20 min. Tubes: Plug the tubes with absorbent. cotton wool. Insert the plug to a depth of 1.25—-1.5 in. into the tubes; enough should project out of the mouth to protect the rim from dust. A tube which will just hold its own weight

when suspended from the tuft of cotton may be considered as properly plugged. Inoculating needles and loops: Sterilize these by flaming, i.e., by passing the atticle through a Bunsen flame or the flame of a spirit lamp. Preparation of Bacteriological

Media

In the preparation of solid media, add the soluble ingredients such as salts, peptone, beef extract, etc. first and the agar last to the required amount of cold water. For dissolving the agar, use a hot water bath rather than an open flame. Adjustment of pH: Adjust the pH by using a pH meter or by using suitable indicators (see Table 35-3). To 10 mlof the solution of unknown pH, add 0.5 ml of 0.02 to 0.04% solution of the indicator. Compare the resulting colour with a set of standards prepared for the specific indicator used. These standards may be coloured liquids in sealed glass tubes or coloured glass discs. To adjust the pH by using coloured standards, pipette 10 ml of the solution into a clean test tube. Use tubes with uniform thickness of wall and bore. Add 0.5 ml of indicator. Mix carefully and place in the central hole cf a six-hole comparator block. Insert a test tube of distilled water behind this unknown. In the slot to the right of the tube with the solution under test, insert a colour standard which is near the colour of the unknown. Behind it place a tube of broth to which no indicator has been added. In the slot to the left of the test solution place another colour standard of + 0.2 pH units of the standard selected for the right slot. Place a tube of broth behind this also. Look through the block at a good source of light (preferably daylight) and see if the colours of the standard andthe solution under test match. Note the pH from the value of the standard that most closely approximates the colour of the test solution. If the broth is acid, add dropwise from a burette 0.1 N solution of NaOH until the colour of the desired pH value is obtained.

648

Analysis of Fruit and Vegetable Products

Note the quantity of 0.1 N NaOH used. Find the amount of 1 N NaOH te-

quired on the basis of the 0.1 N solution used to adjust the pH of a known volume of the media, and add the calculated amount. Recheck the pH of the culture medium to determine the accuracy of the calculation. Filtration of media: Filter liquid media through filter paper or by means of a Buchner suction funnel. Filter melted agar (80—90° C) through ,otton wrapped in gauze. Tubing of media : Fill into test tubes using a glass funnel fixed on a stand and fitted with rubber tubing, pinch cock and tapered delivery tube. Cover the funnel with the lid of a large sterile Petri dish to avoid aerial contamination. The tubes after filling with the medium are plugged with cotton wool or with aluminium caps. Culture bottles having screw caps with rubber or screwcapped jars may also be used. Sterilization of media : Sterilize such m2dia which contain sugars by discontinuous or intermittent heating for short periods (20-30 min) at 100° C or less, depending on the particular media on 2 or 3 successive days. This permits spores to germinate in the interval, and on the second or third heating, the remaining vegetative cells are killed. The Arnold sterilizer also called “‘steamer”’ is best suited for this. The autoclave may be similarly used by leaving

the steam escape valve wide open to prevent the development of ste2m pressure. Use an autoclave for sterilization by steam under pressure. Usually 20 min at 15 psi is sufficient. Do not sterilize media containing sugars longer than the required time at reduced pressure (10 lb), because prolonged heating will hydrolyze many carbohydrates, especially in alkaline media or in very acidic media. When using the autoclave, expel all air before the pressure is allowed to rise. Increase or decrease the pressure slowly. At the close of the sterilization period, release prevent the plugs blowing out or moistening of media. Storage : Store culture media in bottles or flasks tubes. Bottles with screw caps of suitable size may

the pressure gradually to

the cotton by the boiling in bulk, or distribute into be used. Sterilize the bot-

tles with the caps screwed on tightly. Plating: Pour melted agar medium into a Petri dish by lifting the lid just enough to permit pouring and allow to solidify in the form of a thin layer. For a dish of 10 cm diameter, about 10-15 ml of the medium is sufficient. Allow the medium to set for a few hours.

Inoculation

Simp le streak: Use a platinum loop which consists of a piece of platinum wire No. 23 S.W.G., 2.5 in. long with one end fused into a glass rod, or

fixed to a suitable holder, and the free end bent in the form of a closed loop. Nichtome wire may also be used, The loop is bent so that the wire makes

General Instructions for Microbiological Examination

649

an angle of 60° with the perpendicular. Sterilize the loop ina flame so that the whole length becomes red hot, and dip it into the broth culture. Remove a small quantity of the culture (thé inoculum) and streak the inoculum over the plate by touching the surface lightly with the loop (Fig. 20.1a). Hold the wire so that the whole loop is in contact with the surface of the medium.

=e

Simple Streak a

Cross Streak b

Fig: 20.1 Method of preparing streak plates.

Cross streak: With a previously sterilized bent wire loop, transfer one loopful of the inoculum near the rim of the plate and make parallel streaks over a segment (Fig. 20.1b). Flame the loop, cool, and make streaks from area A over

area B with parallel streaks after touching the path made by the loop in A. Take care not to let streaks overlap. I'lame the loop again, cool and repeat streaking through the area C and so on. Each looping out dilutes the inoculum. Two loops may be used instead of one; while one is cooling the other is used. Rotating plate :Use a bent glass wid3 mm in diameterand bent at right angles for spreading the inoculum on the surface of the medium. Sterilize the rod by dipping in alcohol and by flaming (repeat twice). Transfer a loopful of the inoculum to the surface of one plate. Sterilize the glass rod, cool, remove the cover of the dish and, while rotating the dish with one hand, move the bent short limb of the rod over the surface of the media in a to and fro motion with

the other hand. Replace cover. Without flaming the rod, streak a second plate in the same manner. With any of the above methods, the resulting culture should show a number of discrete colonies. Pour plate: Melt several tubes containing 15\ml medium. Place in 45-50° C

water bath to cool. Take the material to be examined in a quarter strength Ringer solution (see “Saline and Ringer Solution” in Ch. 23) and prepare 1/10, 1/100, 1/1,000 dilutions in the same solution. To 15 ml of medium at 45° C, add 1 ml of a dilution, mix by rotating the tube between the palms of the hands

and pour into a Petri dish. Make replicate pour plates of each dilution for incubation at suitable temperatures. Allow the medium to set, invert the plate and incubate. Pour plate cannot be used for purposes of counting as some of the inoculum, diluted in the agar, may remain behind in the test tube.

650

Analysis of Fruit and Vegetable Products

For inoculating a liquid medium such as broth from a solid culture, pick the colony from the culture using a long straight wire (mounted on a holder)

without a loop. Hold the tube to be inoculated inclined almost to the hori-

zontal position, deposit the inoculum on the wall of the tube just above the surface of the liquid at the lower end of the tube, and return the tube to the vertical position. The inoculum should be below the surface of the broth when this is done properly. For adding large amounts of liquid inoculum, use a sterile pipette. The nature of the inoculated material and the date should be marked on the tube or the plate with a glass marking pencil or by pasting a label. Shake Tube Cultures ; Tube media dextrose agar or thioglycolate agar in 15—20 ml amounts in bottles or tubes 2—2.5 cm in diameter. Melt and cool to about 45° C. Add 0.1 ml of inoculura to one tube, mix by rotating between palms. Remove one loopful from this, inoculate a second tube and so on. Allow to set and incubate. The shake tube cultures are useful in observing colony formation in deep agar cultures, especially of anaerobic or micro-aerophilic organisms. Obligate aerobes grow only at the top of the medium; obligate anaerobes only

near the bottom; micro-aerophiles grow near but not at the top; facultative organisms grow uniformly. Incubation : The inoculated cultures, tubes and plates are generally incubated at specified temperatures (25°, 37°, 40° or 55° C) for definite lengths of time.

Agar plates are incubated in the inverted position, i.e., the lid of the

plate is underneath. The plates may also be placed inverted on a clean sheet of blotting paper in the incubator.

Bacteria Counting A total count of the organisms may include those dead or indisti nguishable from other particulate matter. A viable count assumes that a visible colony

develops from each organism. In suspensions, bacteria, particularly in actively reproducing stage, remain clumped. Hence, a visible colony may develop from one organism or from hundreds in a clump. Dilution may break up or induce formation of clumps and it is obviously difficult to obtain reproducible results Total counts :Use a Helber counting chamber which is almost similar 7 Howard mould counting cell (see Fig. 24.1). On the platfo rm, an area of 1 sq mm is ruled. There are 400 small Squares and the area of each small s oe 0.0025 sq. mm. The volume over each small Square is 0.02x0.0025 oo ES, = Suspension, add a few drops of formalin. Dilute the suspension such at there are 5 to 10 organisms per small Squar e. Use 0.1% peptone water containing 0.1%, Teepol (if phase contrast or dark field is ce for counting) and 0.1% methylene blue. Filter before use. Place a loopful of careveeine

General Instructions for Microbiological Examination

651

on the ruled area, apply the cover glass, allow 5 min for bacteria to settle and count the number of organisms in 50-100 squares. Divide the total number by the number of squares counted to find the average number per square. Bacteria __ Number of organisms per ml per square

3

Dilution factor

20,000,000

Viable counts: The procedure involves preparation of dilutions, plating and incubation thereafter, prior to counting. To prepare dilutions, use Ringer solution or preferably 0.1% peptone solution in water. If refrigerated diluent is to be used, allow the solution to attain room temperature prior to use to prevent cold-shock which may prevent organisms from reproducing. Pipette 9 ml of sterile diluent into each sterile test tube with aluminium cap. Do not sterilize the diluent in test tubes with aluminium caps as volume changes may occur. Thoroughly mix the sample by shaking, dip a sterile pipette about 0.5 in. in suspension and remove 1 ml. Deliver into the first dilution blank, about 0.5 in. above the level of the liquid. After 3 sec, blow out and discard the pipette. Dip a fresh sterile pipette about 1 in. into liquid, suck up and down ten times to mix, remove 1 ml, pipette into the next dilution blank and discard the pipette. Repeat the process to get the desired number of dilutions. Number the dilutions serially. The dilutions will be as follows :

Tube No.

sate Dilution

Volume of original F fluid per ml

I

1/io

o.1

or

107}

2

1/100

0.01

or

e684

3

1]1,:000

0.001

or

10-8

4

1]/10,000

0.0001

of

10;¢

1/100,000

0.00001

of

TOme

5

°

Use two or more Petri dishes for each dilution. With a pencil, mark the dilution number. Melt nutrient agar or other suitable media tubed in 10 ml tubes. Cool to 45° C. Pipette 1 ml of a dilution into the centre of the Petri dish, transfer the contents of one agar tube to the dish, and mix thoroughly (by moving clockwise, counter-clockwise, back and forth and to-and-fro;

six times each). Allow the medium to set, invert and incubate for 24—48 hr.

Count plates having 30-300 colonies using a magnifier and a tally counter ot a colony counter. While counting, mark the glass above each colony with pen and ink. Colony count

_ Number of colonies

*per ml

-

per plate

Reciprocal of the

dilution

652

— Analysis of Fruit and Vegetable Products

Monochrome

Staining

To observe the morphology (shape, ‘size, etc.) of the micro-organisms, use has been made of the property of certain dyes to stain cell walls, and increase the contrast under a microscope.

Preparation of Stains 1. Ziehl’s carbol fuchsin: Prepare solution A by mixing 0.3 g of basic’ fuchsin and 10 ml of ethyl alcohol (959%), and solution B by mixing 5 g of phenol with 95 ml of distilled water. Mix solutions A and B. 2. Loeffler’s methylene blue: Dissolve 0.3 g of methylene blue in 30 ml of ethyl alcohol. | 3. Safranin: Prepare a 2.5% solution of safraninO in 10 ml of ethyl alcohol

(95%) and 100 mi of distilled water. PROCEDURE

Clean the glass slides (3x 1 in.) thoroughly and wipe them with a dry cloth. (On a clean grease-free slide, a drop of water can be spread as a thin film.) By means of a sterile wire loop (heated to redness in flame and cooled), transfer a loopful of the bacterial suspension from the broth to the centre of the slide. If preparation is made from solid medium, touch the sterile loop lightly upon the colony and transfer to a drop of water placed on the slide. Reflame loop and cool. Spread the drop into a thin film on the slide and allow to air dry. Fix the dry smear by passing the slide rapidly through the low flame of a Bunsen burner or spirit lamp. Apply just enough heat so that the slide can be tolerated on the back of one’s palm. Cover the smear with several drops of any one of the dyes and allow 15-20 sec in the case of carbol fuchsin and 3—5 min in the case of methylene blue. Wash carefully with water, wipe dry by pressing with a filter paper and examine in a microscope using oil immersion lens. Note the shape of the cells, nature of staining, etc.

Negative Staining Negative staining is a rapid method for studying micro-organisms against a dark background. It enables measurement of the size of bacteria, since the cells are not distorted as in the usual staining procedure. Dorner’snigrosin solution : Add 10 g of nigrosin to 100 ml of distilled water and dissolve by heating for 30 min on a boiling water bath. Replace water lost by evaporation and add 0.5 ml of formalin. Filter twice through double filter paper. PROCEDURE

Place 2 loopfuls of the suspensioon n a slide and add 1 loopful of the nigrosin solution. Mix thoroughly. To mak- a desirable film, sptead the material

General Instructions for Microbiological Examination

653 ~

during the process. Air dry, but do not fix with heat. Examinethe preparations under the oil immersion objective ina microscope. Gram Staining The Gram staining technique helps to differentiate bacteria into two groups, namely, the Gram-positive (those retaining the blue colour) and the Gram-

negative (those which can be decolourized and counterstained red).

This

_ technique is an aid or tool in classifying bacteria. Preparation of Stains Primary dye: Prepare solution, A by dissolving 2 g of crystal violet (85% dye content) in 20 ml of ethyl alcohol (95%), and solution B by dissolving 0.8 g of ammonium oxalate in 80 ml of distilled water. Before use, mix equal quantities of solutions A and B. If this mixture of stains is too concentrated and difficult to decolourize, dilute solution A as much

as 10 times, and mix the

diluted solution with equal quantity of solution B. Mordant: Prepare Lugol’s iodine solution (Gram modification) by dissolving 1 g of iodine and 2 g of potassium iodide in 300 ml of distilled water. Counter stain: Mix 10 ml of a 2.5% solution of safranin in 95% ethyl alcohol with 100 ml of distilled water.

PROCEDURE Clean the glass slides thoroughly and wipe them with a dry cloth. Handle the slides only by grasping them at the edges. Flame the surface to remove any grease and make a mark on one side witha grease pencil, where the smear is to be made. By means ofa sterile loop (heated to redness in a Bunsen flame and allowed to air-cool), transfer a loopful of bacterial suspension, spread it evenly over a small circular area and allow it to air dry. Reflame loop to burn the excess of inoculum. Fix the dried smear by passing the slide rapidly 5 to 6 times through the flame. Keep the film-side up. Just evens heat should be

applied so thatthe slides can be tolerated'on the back of one’s palm. Put crystal violet solution and allow to remain for 1 min. Wash in tap water. Apply the iodine solution (mordant), allow to remain for 1 min and wash in water.

Decolourize by adding alcohol dropwise on the tilted slide until all free colour (blue) has been removed. Wash in water. Flood the slide with safranin (coun¢ ter stain) for 10 sec. Wash in water and air dry. Examine the mounts under-

the oil immersion objective of a microscope. Cells which do not decolourize, but retain the violet dye are Gram-positive. Cells which decolourize and accept the safranin stain are Gram-negative.

Spore Staining

Certain bacteria form endospores or spores within the cell. These spores ate remarkably resistant to conditions which quickly kill vegetative cells c

654

~=Analysis of Fruit and Vegetable Products

bacteria. The spores resist the usual stains and appear as relatively transparent spots in the cell. In the older cultures, the spore-bearing cell tends to disintegrate leaving the “naked spore.” Size, shape and position of the spore are used as aids in identifying and classifying the various species of Bacillus and Clostridium. A special staining procedure must be used to stain the spores properly. Preparation of Stains 1. Ziehl’s carbol fuchsin: Prepare as given earlier in this chapter. 2. Dorner’s nigrosin: Prepare as given earlier in this chapter. 3. Malachite green: Dissolve 5 g of malachite green in 100 ml of distilled water. 4. Safranin: Prepare a solution as given earlier in this chapter. PROCEDURE

1. Dorner’s method: Make a heavy suspension of the organism in 2 or 3

drops of distilled water in a small test tube. Add an equal quantity of carbol fuchsin. Allow the mixture to stand in boiling water for 10 min or longer. On a slide, mix 1 loopful of the stained preparation with 1 loopful of the nigrosin solution. Smear as thin as possible and dry rapidly. Slow drying causes thick films of nigrosin to crack. Examine thin portions of the preparation with the oil immersion objective. The background appears greyish, the cells colourless, and the spores a brilliant red. Note the presence of free spores in the field. 2. Wirtz’s method (modified): Make a smear of the culture on the slide and fix by passing over the flame 5 or 6 times. Flood the smear with malachite green and bring to steaming point. Allow to remain for 2 or 3 min and wash in tap water. Apply the safranin solution. Allow to remain for 30 sec. Wash, dry and examine under the oil immersion objective. The spore stains green, the remainder of the cell a light red.

Motility of Bacteria

The motility of the organism may be observed by making a hanging drop preparation. MATERIALS

1, Hollow or depression slide or slides with glass or rubber ring about 1 mm high. 2. Clean cover glasses, 3. Young (not more than 24 hr old) broth culture of the organism. 4. Vaseline.

jp General Instructions for Microbiological Examination

655

PROCEDURE

Make a square or circle with vaseline on the slide around the concave depression as centre (or apply vaseline to the surface of the ring, if such a slide is used). A film of vaseline about 1/16 in. in height is desirable. By means of an inoculating loop, place a small drop of the young broth culture in the centre of a clean coverslip which is placed near the edge of the table. Take the hollow ground slide, which has previously been prepared as above, and place it on the coverslip so that the drop of culture will be in the centre of the hollow depression. Turn the slide over. A drop of the young broth culture will then be suspended in the hollow of the slide and is protected against evaporation by the vaseline seal. Place the slide on the microscope stage, the coverslip being uppermost. Focus with the low power objective of the microscope on the edge of the drop and then with the high power. Observe whether the organism is |

active or sluggish.

CHAPTER

21

Bacteriological Examination of Water

THE main objectives of bacteriological examination of water required for the cannery are to determine : (i) Sanitary quality of water for human consumption, (ii) treatment measures necessary, (iii) performance of treatment, and (iv) the degree of pollution of river waters, The tests carried out for estimating the bacteriological quality of water are: (i) plate count, (ii) coliform count, (iii) faecal Streptococci test, and (iv) Clostridium welchii test. Under special circumstances, it may be necessary to test for the presence of bacterial organisms capable of reducing inorganic sulphates, iron and sulphur bacteria and other organisms. In routine bacteriological examination of drinking water, search for the preseace of disease-producing microorganisms is not made because of the difficulties involved in the procedure. The plate count is carried out for determining the general bacterial purity of the water and for treatment purposes. Organisms of faecal origin, viz. Escherichia coli, Streptococcus faecalis and Clostridium species can be taken as an indicator of pollution. They do not multiply in water, but survive for longer periods than the pathogens and can be readily isolated from water. Hence the coliform count is carried.out to determine the presence of faecal contamination. The faecal Strepfococci and C/. welchii tests are carried out to obtain additional evidence of faecal contamination and its duration.

Sampling The reliability of the analysis and the interpretation of the results depend largely on the correct manner in which the sample is taken. The sample should be a true representative of the supply and collected without extraneous contamination.

Collect the samples for bacteriological examination in clean sterilized bottles made of neutral glass of capacity 200-250 ml and provided with ground glass stoppers with an overlapping rim. Relax the stopper by an intervening strip of paper to prevent breakage during sterilization or jamming of the stopper. Protect the stopper and the neck of the bottle by a paper or parchment cover. If the water to be sampled

contains

or is likely to contain chlorine,

add a small quantity of sodium thiosulphate (0.1 ml of a 3% solution or a small crystal of the salt) to the bottle before sterilization. Do

not open

sampling bottle until the moment at which it is required for filling.

Bacteriological Examination of Water

657

Collection of sample from a tap : When the sample is to be taken from a tap in regular use, open the tap fully, and allow the water to run for at least two minutes to flush the interior of the nozzle and to discharge the stagnant water in the service pipe. If the tap has not been in regular use, sterilize the tap by heating it either with a blow-lamp or with an ignited piece of cotton soaked

in methylated spirit, until it is unbearably hot to the touch. Cool the tap by allowing the water to'run before collecting the sample. Fill the sample bottle from a stream of water from the tap, avoiding splashing. Avoid collection of samples from leaky taps as the water which might run down the outside of the tap may enter the bottle and cause contamination. If this cannot be avoided, take special precautions to clean the outside of the tap and to flame it sufficiently to ensure sterility. Collection of samples from rivers, lakes, reservoirs, and wells, etc.: Samples from rivers and streams should not be taken too near the bank or too far away from the point of draw-off. For collecting samples directly from rivers, lakes, tanks, wells, etc., attach a string to the neck of a bottle, wrap completely in paper and sterilize. Before taking the sample, remove the paper cover and take care not to allow the sides of the bottle to come in contact with anything. Tie another long clean string to the end of the sterilized string, lower the bottle into the water and allow it to fill up. Then raise the bottle and replace the stopper. Alternatively, hold the bottle at the bottom and plunge it neck downwards below the surface of the water. Then turn the bottle until the neck points slightly upwards and direct the mouth towards the current. If no current exists, as in a reservoir, create a current ‘artificially by pushing the bottle horizontally forward away from the hand. When full, raise the bottle rapidly above the surface and replace the stopper. If the sample isto be taken from a well fitted with a pump, pump the water to waste for about two min and then collect the sample from the pump delivery ot from a tap on the discharge. Transport and storage of samples: The bacteriological examination of the sample should be commenced as soon as possible after collection. Whére this is not feasible, keep the sample in ice until it is taken up for analysis. Analyse all such iced samples within 48 hr after collection. Samples not preserved in this manner are not suitable for bacteriological examination for hygienic quality. Plate Count

The procedure for the quantitative determination of bacteria in water consists in mixing a definite amount of the specimen of water with a sterile, solidifiable culture medium and incubating it for a specified time to permit the formation of visible colonies which may be counted.

APPARATUS

;

.

1. Nutrient agar sterilized in 15 ml quantities in plugged tubes.

658

Analysis of Fruit and Vegetable Products

2. Sterilized Petri dishes of about 10 or 12 cm diameter. 3. Dilution bottles each containing 90 ml of quarter-strength Ringer’s solution or tubes each containing 9 ml of sterile quarter-strength Ringer’s solution

(see page 668) 4. Sterilized 1 ml and 10 ml pipettes. PROCEDURE Prior to use, place the required number of nutrient agar tubes in boiling water until the medium completely melts and then cool to 45°—50° C. Allow the medium to remain in the liquid state at this temperature. Shake the sample vigorously twenty-five times in order to distribute the bacteria fairly evenly throughout the sample. Remove the stopper and flame the mouth. The quantities of the sample taken for test vary according to the anticipated purity of the water. When the water is expected to be unpolluted, withdraw 2 ml from the bottle with a sterile pipette and introduce into the sterile Petri dish. If the water is expected to be polluted, prepare decimal dilutions of the sample, withdrawing 1 ml of the wel!-shaken sample and adding

it to 9 ml of quarter-strength Ringer’s solutior, and so on, using a fresh sterilized pipette for each dilution and ensuring thorough mixing. Inoculate at least two Petri dishes with 1 ml each of the different dilutions. Add not less than 10 ml of melted nutrient agar at 42-45°C to the water in the Petri dishes.

Mix the medium with the sample in the dishes by a combination of rapid to and fro and circular movements of the dishes, keeping them flat on a table. After mixing, leave the plates on the table until the medium solidifies, and then place them inverted in the incubator. Incubate at 37° C for 24 + 3 hr.

RESULTS

Remove the plate from the incubator after 24 hr incubation and count the colonies with the aid of a magnifying glass. A suitable colony counter may

be used. The best plates to count are those with 30-300 colonies. colonies in these. In plates with

less than

Count all the 30 colonies, count the plate

inoculated with the undiluted water. Count each spreader as one colony. Discard plates with spreaders covering half of the plate or more. In'such cases, where all the plate counts for the diluiions inoculated are over 300,

a fair approximation may be obtained by counting the colonies in a few sectors and calculating the total. Multiply the observed count by the dilution factor and record the results to only two significant figures. Report the result as “plate count per ml at 37° C in 24 hr.” Interpretation : The bacterial colonies growing on nutrient agar are largely derived from soil, dust, sewage and other extraneous materials that have gained access to the water.

The number thus indicates the ‘‘cleanness” or

“dirtiness” of the sample. The presence of a variety of colonies of different sizes and shapes, presenting a disagreeable appearance and an ynpleasant

Bacteriological Examination of Water

659

odour, is suggestive of undesirable pollution. Surface waters generally show a high plate count, especially after rainfall following a period of dry weather. High counts may also be obtained with waters from newly sunk wells which gradually decrease with continuous pumping. A rise in the: plate count of a deep well or treated water may afford an early indication of contamination. A properly filtered and chlorinated water supply will not show a total count exceeding 10 per ml. The determination of variations from a normal value of the plate count for any water furnishes évidence of pollution and fall in the efficiency of the purification processes.

Coliform

Count

The coliform count involves three tests : (i) Presumptive test, (ii) confirmed test, and (iii) completed test. Presumptive Test

E. coli is one of the few bacteria which is able to ferment lactose with the production of acid and gas. If acid and gas are produced in lactose broth inoculated with the water being tested, this becomes a “‘presumptive”’ evidence of sewage pollution. Several non-intestinal bacteria will also produce this result. Therefore, a presumptive test must be “confirmed.” These various tests have been developed as “Standard Methods.” MATERIALS

1. Lactose broth i. Five large test tubes containing 10 ml of double strength lactose broth and inverted Durham tubes. ii. Two test tubes containing 10 mI of single strength lactose broth inverted Durham tubes. 2. Sterile pipettes, 1.1 ml and 10 ml

3. Water samples (known to be contaminated in some degree with intestinal matter) PROCEDURE

Using a 10-ml pipette, transfer 10 ml portions of the water being tested to each of the tubes containing double strength lactose broth. Into the two single strength lactose broth tubes, place 0.1 and 1 ml of water respectively. Incubate at 37° Cand examine after 24 and 48 hr. Report as positive any tubes in which 10% or more of the volume of the Durham tube is occupied by gas. Gas may be found in all the tubes or only in some. Gas in all tubes including those receiving 1and 0.1 lof water indicates gross contamination. On the other hand, a sample showing only one positive 10-ml tube should be classed as questionable. Record the results indicating the probable degree of pollution presumed to be present in the sample.

660

Analysis of Fruit and Vegetable Products

Confirmed Test The materials required for the test are: 1. Pour plates of sterile eosin-methylene

blue

agar

(EMB

agar)

(Levine’s).

2. Tubes of brilliant green bile broth (2%) with inverted Durham tubes.

PROCEDURE

Select a positive presumptive tube, preferably the highest ition showing gas (0.1 or 1 if these are positive) or a tube showing gas in 24 hr in preference to one which shows gas only after 48 hr. Using a straight inoculating needle, flame and then insert into the positive tube holding it in a slanted position to avoid picking up any scum or surface membrane. Streak on the EMB agar plate. Flanie the inoculating needle again and streak a second time to ensure a distribution giving discrete colonies. Incubate at 37° C and examine at the end of 24 hr. Typical E. co/# colonies

will have dark to black centres, button-like in appearance, and will often be surrounded by a greenish metallic shine. After streaking the EMB agar plate, inoculate a loopful from-the same tube, or another positive tube of the series,

into a tube of the brilliant green lactose bile broth. Incubate at 37° C for 24 hr. Appearance of gas is considered confirmatory evidence of the presence of E. coli. Examine plate and broth culture and report colonics on plate as typical E. coli colonies present. Report also any typical colonies. These may be Aéerobacter aerogenes ot intermediate coliforms. Completed

Test

Under some conditions a completed test may be made, particularly if the positive confirmatory test does not give clear-cut results. MATERIALS

1. Nutrient agar slants. 2. Tubes of single strength lactose broth with Durham tube PROCEDUKE

Transfer a well isolated, typical or atypical E. coli colony from the EMB agar

plate to a nutrient agar slant and a tube of lactose broth. Incubate both for 24 hz at 37° C. Make a stained mount of the nutrient agay culture. A completed test should show a pure culture of Gram-negative short rods, and gas should be produced in the lactose broth tube.

Faecal Streptococci Test The genus Streptococcus constitutes a large and diverse group of cocci widely . distributed iin nature. They are spherical, 0. 6-1 p in diameter, and _tend to

Bacteriological Examination of Water

661

be arranged in chains of varying length. They.are non-motile, non-sporing and are Gram-positive. Most species are aerobic and facultatively anaerobic; some

are dangerous pathogens. Those found in faeces are called faecal Strepsococei or Exterococci of which Streptococcus faecalisis typical. Streptococci usually occur in pairs of ovoid cocci, or in short chains. Unlike many other Streptococci, these grow well in ordinary laboratory media in the presence of bile salt. They produce deep red pin-point colonies on MacConkey agar and ferment glucose, lactose and mannitol, producing acid but no gas. While most Streptococci are susceptible to destructive agents, such as heat (55° C for 15--20 min), Sreptococcus faecalis offers a relatively high resistance to heat and can withstand a temperature of 60° C for 30 min. In examining water for the presence of faecal Streptococci,

advantage is taken

of its relative

heat resistance,

its ability to

form acid in MacConkey broth, and grow in the presence of a concentration of sodium azide sufficient to prove inhibitory to most coliform bacteria. Direct azide method : Inoculate tubes of Hanny and Norton’s sodium azide medium with various volumes of the sample or dilutions of the same volume made in quarter-strength Ringer’s solution as in the presumptive coliform test. Incubate at 44—45° C and examine the tubes at intervals of 18,. 24 and 48 hz.

The production of acid in the medium, which is shown by a yellow colouz, indicates the presence of faecal Strepfococci. Those tubes which turn yellow after incubation for a period over 18 hr may contain other organisms capable of producing acidity such as the anaerobic spore-forming baci//i, Confirmatory test for the presence of faécal Streptococci is, therefore, essential.

Confirmatory Test Plate a heavy inoculum from the positive azide tubes on MacConkey agar as soon as acidity is noticed in the azide broth tube. Incubate the plates at 37° C for 48 hr. The growth of minute pin-point red colonies on the plate is a strong evidence of the presence of faecal Streptococci. Pick individual colonies, Gram-stain, and examine

under

the microscope.

The Streptococci

appearas Gram-positive short chains. The faecal Strepsococci occurin faeces in comparatively smaller numbers than the coliform bacteria. The value of the faecal Streptococcz test lies in the fact that these organisms when found in water afford confirmatory evidence of faecal contamination and assist in interpreting the results of the coliform test. The faecal Streptococci survives in water betterthan the E. co/. Therefore, the

faecal Streptococci test also enables the significance of the presence of coliform organisms other than E. co/i to be more readily interpreted. If H. coli is absent and Streptococcus faecalis and the other members of the coliform group are. present, it is strong evidence of faecal pollution. But if E. co/i and Streptococcus faecalis are both absent, the presence of a small number of other coli~: form organisms may be disregarded.

662

Analysis of Fruit and Vegetable Products

Coliform and Faecal Coliform Count by

Membrane Filter Technique Membrane filter may be used to filter bacteria from water and other materials. The trapped bacteria are then grown on the filter by placing it on suitable growth medium. By the use of a selective medium, the number of coliforms and certain other species of bacteria in the sample can be determined. The technique enables direct isolation of particular organisms, e.g., salmonella, even when these organisms are present only in small numbers. Principle Total Coliform Count

A measured volume of the sample is filtered through a thin 150 um bacteriaretaining membrane. The bacterial cells are trapped on the surface. The membrane is then transferred onto a thin absorbent pad which is saturated with the particular medium designed to grow or permit differentiation of the organisms sought. For example, a modified Endo medium is used if coliform organisms are sought. The pad is incubated at 37° C for 18-24 hr in a small Petri dish after which colonies are counted under low magnification. The success of the method is dependent upon the use of effective differential or selective media that will allow easy identification of colonies. This method has advantages over the traditional methods described earlier in being more direct,

quicker (giving results in 18-24 hr), and enables the use of larger volumes of sample which renders the results more representative. EQUIPMENT

Use a seamless funnel which fastens to a receptacle bearing a porous plate for support of the filter membrane. The funnel should be so designed as to be suitable for attaching to the receptacle by means of a convenient locking device. The construction should be such that the membrane filter should be securely held on the porous plate of the receptacle. Without mechanical damage, all the fluid should pass through the membrane during filtration of the sample. Wrap the two parts of the assembly separately in heavy wrapping paper, sterilize by boiling, autoclaving

or ultra-violet radiation. For filtration, mount the receptacle of the filter holding assembly to a filtration (Buchner) flask. Have a trap between the flask and the vacuum source.

Use filter membranes which provide full bacterial retention, stability in use, free from chemicals which are inimical to the growth of organisms, and satisfactory with respect to speed of filtration. For sterilization of the filter membranes, remove the paper separators, and not the absorbent paper pads, place 10 to 12 pads in 10 cm diameter Petri dishes or wrap in paper and sterilize by autoclaving at 15 psig for 10 min. | . Use high grade filter paper free from sulphites or other substances which are inimical to the growth of the organisms. Cut the filter paper into circles of 4.8.cm

Bacteriological Examination of Water

663

diameter and of thickness sufficient to absorb 1.8 to 2.2 ml of nutrient. Sterilize

them along with filter membranes. PROCEDURE.

Sterilize the filtration assembly before use. Attach the filter holder to the rubber stopper, insert in the vacuum flask, and connect the flask to the vacuum source. Using sterile forceps, transfer a sterile filter pad to the platform base of the filter unit. Place the filter with rules side up. Place the matched funnel unit over the filter disc, making sure it is clamped in place firmly by the scissors type clamp. Pour the sample into the funnel and filter by maintaining a slight vacuum. Rinse the funnel with two 30 ml portions of, sterile water. Add 1.5-2 ml of an enrichment medium lauryl! tryptose broth to a sterile absorbent pad (filter paper) in a sterile culture plate to saturate the pad, and carefully remove any surplus

liquid. Aseptically remove the filter membrane from the filtration unit, and place on the filter paper soaked with medium in such a way as to exclude any air between the two discs. Incubate the filter membrane in the enrichment medium for 2 hr at 37° C. Place a fresh sterile absorbent pad ina sterile Petri dish (60 X 15 mm, flat), and pipette onto it 2 ml of modified Endo medium. Transfer the filter disc to the surface of the fresh pad enriched with Endo medium with the help of a sterile forceps. Take care to exclude air. Invert the dish, and incubate at 37° C for 24 hr. In place of liquid medium, if agar-based medium (LES Endo Agar) is used, take out the Petri dishes from the refrigerator, remove the filter membrane

from

the enrichment pad, and roll on to the surface of the agar taking care to prevent entrapment of air. Invert the dish and incubate as before. Counting of Colonies on Filter Pads

A direct estimation of the number of coliform organismsismadeby counting all dark colonies having a greenish, metallic-appearing surface lustre. Counts should be made under a low-power lens (10-15 diameters) and by reflected light. CALCULATION

Total coliform colonies _ coliform colonies counted X 100 per 100 ml ml of sample filtered

The same

method

(through slight variation

in technique and sampling

equipment) has wide potential application in foods, industrial bacteriology and air contamination. Faecal Coliform Bacteria

If the total coliform count is made by membrane filter technique, the faecal coliform count may also be made by the same procedure. Procedure being the same, enriched lactose medium

temperature of 44.5 + 0.2° C.

should be used, and the dishes incubated at a

664

Analysis of Fruit and Vegetable Products

The procedure requires the use of tight fitting 60 X 15 mm plastic dishes. These

may be sterilized by immersion in 70% alcohol for 30 min or by ultra-violet radiation. If sterilized using alcohol, wipe witha sterile techniques.

sterile towel, and close employing

PROCEDURE

The volume of water sample chosen should yield a countable membrane, and in addition, select two additional quantities representing one-tenth and ten times this quantity, respectively. Sample quantities yielding between 20 and 60 faecal coliform colonies result in greater accuracy of density determination. Prepare the assembly and filter as described before. Place a sterile absorbent pad in each culture dish, and pipette approximately 2 ml of M-FC medium. Carefully — remove the surplus liquid from the dish. Place the filter membrane through which the water has been filtered on the pad impregnated with the medium, and cover

the dish. Place the dishes inwater-proof bags for protection, submerge the bag below the water level in a bath maintained at 44.5° + 0.2° C, and incubate for 24

hr. Incubation must be started within 30 min of filtration. The colonies of faecal coliform have blue colour while the non-faecal colonies are coloured grey to cream. The background colour of the filter membrane varies from

a yellowish cream to faint blue depending upon the age of rosolic acid salt reagent. Usually, few non-faecal coliform colonies are observed on M-FC medium because of the elevated temperature used for incubation and addition of the rosolic acid salt reagent to the medium. Count the colonies with the aid of a low-power (10-15 magnifications) binocular wide-field dissecting microscope. The number of colonies should be in the range of 20-60 because of larger colony growth on M-FC medium. Calculate as in the coliform count, and report as faecal coliforms per 100 ml. Reference

Standard Methods for the Examination of Water and Wastewater, American Public Health Association, 1015, 18th Street, N.W. Washington, D.C. 20036, 13th edn., p. 678 (1971),

Preparation of Culture Media and Reagents Nutrient Agar

The materials needed are yeast extract or meat extract 3 g, peptone 5 g, agar (washed, shredded or powdered) 25 g and distilled water 1,000 ml. Dissolve the yeast or meat extract and peptone in distilled water on a water bath and cool. Adjust the pH to 7.4, using phenol red as the indicator. Weigh the agar. If not shredded, cut into pieces, tie ina muslin bag, wash in running water for 15 min, squeeze out excess water, and add to the yeast peptone

mixture, Autoclave at 15 psig for 20 min, and immediately filter through paper pulp in a Buchner funnel or through a wad of cotto .

Bacteriological Examination of Water

665

Test the pH of thefiltrate at 50° C and adjust the pH,if necessary, to 7. Add 15 ml quantities to each tube, plug the tubes and autoclave at 15 psig for 20 min. The final pH of the medium at room temperature should be 7.2. When required for use, place the tubes in boiling water till the agar completely melts and then cool to 42—45° C. Unless the tubes: are used within one week of preparation, store in the cold storage (refrigerator) to retain the moisture in the medium. Lactose Broth

Take beef extract 3 g, peptone 5 g, and distilled water 1,000 ml. Heat slowly in a water bath stirring until solid materials are dissolved. Add 5 g of lactose and make up the volume to1,000 ml with distilled water. Adjust the pH of the medium so that after sterilization it is between 6.8 and 7.0. Transfer to fermentation tubes containing Durham tubes and sterilize at 15 psig for 15 min. Remove from autoclave and cool rapidly. Repeat this procedure on three successive days. The total time of exposure to heat in the autoclave * should not exceed 60 min.

MacConkey Broth (Single Strength) Mix commercial sodium taurocholate or sodium tauroglycocholate 5 g, peptone 20 g, sodium chloride 5 g and distilled water 1,000 ml. Steam for 2 hr, cool, and keep in the refrigerator overnight. Add 10 g of. lactose and when it dissolves, filter throuph a filter paper while still cold. Adjust the pH to 7.4 using phenol red as the indicator. Add 1 ml of a 1% alcoholic solution of bromocresol purple, or 5 ml of 1% aqueous solution of neutral red and transfer 5 ml each into 6 x 5/8 in. test tubes provided with Durham fermentation tubes. Sterilize in the autoclave at 10 psig for 15 min. MacConkey Broth (Double Strength)

Prepare in the same way as the single strength MacConkey broth using double the quantities of the ingredients for the same quantity of water. Transfer 10 ml of this medium into each 6 x. 3/4 in. test tube. If 50 ml quantities of water are to be tested, 50 ml of the double strength broth should be put

into tubes or bottles of suitable size. Sterilize at 10 psig for 15 min. Brilliant Green Lactose Bile Broth

Prepare solution A by dissolving 10 g each of peptone and lactose in 500 ml of water. Dissolve 20 g of dchydrated ox-gall in 200 ml of distilled water and adjust the pH of the solution between 7 and 7.5 (solution B). Mix solutions A and Band make up with distilled water to approximately 975 ml. Adjust pH to 7.4. Add 13 ml of 0.1% solution of brilliant green in distilled water and make up to 1000 ml. Filter through cotton, distribute in 5 ml quantities in fermentation tubes (6 x 5/8 in.) and sterilize at 10 psig for15 min. The PH should be not less than 7.1 and not more than 7.4.

666

Analysis of Fruit and Vegetable Products

Eosin Methylene Blue (EMB) Agar Mix peptone 10 g, dipotassium phosphate 2 g, and agar 15 g, with 1,000 ml of distilled water. Note the volume by marking with a glass marking pencil. Heat on a water bath until dissolved and make up loss due to evaporation with distilled water. Transfer aliquots (100 or 200 ml) into flasks or bottles and sterilize in the autoclave at 15 psig for 15 min. Just before use, add 5 ml of 20% sterile lactose solution, 2 ml of 2% aqueous eosin yellowish solu-

tion, and 1.3 ml of 0.5°%% aqueous methylene blue solution to each 100 ml of the melted agar. Alternatively, all ingredients may be‘added to the stock agar at the time of

preparation, placed in tubes or flasks ‘and sterilized. Decolourization occurs during autoclaving, but the colour reappears on cooling.

MacConkey’s Agar Mix commercial sodium taurocholate or sodium tauroglycocholate 5 g, peptone 20 g, NaCl 20 g, and washed shredded agar 25 g with 1,000 ml of distilled water. Steam until the solids dissolve. Cool to 50° C and, at this temperature, adjust the pH to 7.6—7.8. Add egg white (well-beaten white of one egg for 3 litres of the medium), autoclave at 10 psig for 15 min, and filter while hot through a good grade filter paper. Adjust the pH of the filtrate to 7.3 at 50° C or 7.5-at room temperature. Add 10 g of lactose and 5-10 ml of a1% neutral red solution. Mix thoroughly, distribute into flasks or sctew-capped bottles and sterilize in the autoclave at 10 psig for 15 min. For use, melt by heating on a water bath and pour into Petri dishes using 15 ml for each dish. Formate Ricinoleate Broth

Add 5 g each of peptone, lactose, sodium formate, and sodium ricinoleate to 1,000 ml of distilled water and dissolve by heating slowly on a water bath

stirring constantly. Add distilled water to make up to 1 litre. Adjust the PH so that after sterilization it will be 7.3-7.5. Distribute into fermentation tubes and autoclave at 10 psig for 15 min. Sodium Azide Medium

Dissolve 10 g of peptone, 5 g each of dipotassium phosphate, NaCl and glucose; 2 g of potassium dihydrogen phosphate; 3 g of yeast extract; 0.25 g of

sodium azide and 2 ml of 1.6% alcoholic solution of bromocresol purple in 1,000 ml of distilled water.

Transfer

5 ml to each tube and sterilize in the

autoclave at 10 psig for 15 min. For use with inocula of 10 or 50 ml of water, prepare a medium of double this strength and distribute in 10 and 50 ml quantities. Andrade Indicator

Dissoive 0.5 g of acid fuchsin in 100 ml of distilled water. Add 17 ml of 1 N NaOH solution and allow to remain overnight at room temperature. The colour of the solution should be straw yellow. If brownish, add a little

Bacteriological Examination of Water

667

more 1 NV NaOH and allow to stand again. This solution is highly alkaline. So the media to which it is added should be standardized previously to a pH of about 6.8. Glucose Phosphate Medium

Mix 5 g each of peptone and dipotassium hydrogen phosphate with 1,000 ml of distilled water. Steam until the solids are dissolved, and filter while hot

through a good grade of filter paper. Adjust at room temperature to pH 7.5. Add 5 g of glucose, mix well and distribute 4 ml quantities into 15x1.5 cm test tubes. Autoclave at 10 psig for 10 min. Test for sterility by incubation

at 37° C for 24 hr.

:

Lauryl Tryptose Broth Dissolve 20 g of tryptose, 5 g of lactose, 2.75 g each of dipotassium hydrogen phosphate (K2HPO,) and potassium hydrogen phosphate (KH2PO,), 5 g of NaCl

and 0.1 g of sodium lauryl] sulphate in 1,000 ml of water. Adjust the pH so as to be approximately 6.8 after sterilization. Transfer 10 ml to each tube and sterilize at 10 psig for 15 min. Endo Medium

Weigh 10 g of tryptose or polypeptone, 5 g of thiopeptone or thiotone, 5 g of cositone or trypticase, 1.5 g of yeast extract, 12.5 of lactose, 5 g of NaCl, 4.375 g of dipotassium hydrogen phosphate (K2HPO,), 1.375 g of potassium dihydrogen

phosphate

(KHe2PO,), 0.05 g of sodium lauryl sulphate, 0.1 g of sodium

desoxycholate, 2.1 g of sodium sulphite and 1.05 g of basic fuchsin. Dissolve in 1

litre of water containing 20 ml of 95% ethanol. Heat the medium to boiling point, promptly remove from heat, and cool to below 45° C. Do not sterilize by autoclaving. The final pH should be between 7.1 and 7.3. Store the finished medium in the dark at 2-10° C, and discard the unused medium after 96 hr.

LES Endo Agar Weigh 1.2 g of yeast extract, 3.7 g of casitone or trypticase, 3.7 g of thiopeptone or thiotone, 7.5 g of tryptose, 9.4 g of lactose, 3.3 g of dipotassium hydrogen phosphate (KgHPOs,), 1.0 g of potassium dihydrogen phosphate (KH2PO,), 3.7 g_

of NaCl, 0.1 g of sodium desoxycholate, 0.05 g of sodium laury] sulphate, 1.6 g of sodium sulphite, 0.8 g of basic fuchsin and 15 g of agar. Rehydrate in 1 litre of distilled water containing 20 ml of 95% ethanol. Bring to a boil, and cool to 45-50°

C. Pipette 4 ml each into 6 cm diameter Petri dishes. To dishes of other size, add sufficient volume to give an equivalent depth. Store in the dark at 2-10° C. The plates may be stored up to 2 weeks. Do not expose the plates to direct sunlight.

M-FC Broth Weigh 10 g of tryptose, 5 g of proteose peptone No. 3 or polypeptone, 3 g of

668

Analysis of Fruit and Vegetable Products

yeast extract, 5 g of sodium chloride, 12.5 g of lactose, 1.5 g of bile salt No.3, or bile salts mixture and 0.1'g of aniline|blue. Dissolve in 1 litre of water containing 10 ml of 1% rosolic acid in 0.2

N NaOH. Heat the medium to the boiling point, and

cool quickly to 45°C. Do not autoclave as the rosolic acid decomposes. The final pH should be 7.4. Store the finished medium at 2-10° C, and discard the unused

medium after 4 days.

4

.

The stock solution may be stored at 2-10° C for 2 weeks. Discard earlier, if the

colour changes from dark red to muddy brown.

Ringer’s Solution (Full Strength) Prepare a solution of NaCl 9 g, potassium chloride 0.42 g, calcium chloride 0.48 ¢ and sodium bicarbonate 0.2 ¢ in 1,000 ml of distilled water. To

prepare one-quarter strength solution, dilute 250 ml of thissolution with 750 ml of distillcd water and sterilize by autoclaving at 15 psig for 20 min. References

1.

Manual of Methods for the Vixamination of Water, Sewage and Industrial Wastes, Special Report No. 47, Indian Council of Medical Rescarch, New Delhi (1963).

2. Salle, A.J., Fundamental Principles of Bacteriology., 6th edn., MCGraw-Hill Book Co., New 3.

York, p. 528 (1967). Standard Methods for the Examination of Water and Wastewater, Health Association, Washington, D.C. (1971).

13th edn., Amcrican Public

4. Taylor, E.W., The Examination of Water and Water Supplies, 7th edn., J. & A. Churchill Ltd., London, p. 426 (1958).

CHAPTER 22

Assessment of Surface Sanitation

BACTERIAL

counts on utensils, equipment, working surfaces, walls, floors

etc., are uscful means of assessing the standard of hygiene and the efficiency of cleaning procedures in food factories. The “Swab Rinse Method” may be

used for this purpose. Cotton wool swabs : Wind non-absorbent cotton wool on wooden sticks or

glass rods about 7-8 in. long to form a swab of about 0.5 in. in diameter. Place in pairs in test tubes (6 X 1 in.) so that about 1 in. of the sticks projects from the tube. Plug with non-absorbent cotton wool and _ sterilize

in a hot-air oven for 90 min at 150° C. PREPARATION

OF MEDIA

Ringer solution : Dissolve NaCl (AR) 2.15 g, potassium chloride (AR) 0.075 g, anhydrous calcium chloride (AR) 0.12 g, and sodium thiosulphate 0.5 g in 1,000 mlof distilled water. Sterilize in an autoclave for 20 min at 15 psig. A 0.85% NaCl solution in water has the same osmotic pressure as microorganisms. Ringer solution used as diluent is ionically balanced. The toxic

effects of the metallic ions neutralize one another and are better than saline for making bacterial strength.

suspensions.

It is used

at one-quarter of the original

Nutrient agar : Mix Lab Lemco 10 g, peptone 10 g and NaCl (AR) 5 g with 1,000 ml of distilled water. Autoclave at 5 psig for 10-15 min and adjust the PH to 8. Filter if phosphates are precipitated. Add 1% agar, steam or autoclave to dissolve, bring pH back to 7.4, and bottle. Autoclave at 10 psig for 10 min. Yeast extract agar: The ingredients

required are given on page 670. The

medium is used for plate counts on food, water, etc.

Heat and dissolve the ingredients and adjust the pH to 7.3. Filter, if necessary.

Bottle or tube and autoclave at 10 psig for 10 min.

670

Analysis of Fruit and Vegetable Products Routine work

Richer medium

(g)

(g)

=

5

Peptone

5

_

Yeast extract

r}

£58)

Dextrose

_

I

Agar

10

12

1,000 ml

1,000 ml

Tryptone

Water

MacConkey’s single strength broth : This is used to detect coliform bacilli in water samples, ctc. Other organisms are discouraged by the bile salt. Coliforms ferment the lactose and produce acid and gas. The acid changes the colour of the indicator and the gas produced collects in the Durham’s tube. Mix sodium tauroglycocholate 5 g, peptone 20 g and NaCl 5 g in 1,000 ml of water. Dissolve by steaming. Add 20 g of lactose and dissolve. Adjust the pH between 7.0 and 7.5. Filter, if necessary. Add about 10 ml of 1% aqueous neutral red to give a satisfactory red colour. Take 5 ml in each test tube with Durham’s tube and sterilize by autoclaving at 5 psig for 10 min. Avoid excess of heat after adding lactose to prevent hydrolysis of the sugar. MacConkey’s double strength broth : Mix 10 g each of sodium tauroglycocholate

and NaCl, and 40 g of peptone with 1,000 ml of water and proceed as for the single strength broth. Add 40 g of lactose and 20 ml of 1% neutral red solution. Add 10 ml to each test tube (6 x 0.75 in.) and 50 mil for each bottle (4 oz) with Durham’s tubes in each. Add 10 and 50 ml of sample to tube and bottle respectively which reduces the medium to single strength. Instead of neutral red, some prefer to use bromocresol purple (1.5%) for MacConkey’s broth.

Add sufficient dye solution so that its concentration is

0.04%. MacConkey’s agar for roll ‘nbes : Add agar 12 g, sodium tauroglycocholate 7.5 g, peptone 20 g and NaCl 5 g to 1,000 ml of water and dissolve by heating. Adjust the pH to 7.6. Then add 20 g of lactose and about 10 ml of 1% neutral red. Sterilize by autoclaving at 5 psig for 10 min. The coliform bacilli ferment lactose and give pale pink or slightly yellow colonics. MBS medium : Mix Lab Lemco

10 g, yeast extract 5 g, peptone 10 g, di-

potassium hydrogen phosphate 2 g, sodium acetate 5 g, magnesium sulphate (MgSO,.7H,O) 0.2 g, manganese sulphate (MnSO,.4H1,O) 0.05 g and Tween 80, 1 ml with 1,000 ml of water. Dissolve by heat, bottle and autoclave. For use, melt and add 2% sterile (filtered) dextrose solution. This medium is

Assessment of Surface Sanitation

used for the growth organisms.

of Lactobacilli and

its low pH

inhibits

671

ayy other

Examination?.*

Dip a swab in sterile Ringer solution and rub overa definite area of the surface to be tested. Use one swab for a predetermined area of five such articles, like a cutting table, chopping board and both sides of knives, ladlcs, etc.

Return this swab to the tube and swab the same surfaces again with another dry swab.

To the tube containing both swabs, add 10 ml of sterile Ringer solution. Shake and allow to stand for 20--30 min. Make plate counts with 1 and 0.1 ml amounts using yeast extract agar. Divide the count per millilitre by 5 to obtain the count per article. Inoculate three tubes of single strength MacConkey’s broth with 0.1 ml amounts. Coliform organisms should be absent. Rinse Method

For bins and large utensils, add 500 ml of sterile Ringer solution to the vessel. Rotate the vessel to wash the whole of the inner surface and then transfer the rinse into a screw-capped jar. Make counts with 1 and 0.1 ml amounts of

the rinse in duplicate. Incubate one pair at 37°C and the other at 22°C for 24 hr. Take the mean of the 37 and 22° C counts 500. The product gives the count per container.

per ml and

multiply by

If bottles are sampled after they have been washed through a washing machine where a row of bottles travels abreast, test all the bottles

in one such

row. This shows if one set of jets or carriers is out of alignment. In any case, do not examine less than six bottles. Cap or stopper them immediately. To each bottle, add 20 ml of sterile Ringer solution. Close with sterile rubber corks and roll the bottles on their sides so that whole of the internal surface is rinsed. Leave them on their sides and roll at intervals of 30 min. Pipette 5 ml from each bottle into each of two Petri dishes. Add 15—20 ml of yeast extract agar, mix

and incubate one plate from each bottle at 37° C and one at 22° C for 48 hr. Pipette 5 ml from each bottle into 10 ml of double strength MacConkey’s broth and incubate at 37° C for 48 hr. Take the mean of the 37 and 22° C plate counts and multiply by 4. ‘This is the count per bottle. Find the average of the counts, omitting any figure whith is 25 times greater than others, which in-

dicates a possible fault in that particular line. ‘The figure obtained is the average colony count per container. No standards have been laid down for the beverage bottles. In the case of milk bottlcs, however, the UK Ministry of Agriculture and Fisheries (1947) Classification prescribes average colony counts of 200 or less as satisfactory; counts from 200 to 600 as fairly satisfactory, but over 600 as unsatisfactory. Coliform bacilli should not, however, be present in 5 ml of the rinse.

672.

— Analysis of Fruit and Vegetable Products

Use nutrient or similar agar for most bacteria, MRS medium for Lactobacilli,

RCM agar for Clostridia and buffered yeast agar for yeasts. Increase the agar concentratidn by 0.5%. Use the roll tube MacConkey’s\agar for coliforms.

Mclt the medium and cool to 55° C. To quart or litre bottle, add 100 ml of medium and proportionally less to smaller bottles. Stopper the bottles with sterile rubber corks and roll them under a cold water tap to form a uniform film of agar all over the inner surface. Incubate vertically and count the colonies. For Lactobacilli and Clostridia, replace the bung by a cotton wool plug, and incubate

anaerobically.

Examination of Sink Waters

To study unhygicnic conditions and the necessity for frequent changes of washing water, take 100 ml of washing or rinse water by immersing a water sample bottle (containing sodium thiosulphate in case hypochlorites are used in washing) into the sink and allow it to fill. Stopper and cool undcr the tap. Carry out plate and coliform: counts. Test at the beginning and at intervals

during the washing. Swab Counts on Working Surfaces, Floors, Walls, etc.

These are used to assess hygienic conditions. Cut card or cellophane into 10 cm squarcs and again cut 5 cm squares in them. Sterilize these squares in envelopes. Place a square piece on the surface to be examined and swab the area within the 5 cm square with a cotton wool swab dipped in Ringer solution. Carry out plate connt’as described before. The count per 25 cm? is given

by the number of colonies per millilitre of rinse or solvent multiplied by 10, The Scotch Tape Method

Cut strips of scotch tape or similar material about 2 in. long from a reel 1 in. wide. Without handling the sticky surface, place it firmly on the surface to be counted. Peel off, press on the surface of agar medium in a Petri dish. Peel off and discard the tape. Incubate the culture medium and count colonies which grow from the transferred bacteria. References 1. 2.

De Man, J.C.,M. Rogosa, & M. E. Sharpe, J. Appl. Bach» 23, 130 (1960). Knox, R. & J. Walker, j. Hezg. Camb., 45, 187 (1947).

3. Gos) C.H., Microbiological Methods, 2nd edn., Butterworth & Co. Ltd., London, p. 347 1997

CHAPTER 23

Microbiological Examination of Spoilage CANNED

FRUITS AND VEGETABLES

MICROBIOIOGICAL spoilage in canned fruits and vegetables is due either to underprocessing or leakage. Underprocessing is the failure to destroy during the heat process all bacteria capable of subsequent growth in the product. Leakage is due to the contamination of the product after an adequate heat process, either due to faulty seam or damage to the canafter sealing. To clearly visualize the spoilage relationships, fruits and vegetables, according to the pH value, can be classified into four arbitrary groups (Table 23-1).

TABLE 23-1 : pH Classification of Foods

Class

Low acid

pH range

5.3 and higher

Products

Vegetables such as corn, peas, lima bean, asparagus, cauliflower,

potato, spinach, French

beans and beet.

Medium acid

5.3-4.5

Cabbage, turnip, pumpkin, carrot, okra, green

beans, beet, etc., and products like soups and sauces, Acid

4-5-3-7

Tomato, pear, pineapple, swcet cherry, banana,

mango, apple, jackfruit, peach and other fruits High acid

3.7 and lower

Sauerkraut, citrus juice, rhubarb, prunes, etc.,

and pickles, chutncys, etc.

Organisms Causing Spoilage

The organisms commonly involved in the spoilage of canned foods have

definite relationship to these four acidity groups as shown in Table 23.2. There are two groups of spoilage organisms—a thermophilic group and a mesophilic group. In each group, at least three types of organisms have been found to produce spores highly resistant to heat.

674

Analysis of Fruit and Vegetable Products TABLE 23-2 : Microbiological Spoilage Relationships in Canned Foods pH Group

Spoilage type

Product

Thermophilic Flat sour*

5.3 and higher

Corn,

peas

Thermophilic anacrobe*

4.8 and higher

Spinach, corn

Sulphide spoilagc*

5.3 and higher

Corn, peas

Mcsophilic Putrefactive anacrobes*

wer and higher

Corn, asparagus

Butyric anacrobcs*

4.0 and higher

Tomatoes, pears

Aciduric flat sours*

4.2 and higher

Tomato

Lactobacilli

4-5-3-7

Fruits

Yeasts

3.7 and lower

Fruits

Moulds

3.7 and lower

Fruits

juice

*Spore-forming bacteria.

In most canned fruits, the pH of which ranges from 3.7 to 4.5, aerobic and anaerobic spore bearers may cause spoilage, but this is not common. Lactobacilli and Leuconostocs have been reported. The butyric anaerobes, which af-

fect tomatoes, and the aciduric flat sours found in tomato juice are important in this category. No spore formers have been found to spoil highly acidic canned fruits (pH. < 3.7). Osmophilic yeasts and the mould Byssochlamys are sometimes found. Spoilage is rare in high acid canned fruits, fruit juices or pickles with a pH of 3.7 or less. In severe underprocessing, yeasts may occur. In low acid foods such as canned soups, vegetables, etc., the pH of which is 4.5 or more, one of the following thermophiles is usually found in spoilage due to underprocessing.

1. B. stearothermophilus causing flat sour spoilage. 2. Cl. thermosaccharolyticum causing hard-swell. 3. Cl. nigrificans causing sulphur stinkers. 4, Mesophilic spore bearers, putrefaction.

obligate or

facultative

anaerobes

causing

Leaker spoilage in products with normal pH values above 4 may be due toa variety of organisms: aerobic and anaerobic spore formers, Gram-negative nonsporing rods and various cocci including Lesconostoc. and micrococci

may be found. The presence of micrococci and/or yeasts is almost certain evi-

dence of leakage.

Microbiological Examination of Spoilage

675

Investigation and Diagnosis In any investigation of spoilage causes, note aa: Extent of spoilage. Zi Number of.cans involved. Be Ingredients used in the case of a formulated product. 4. Canning procedure used, including the duration and the temperature of the process. . Cooling method employed, speed of cooling and the temperature to which cooled.

. Storage period at the time of spoilage. a . Temperature of storage at which spoilage occurred. . Any deviation from the normal procedure which might correlate with the outbreak of spoilage.

Sampling of Cans When a can is examined for sterility, a negative result on a particular sample is only significant for that particular sample and is not indicative of the whole batch. Unless the extent of spoilage is widespread, the number of cans required to detect spoilage is large. Scott,! Thomas and Cheftel,” and

Mossel

and

Drion*

have discussed in detail the subject matter of sam-

pling of cans for sterility.

:

In the examination of canned foods for spoilage which is widespread, if the cans are blown or swollen, examine 6 cans and take 6 normal cans from

another batch, as controls. In suspected underprocessing, examine 6-12 cans from each batch. Leaker spoilage is likely to occur in only a very small number of cans in a batch. Hence, examine as many as possible. Physical Examination

Examine

the cans and record the following information.

1. Name and nature of the product . Code mark . Container size . Gross weight . Physical condition of cans OND mb i. Mechanical defects—dents, particularly near the double scams ii, Pinholes or rusting

iii, Buckling or panelling iv. Can ends—flat, flipper, springer, soft swell or hard swell . External seam

dimensions Incubation

Incubate sound cans of fruits and vegetables prior to examination. Temperature and time of incubation are given below.

676

Analysis of Fruit and Vegetable Products

Fruits and fruit products (pH 4.5 and lower) Whole

tomatoes

37° C for 3 days At

55°C

for

certain rare

thermophilic

spoilage types if indicated or at 32°C if butyric anaerobes are suspected‘.

Vegetables and vegetable products (pH 4.5 and higher)

Some at 37° C and others at 55°C for 3 days

Incubation encourages the multiplication of a small number of surviving organisms which might otherwise be missed in the sampling of cans. American Public Health Association’ recommends an incubation period of 14 days. During incubation, examine the cans periodically and remove the swollen cans before they burst. After incubation, cool the cans to room temperature before opening.

Opening

the Container

Flat, flipper or springer cans : Clean the cans with soap and water, or with petroleum ether if greasy. Swab the top of the can with cotton wool and methylated spirit. Grasp the can in the hand, invert over a Bunsen burner and distribute the heat uniformly by making a circular motion. Puncture the flamed surface with a sterile can opener (Fig. 23.1) by giving a sharp blow on

Fig. 23.1. Tapered punch for cutting hole in the centre of the can.

the opener with a hammer. (Sterilize the opener before use by washing with

soap and water, swabbing with alcohol and flaming in the burner. ) Soft-swollen and hard-swollen cans: These cans,

if flamed,

may burst.

Wash

such a can with soap and water. Keep the can upright in a clean shallow tray

Microbiological Examination of Spoilage

. 677

of dilute hypochlorite to receive any material which may drip. Place a clean

cloth over the lid of the can. Pour disinfectant (5° phenol ora solution containing 200 ppm chlorine) liberally on the towel so as to cover the top. Allow the disinfectant to act for at least 15 min before opening the container® . On opening, due to pressure inside, the contents of the can may spew out and if contaminated with C/. botulinum, may be toxic. Flame the metal can opener (Fig. 23.1) as described in the case of normal cans. Keep the disinfectant towel in place firmly with one hand and puncture the container lid with the sharp can opener through the cloth. This will relieve the internal pressure. Then wipe the surface with the disinfectant towel and replace this. towel with another clean towel which has been soaking in the disinfectant. The container is now ready for opening and sampling.® Alternatively, a sharp metal can opener with a plate slightly above the sharp point (Fig. 23.2) may be used. The diameter of the plate should be slightly

Fig. 23.2. Opener for swollen cans

larger than the diameter of the can. Swab the can opener, pour 1-2 ml of methylated spirit, and flame before use. A large glass filter funnel, firmly plugged in the stem with cotton wool through which is passed. a thin steel tod with a sharp tip, may = used for opening the can. This gives excellent protection to the operator.’ Wrap the funnel/and the rod prepared as above in stout brown paper and sterilize. Invert the funnel over the previously cleaned and sterilized can. The sharp tip of the rod should rest on the can end. Give a blow on the blunt end of the tod, which will force the tip through the can. The funnel prevents the spewing contents from. contaminating the operator. In all these methods of opening

swollen cans, push the rod in and out

of the hole in the can several times before finally removing it to ensure that the hole is not blocked with a piece of food due to internal pressure.

678

Analysis of Fruit and Vegetable Products

Sampling of Can Contents The can may be opened in a number of ways for sampling of the contents, depending on the nature of the product.

Liquid products8: Cut a hole of about 0.5 in. diameter with a tapered punch as shown in Fig. 23.1. Sterilize the punch before use by flaming. While opening, hold a’ Bunsen flame near the opening. Remove the sample with a length of sterile glass tubing of 7-8 mm outside diameter or with wide-mouthed pipettes (20 ml capacity). Tubes are more suitable for semi-solid or viscous products. Inoculate directly into the media or transfer intoa sterile cottonplugged test tube for subsequent subculturing.

Solid or semi-solid products: Sterilize the opener and cut out a circular disc around the central puncture. With solid packs, remove a cylinder with a sterilized cork borer and expel from the borer with a rod,ifnecessary. Use a fresh

sterile borer for each can. In case of underprocessing, the organisms are most likely to be confined to the centre of the can while in the case of leakage, in the exterior ends of the can. In the former case, take the sample from the geometric centre of the can while in the latter, push the borer obliquely in several directions and collect the sample. (Instead of a cork borer, a spatula may also be used.) Remove 10-20 g of sample. Expel the samples from the borer or spatula into a wide-mouthed sterile bottle with a screw cap or conical flask containing 50 ml of water and a few glass beads. Plug the latter tightly with cotton. Shake to distribute the sample. Use this for subsampling.

Products containing liquid and solid constituents as in canned fruits and vegetables: Sample as in the case of liquid or semi-solid products. During the examination, to prevent contamination from air, cover the can with sterile Petri dish, sterile new can lid or disinfectant moistened towel.

Direct Microscopic Examination

After the contents of the spoiled can have been transferred into a sterile container for subculturing, the next step is direct microscopic examination. Make smears with a sterile inoculating loop. Stain with methylene blue or carbol fuchsin, which will give the general morphology of the organism(s) present. Also stain with Gram-stain. The presence of Gram-positive rods

| Suggests underprocessing, while cocci, yeasts, etc., suggest leaker spoilage. If spoilage is due to bacteria surviving heat process, not more than one ot two types would be present, usually only one, except in gross underprocessing. In the case of container leakage in products of pH above 4, the microflora would usually be a mixed flora. The presence of micrococci and/or yeasts is a sure evidence of leakage. The organisms seen may be dead or killed during processing. Normal unspoiled cans may contain nonviable organisms in large numbers. This may

be due to poor raw materials or development of the organism during the time

Microbiological Examination of Spoilage

679

lag between preparation and processing. The food may contain alarge number of microorganisms when examined microscopically but cultures may be sterile because of auto-sterilization of organisms. Such organisms stain poorly or unevenly and may show other signs of degeneration. pH Determination Determine the pH of normal and spoiled cans from the same lot using a pH meter with glass electrode. pH determination is of particular importance when flat-sour is suspected. Variations in pH, even as small as 0.15 to 0.2,

may often be significant. Product and Can Examination

Do not taste spoiled foods. Note the appearance, texture and odour. Determine net and drained weight of leafy vegetables and small diced vegetables as overfilling may diminish the rate of heat penetration during processing and result in underprocessing. As regards the cans, cut open the seam and examine

the cover hook and the body hook for any abnormalities (see Ch. 14) Gas Analysis When the can is swollen, but the microbial count is low, analyse the head-

space gas for the detection of hydrogen swells. The combustible nature of the gas can be demonstrated by making a small puncture in the can while being held adjacent to a flame or by a differential analysis using an Orsat type gas analyser. In the case of hydrogen swells, the internal surface of can would show signs of heavy corrosion. Culturing of Can Contents Proceed to culture the contents according to the nature

of the product

to be examined as given in Table 23-3, unless otherwise indicated by microscopic examination. If visual observations, PH and microscopic examinations indicate special type of spoilage like sulphide spoilage in vegetables or flat-sour spoilage in low-acid vegetables or tomato juice, or yeast or mould

spoilage in acid fruits, additional inoculation suggested (Table 23-4).

into

specific medium

As soon as growth appears, note

1. Visual appearance of growth 2. Presence or absence of gas, odour, etc. 3. Microscopic examination i. ii. iii. iv.

Morphology—stain with methylene blue or crystal violet Gram-positive or negative—by Gram staining Presence or absence of spores—by spore staining Purity of culture

is

Analysis of Fruit and Vegetable Products

680

TABLE 23-3 : Culturing of Can Contents Vegctables Low and medium acid ii (pH 4.5 and higher)

Fruits Acid products (pH 4.5 and below)

aa

Routine inoculation media—

Aerobic

Dextrose-tryptone broth with bromocresol purple

Orange serum broth or broth of test fruit

Anaerobic

Tryptone broth or liver broth

Orange serum liver broth with tomatoes

6 tubes

6 tubes

2 g or ml

2 g orml

476

2 tubes 2 tubes

2 tubes 2 tubes

55 C

2 tubes*

2 tubes*

Period of incubation

2-3 days

3 days

No. of tubes to be inocu-

agar or crushed

lated

Quantity of material to be used for inoculation

Incubation temperature Room temperature

Special media Suspected sulphide spoilage in canned vegetables

Sulphide agar Inoculate 4 tubes, incubate 2 tubes at

37° C and 2 tubes at 55° C* for 2-3 days Flat-sout spoilage in tomato juice

Proteose-peptone acid agar. Take4 plates. To two plates add

1 ml of juice and to another two add o.1 ml. Incubate 1 plate in each set at 37° C and the other

plate at 55° C. Yeasts and moulds

Malt extract medium

Osmophilic yeasts

Honey medium

Demonstration of spore formation

Sub-culture from the aerobic broth tubes on nutrient agar

slants and incubate at 37° C and 55° C for 3-4 days.

*Preheat all tubes intended for 55° C incubation to 55° C before keeping in incubator.

pry

spooy jo

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poysnys WM

S¥>H¢

qI0Iq WMIES DBuVIO

reese yseaA osu03dad-asoaj0rg prow

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MoT pur umIpsw prog spoo3 JO Se citric > acetic. - Based on pH, the order is acetic > citric > lactic.

726

Analysis of Fruit and Vegetable Products

Non-sporing bacteria have been found to have maximum heat resistance at

a pH value at which the resistance to disinfection by alcohol and stability in suspension is at a-maximum.'® Effect of sugar, salt, fat and protein: Heating time required to kill yeasts,

moulds and spores of bacteria increases with increasing concentration of sugar. Thermal resistance of C/. botulinum increased with the concentration of sugar, some protection being afforded in concentrations as low as 12.5%. Sodium chloride protects heat resistance of spores up to 2.5%," and decreases the resistance with further increase in concentration’®’. High concentration (8%. or more) of salt considerably reduces the resistance. Desrosier!® reports that the phosphate level of soil and medium influences the heat resistance of bacterial spores. Phosphate ions are important in spore formation, spore germination, and the heat resistance of spores. - High concentrations of fat have, in some instances, been found to increase resistance, but this factor is not of significance in the canning of fruits and vegetables, except probably in the case of canned curried vegetables and soups. Destruction of spores of bacteria in oil appears to be similar to dry heat sterilization, when they are more resistant than when heat processed in relatively high water contents. The spores of C/. bo/ulinum have been found to survive beyond all reasonable expectations when heated in oil suspensions. Heat resistar:ce of non-sporing bacteria does not increase,while that of yeast

increases in the presence of oil or fat. Starch in medium permits the growth of greater number of spores, probably due to its ability to absorb inhibitory substances. Proteins may offer some protection, while essential oils and spice oils may have inhibitory effect and reduce spore resistance to heat. Effect of medium :'The medium in which the TDT is determined is an important factor. Neutral phosphate or peptone solutions have been tried. From the discussion in the previous paragraphs, it is obvious that the characteristics of the food under investigation may considerably affect the TDT of any organism. Thermal resistance of the spores suspended in two different media may be identical at one temperature, but may differ at other temperatures which will affect the final process calculation. In view of these and other causes which influence thermal resistance, usually TDT determinations are made by suspending the test organism in the food for which the heat process is calculated.

Determination of Heat Resistance of Spores The methods which are now in use to establish TDT are usually based on determination of the ability of a spore suspension of known concentration to survive or not to survive the given heat treatment. The principal methods used for measuring thermal resistance of bacteria are the following: TDT tube method,’® TDT can method,”® tank method, flask method,” thermo)

Determination

of Thermal

Process Time

727

resistometer method,” unsealed tube method,”* and capillary tube method.?® These methods have been discussed in detail by Hersom and Hulland,?* Stumbo” and National Canners Association.® Of these, the first two methods are most commonly used and are given below. TDT Tube

Method

' Heating suspensions of microorganisms in sealed tubes was first described

by Bigelow and Esty’® and revised by Esty and Williams.2? This method is widely used. Tubes : Make the tubes from 9 mm outside diameter Corning tubing having a 1-mm wall. Cut into approximately 12—13 cm pieces and seal at one end ina flame. Do not use soft glass as its high soda content may change the pH of the suspension and affect the heat resistance of the organism. Wrap the TDT tubes in paper in bundles of ten, and sterilize dry. Prepare sufficient quantity of the product to add 2—4 ml to each TDT tube. Brine from canned vegetables may be made use of. Sterilize the medium and inoculate with sufficient spores (prepared as described before) of the test organism to give the desired number per tube. In ordcr to avoid erratic results, use at least 10,000 spores per tube. Townsend e¢ al.4 made use of 2,000 to 1,200,000 spores of PA 3679 and 10,000,000 to 200,000,000 spores of C/. botulinum

per tube. Mix thoroughly. When very large numbers of spores are required, the large volume of mother liquor added with the spores may influence the heating medium. In such a case, where the suspension to medium ratio is more than approximately 1 : 25, centrifuge sufficient suspension in sterile tubes containing glass beads, and discard the mother liquor. Add sterile water, shake to wash the spores, and again centrifuge. Pour off the water and resuspend the spores in the heating medium. Pipette 2 to 4 ml of inoculated product into each of the required number of TDT tubes, and use a part of the suspension for counting. If the test material in which the microorganisms are to be suspended for heating is not sufficiently thin for pipetting into the tubes, dilute with minimum quantity of water. In the case of thick products like puree, use a grease gun or a syringe fitted with a large artery pumping needle for distributing

products into TDT tubes. Such a material is difficult to subculture and is usually incubated in the tubes after heating without subculturing. Growth is indicated by gas formation in the faod. After filling, seal the tubes as close to the end as possible in an oxygen flame. After sealing, there should be at least 4.5—5 cm head-space above the level of liquid for ease in breaking the tubes and subculturing the contents. Number of tubes: At each time interval, three or four tubes are sufficient. Make a preliminary run using two tubes per time interval. If destruction rates

are to be estimated from the number of positive tubes in each set, use ten or more tubes for each time interval. Colony counts may be made on suspen-

728

Analysis of Fruit and Vegetable Products

sions from the heated rates.

TDT

tubes

as a means

of deterniining

destruction

Heating procedure : For temperatures of 212° F-and jess, use constant temperature water or oil bath, and oil bath or TDT retort, for temperatures above

212° F. To suspend in a water or oil bath, insert the tubes in a metallic circular or square test tube holders which can be slipped over a hollow rod of sufficient length to more than span the bath. Insert a solid rod of smaller diameter in the hollow rod. Ensure that the entire tube is immersed in the heating medium. Have the bath a few degtecs high in temperature before the tubes are put in, to compensate for the drop in temperature which occurs when a large number of tubes are immersed. Alternatively, use a large-sized bath. For determination of TDT, a convenient size of retort is about 9 in. high

and 6 in. in diameter.*® Lugs for fastening the lid should be constructed so

that the retort can be quickly opened and closed. Use a mercury-in-glass thermometer graduated in 0.5° F or a copper-constantan thermocouple connected to a potentiometer for noting the temperature. Connect the steam line to a small retort or steam reservoir equipped with the thermometer,

an automatic temperature controller and recorder, a pressure

gauge, a 0.5 in. vent valve, 0.125 in. bleeder and a safety valve. Five or six retorts can be mounted directly over the steam reservoir, or connected to a steam line from a retort equipped with an automatic temperature controller (Fig. 25.1). The steam and water valves provided in the retort should

be of the quick-opening type. Tubes may be placed in a small basket before

placing them in the retort.

Fig.

25.1. Special thermal death time apparatus, designed and built by American Can Co., for use in process determination work. Each chamber is connected through quick-acting valves to a large high-pressure steam reservoir and a cool water supply. Precise measurements are-assured by almost instantancous rise to exact processing temperature, followed. by rapid cooling.

(Courtesy:

American Can Co.)

Determination of Thermal Process Time

729

To operate the retorts, thoroughly vent the steam reservoir and bring it to the desired temperature. Close all outlets from the TDT retorts and draw a vacuum of 25 in. oneach TDT retort by connecting to a high vacuum pump. Open the steam valves to individual TDT retorts widely and partially open the 1/8in. blecders in the lids. Start counting time the moment the steam is turned on. At the end of the heating period of each retort, close the steam valve and open the water and overflow valves immediately. The water should be allowed to run through until the retort is cool. An accurate timer is essential. Use a chronometer. with a large and easily readable dial, preferably graduated in minutes and hundredths of a minute. Opening and subculturing of tubes or direct incubation : Scratch the tubes about 0.5 in. from the end with an emery wheel or a steel glass-cutting wheel and break them open. Open the tubes aseptically and pour the contents into tubes or plates of'an appropriate culture medium or culture a portion of the contents

quantitatively,

to determine

the number

of spores

surviving

at each

. interval. TDT tubes can be houbated directly, without subculturing, if the heating medium is a favourable one, and change in pH or gas foriiatone is an adequate criterion of growth. Incubate such tubes for the necessary time, break them open and determine the pH. If incubated directly, do not stratify anaerobic

cultures with vaseline before sealing as the vaseline may have protective action on the microorganisms during heating. Culture media: The culture media need not necessarily be the same as those used to obtain spores. Media which have been found satisfactory for subculturing of the various groups of microorganisms are given in Table 25-2. Tubes of all media for gas-producing anaerobic bacteria should be boiled before inoculation, and should be layered with vaseline or other sealing material after inoculation to provide anaerobic conditions. Examination of cultures ; Cultures must be examined daily during the first week of incubation. Note the date when the culture shows signs of positiveness by the appearance of cloudiness, gas evolution, or a change in colour if the medium contains a pH indicator. As incubation may slightly modify the pH without any growth of microorganisms, incubate some control tubes also and note the change in pH, if any. Cultures which appear positive on visual examination should be examined

microscopically when in doubt. When C/. botislinum is suspected, it is advisable to verify the presence or absence of toxicity in the negative cultures which have been subjected to the shortest heating time as well as in doubtful cultures by

intraperitoneal injection into guinea pigs or mice.

|

When cultured in plates or in deep agar, establish the mortality rate according to the number of spores or microorganisms surviving after ditferent duration of heating. Count the colonies after 2 and 5 days for thermophilic bacteria,

730

Analysis of Fruit and Vegetable Products TABLE 25-2 : Media for Subculturing from TDT Tubes ———

Incubation

Time

Microorganism

Media

Temperature

Dircct Subincuba- _—cultured

tion of TDT

—_ (days)

tubes

a *

(weeks)

Dextrosc-tryptone broth or agar

37

4-6

714

B. stearothermophilus and other thermophilic flat sours

Desxtrose-tryptone broth or agar

50-55

2-3

4-7

B. coagulans

~Thermoacidurans agar

55

Li)

37

3-5

B. subtilis and related meso-

philic aerobic sporeformers

Liver broth, Tryptone-ycast extract broth, Liver-tomato broth

27-30

Dextrose-tryptone broth or agar, Orange serum agar

27-302

Cl. botulinum

Liver or beef heart broth

27-30

6

Tid

Other puttefactive anacrobes (c.g. Cl. sporogenes, Cl. nigrificans)

Liver broth, pork broth or pork infusion agar

59-55

2-3

4-7

28-30

2-3

4-7

Yeasts and moulds

Dextrosc-tryptone agar

27-30

2

ai

Butyric anacrobes (e.g. Cl. pastenrianun,

Cl. thermosaccharolyticum)

Non-spc >-forming bacter, (mainly

Lactobacillus and Leuconostoc specics)

6

3-5

3-5

Potato-dextrose agar Crook’s No. 2 agar

Malt extract medium — NOTE : Incubate mesophilic anacrobes at 27-30° C for at least 6 wecks and at Icast 2 weeks after appearance of the last positive tubes. Incubate moulds at 21-30° C, Duration may vary from two wecks to several months.

Determination of Thermal Process Time

731

and after 4 and 7 days for mesophilic bacteria. However, observation of deep tubes must sometimes be continued for several months.* TDT Can Method

Townsend ef a/.4 made use of small cans (208 x 006) of maximum capacity 16 ml to determine TDT in raw food products. The use of cans enables determination under conditions which do not require pureeing and foods. Foods containing small pieces (0.25 in.) and such foods be readily introduced into the tubes could be used. The cans advantage that they are not suitable for experiments on foods test organism docs not grow readily. The procedure for TDT using the cans is given bclow.

juicing of the which cannot have the dis-

in which the. determination

Wash the cans and sterilize in steam before use. If the test organism is of | low heat resistance, wrap the cans with lids inverted and sterilize in an auto-

clave for 30 min at 250° F before use. Pipette (not to exceed

13 ml) or weigh

the inoculated sample into each can and seal the cans in a vacuum double seamer under a vacuum of 20-25 inches.

To inoculate, mix sufficient quantity of test organism directly with the

material to give the desired load of organisms per can and distribute equal quantity into each. If the product contains solid pieces as in the case of canned vegetable, the ratio of solids to liquids must be the same as in commercial

packing. Mix the inoculum with the brine or syrup before adding into cans or after dissolving in sterile water, pipette into.each can (not more than 0.2 ml). Number of cans and the heating procedure are the same as in the case of tubes. Culturing from cans after heating is difficult and hence incubate directly. If required to be cultured, cut the can cither with a cutting wheel or a sharp blade. After cutting cach can, stcrilize the cutter with a flame. Transfer the contents to a suitable medium (sce tube method) and stratify with vaseline, if necessary, to provide anaerobic conditions. Incubate and cxamine as in tube method. When the cans are incubated directly, swelling of cans indicates gas

formers. In the case of organisms which produce little or no gas, open the cans at the end of the incubation period, determine the pH and note the appearance of the product. If C/. botulinum is suspected, make toxicity test as in tube method using cans heated for shortest period with no gas formation or the cans showing growth after longest heating. Heating Time and Temperature Since the death of organisms follows a logarithmic rate, choose time intervals which represent a definite logarithmic cycle apart.? For example, at 250° F the suggested time intervals are 1.0, 1.4, 2.0,

2.8, 4.0, 5.6, 8.0, etc. To de-

termine time at other temperatures, plot shortest and longest time selected

(logarithmic scale) against

temperature (linear scale) on a semilogarithmic

732.

Analysis of Fruit and Vegetable Products

paper. Draw two lines with z= 18 (or other x values) through these points. The estimated heating times for any other temperatures will be covered by

these lines.

a

In determining TDT, make an exploratory run using two cans or tubes

and subsequently determine the exact TDT at narrow time intervals. mine TDT of an organism at least at four temperatures.

Deter-

Phosphate buffer as a reference medium : As a wide variation may occur in the resistance of spore suspensions prepared under apparently identical conditions, and also variations in the composition of a given product, it is customary to heat a suspension of spores in phosphate buffer parallel with each determination of mortality. For this purpose, use a phosphate buffer of pH 7, , prepared by mixing solutions of M/15 disodium hydrogen phosphate (Na, HPO,) and M©/15 potassium dihydrogen phosphate (KH,PO,). Prepare solutions by using very pure quality salts. Autoclave the buffer at 250° F for 20 min, cool, inoculate and heat for selected time intcrvals. Cool and subculture the tubes.

Addition of phosphate buffer to a culture medium has a tendency to slightly inhibit the germination of spores and the development of culture. Since the buffer added can vary slightly between one procedure and another, the results obtained by different techniques may not always be comparable.? Correction for lag: The duration of heating of either the tube or of the can in the determination of TDT must be corrected to account for the necessary delay in bringing the contents to the temperature of the heating medium. The value of these corrections will depend on the consistency of the product, temperature, and the parameter x which determines the slope of the TDT curve. If a retort is used, one must also take into account its coming-up time. These corrections will vary between 0.7 and 1.3 min for tubes and 0.45 and 2.1 min for TDT cans. It is not merely the determination of the time lag in heating and cooling which is important, but also the sterilizing value of this time. To determine this, in addition to noting the time lag, it is necessary to know the parameter x. ‘The procedure is given below. On a semi-log paper, preferably having 3 cycles, note the time in minutes on the log scale and the temperature in °F on the linear scale. Without correcting for lag, plot the longest survival time and destruction time for each temperature used in the run. Even if a skip is noted, plot the longest survival time. Above all survival points and below as many destruction points as posvible, draw the best straight line. Determine the preliminary z value by noting the temperature in °F required for the curve to traverse one logarithmic cycle. Read from Table 25-3, compiled by Sognefest and Benjamin,” the corrections to be applied to experimental heating periods. A more precise method involves recording the heat penetration and cooling curves of the tubes ot the cans by the general method of Bigelow ef a/.° (see page 766),

the sterilizing value which

cofresponds

to these curves

and

then correcting the experimental heating periods. For example, if the experi-

Determination. of Thermal Process Time

733

TABLE 25-3: Corrections for Experimental Heating Times in Steam to give Actual TDT Times*

Run ConNo. _ tainer

Product

2-40

Water

Tube

2-65

‘Tube

50% Sugar solution

2-69

Tube

60% Sugar solution

2-61

‘Tube

Pea puree

2-34

2-63

2-67

Can

Can

Can

2-38! Can

2-58

2-71

Can

Can

Water

50% Sugar solution

60% Sugar solution Spinich puree

Pca putce

Pork luncheon meat

Exp. Corrections to be subtracted Corrected times heating times oo. y=14°F z=18°F y=22°F | z=14°F p=18°F e=22°F min 1.0 TAS

min 0.80 0.95

min 0.75 0.85

min 0.70 0.75

min 0.20 0.55

min 0.25 0.65

min 0.30 0.75

2.0 3.0

0.95 0.95

0.85 0.85

0.75 0.75

1.05 2.05

1.15 Zaks

1.25 225

1.0 aS5 2.0 3.0 1.0 Lay 2.0 3.0 1.0 1.5

0.90 1.05 1.05 1.05 0.95 1.10 1.10 1.10 0.97 1.20

0.88 1.00 1.00 1.00 0.90 1.05 1.05 1.05 0.95 Tel §

0.85 0.95 0.95 0.95 0.85 1.00 1.00 1.00 0.90 1.10

0.10 0.45 0.95 1.95 0.05 0.40 0.90 1.90 0.03 0.30

0.12 0.50 1.00 2.00 0.10 0.45 0.95 1.95 0.05 0.35

0.15 0.55 1.05 BOY 0.15 0.50 1.00 2.00 0.10 0.40

2.0 3.0

1.30 1.30

1.20 1.20

1.10 1.10

0.70 1.70

0.80 1.80

0.90 1.90

0.5 1.0 15 2.0 1.0 L.§ 2.0

0.48 0.60 0.60 0.60 0.90 0.95 1.00

0.46 0.55 0.55 0.55 0.85 0.90 0.90

0.45 0.50 0.50 0.50 0.80 0.80 0.80

0.02 0.40

0.04 0.45

0.90 1.40 0.10

0.95 1.45 0.15

0.55

0.60

1.00

1.10

0.05 0.50 1.00 1.50 0.20 0.70. 1,20

3.0

1.00

0.90

0.80

2.00

2.10

2.20

1.5 2.0 3.0 1.5

1.20 1.30 130 1.40

se} 1.20 1.20 T2235

1.05 1.10 1.10 1.30

0.30 0.70 1.70 0.10

0.35 0.80 1.80 0.15

0.45 0.90 1.90 0.20

2.0

1.65

1.55

1.45

O35

MO.ase?

Whorss

3.0

Ths

1.65

1.55

125

L535

1.45

4.0

TTS

1.65

1.55

2.25

2.35

2.45

BeO

1.80

1.70

1.60

0,20

0.30

0.40

2.5 3.0

1.95 2.00

1.85 1.90

1.75 1.80

0.55 1.00

0.65 1.10

0.75 1m2o

4.0

2.10

1.90

1.80

1.90

2.10

2.20

2.0

1.80

1.70

1.65

0.20

0.30

0.35

3.0

2.10

1.95

1.85

0.90

1.05

1.15

4.0

2.10

1.95

1.85

1.90

2.05

Brals

5.0

PySoy,

SEBO):

1.85

2.90

3.05

3.15

|

*Fot experimental heating times greater than those shown,the correction factor is obviously the same as that shown fcr thc maximum time. (Reprinte

from “Thcorctica)

Considerations

in the Sterilization of Canned Foods”,

Sterilization of Canned Foods, with permissiun of the American Can Company.)

734

Analysis of Fruit and Vegetable Products

mental heating period at a temperature, say 250° F, is 4.5 min and the coming up and cooling time, each 1 min, the corrected time is 2.5 min plus the time corresponding to the sterilizing value of the rise in temperature and of the cooling. In practice, the sterilizing value is subtracted from the total heating period. A more convenient way instead of subtracting the correction from the effective heating periods is to heat for the length of time desired plus corrections.

This will yield times in round figures which will make for easy correction. Interpretation of Results TDT curve: Inthe method described above for the determination of TDT, at various heating times for each of the temperatures studied, if the number

of spores which survive the shorter heating periods are not counted, a series of paired results will be obtained as given in Table 25-4. When

the survival

and destruction times in minutes are plotted on the logarithmic scale against temperature in °F on the linear scale, a straight line of best fit drawn above all survival points and below as many destruction points as possible will result in the TDT curve. A typical TDT curve is shown in Fig. 25.2. Such a curve may be characterized by the TDT at some particular temperature and the slope. A temperature 1000

a| Ee Le |3|a

7

7 RE Pa Ta TMP a AT | 0 Oe eT Lae

kk Pikmi ed Bs =I BS ! a fedaa!AJbs mek@) 200-215 230 245 260 Temperature

°F

Fig. 25.2 Thermal death time curve of Clostridium botulinum in neutral solution. Points below line represent survival timcs; those on or above, death. times.

of 250° F (121° C) is taken as the reference and the symbol F is used to designate the TDT at this temperature. Therefore F value is the time in minutes

requircd to destroy the organism at 250° F. The slope of the TDT curve is re-

Determination of Thermal Process Time

735

presented by x, and its value is the number of degrees Fahrenheit required for the TDT curve to traverse one logarithmic cycle, i.e.,to produce a ten-fold increase or decrease in TDT. These two values, F and x, establish and describe the TDT curve and are a quantitative measure of the heat resistance of the spores over a range of temperature. TABLE 25-4 : Survival and Destruction Times of Cl. botulinum Strain 62, Type A in the

Peas and Phosphate Buffer Subcultured in Tubes‘

(2 ml per tube, i.e., 10,000,000 spores per tube) Duration of heating SS Survival Destruction (min) (min)

Product

Heating temperature

Peas

212° F (100° C)

160.0

180.0

221° F (105° C) 230°F (110° C) 239°F (115° C)

29.6 3.6 1.6

34.6 5.6 2.6

Phosphate buffer

212° F (100° C}

160.0

180.0

(pH 7)

221° F (1os° C) 230° F (110° C) 239° F (115° C)

34.6 5.6 1.6

40.0 7.6 “ 2.6

(pH 5.42)

Fi

4

0.28

13.6

0.28

13.6

(Reprinted with permission of the Institute of Food Technologists, USA. Copyright©). “

Plotting of TDT curve : The end point of destruction at any given temperature is generally considered as the shortest time in which no visible growth is observed in any of the replicate samples. To plot TDT curve on a semi-log paper, note the time on log scale and the temperature in ‘F on the linear scale. Plot the corrected survival and destruction times. As a rule, the TDT curve

must be above all survival points and below the largest possible number of destruction points. It is not always the case even when many repetitions of the same experiment are made as even a single result indicating exceptional resistance may prevent adhering to the above procedure. Some latitude is, therefore, necessary in the drawing of the TDT curve, even when it is above all the survival points and below most of the destruction points. The National Canners Association® recommends drawing of two straight lines which show

the maximum divergence in slope, but are still above all survival points and below most of the destruction points. Determine the slope of each by measuring the number of minutes required to traverse one logarithmic cycle and

testestetennnennaennbentindaronlennadinibed a pdmo

Analysis of Fruit and Vegetable Products

736

average the z values obtained (Fig. 25.3). The F values for any temperature may be averaged in the same way. The TDT curve so constructed is c alled

the “end-point” curve.

:

6E

oS

a

@

Destruction

Time

seeCCSBane

Sornuype

mom

20g) sy

sz0og) OnTeA (9asnd LOL mowed

(aaznd LOL

a 217.3

iii! '

A

SaTL

_PHANTOM

Temperature

in

Degrees ¥

Fig. 25.3 Thermal death ‘time curve.

(Reprinted from Laboratory Manual for Food Canners and Processors with permission of thc National Canhers Association, USA)

Thermal Death Rate (or Survival) Curve®?® When bacteria are subjected to moist heat, generally the number of viable cells reduces exponentially with time of exposure to a lethal temperature. Consequently, if logarithms of the numbers of survivors are plotted against times of exposure, a straight line will be obtained which is commonly referred to as a logarithmic order of death. Although a number of exceptions have been re-

Determination of Thermal Process Time

737 |

ported, generally the death of vegetative cells as well as of spores is logarith-

mic.'”s*? Tt means that in a given time interval, the same percentage of bacterial population will be destroyed irrespective of the number present. Consequently, in the heat processing of foods, if the number of microorganisms increases, the heating time required to sterilize the food also increases. Fur-

ther, it is theoretically impossible to achieve complete sterility, as the curve is logarithmic, tending asymptotically to zero. The TDT curve described earlier has the disadvantage of using only the two final points of each experiment, viz.,the longest duration of heating which still permits survival and the subsequent duration of heating which ensures

destruction. The F value so obtained is valid only for the number of organisms used in the particular run. It is, therefore, preferable to measure at each temperature, the proportion of surviving spores as a function of the heating time. ‘This gives the decimal reduction time D, i, e., time required to reduce

the bacterial population by 90% at temperature T (°F). From the D values at different temperatures 7 can be deduced. To do this, carry out the studies in such a way as to allow the evaluation of the number of surviving spores. After each heating,count the number of survivors cithcr by subculturing on a solid medium or by dilutions. However, if the medium is not suitable for counting by subculture as in the case of solid products, incubate the tubes or cans directly. Take the number of containers showing survival at the longest time of heating as equivalent to the number of surviving organisms. Plot the duration of heating in the linear scale and the number besurvivors on the logarithmic scale (Fig. 25.4). Draw a straight line to fit the points obtained. The D value (defined earlier) is given by the slope. Like the TDT, to which it is mathematically related, the D value depends on the conditions under which the organisms are produced, the medium in which they are heated, and the temperature at which it is determined, but not upon the initial number of cells. It may be read off from the diagram or calculated using the

formula*>® given below. U D= log, eae log,

where,D is death rate in min, U is heating time in min, a is initial number

of microorganisms, and ) is number of microorganisms which survived the heating time U. The example in Table 25-5 is given to calculate the D value (Fig. 25.4), at 240° F.

Analysis of Fruit and Vegetable Products

738

iB

=

$

$8

MT ET B A MT Tee UTa SU LST

10. 000

DEATH

RATE CURVE

P A 3679 in Strained Temperature

Pees

= 240°F

—]

‘ ~«a wn

SAOATAINS

Lita

.

= HLL

° Ss on o o° 3° on

saquiny jo

Initial No. (a)=10,000 spores After 18 minutes heating (U=18)

PELE

2 spores survived (b=2)

‘a

18

iid

log 10,000 - log 2

ania

4.85

UT

= Ng =. £

= ry oe Time

in

Minutes

Fig. 25.4 Death rate curve

(Courtesy : National Canners Association, USA)

4- 0.301

Determination of Thermal Process Time

739

TABLE 25-5: Survival of PA 3679 at 240° F in Strained Peas in TDT Cans? (12 g of product, 10,000 spores per can)

Surviving sporés Duration of

Number of

heating

cans per ,

(corrected)

series

Number per can

Logarithms

(min) 10

6

14

6

12

18

6

2

22

6

0.33

0.§2

23

6

°

=

36

6

°

c=

‘Dose pe

Dio gets

pendant

18 im baton log 10,000 — log, 4—0.3 aay

—

cara

wit

log 10,000 —log,,

—

ened =

4—1.08

4--1.95

The valucs of D similarly determined

1.95 1.08 \

0.30

= 4.865

fl}

tae Beate es

log 10,000—log,,

10.47, 4.92, 2.56 and 1.23 at

aes

4,794

4089

at other temperatures

are 19.65,

230, 235, 240, 245 and 250 °F respectively.

The example in Table 25-6 is given to illustrate the calculation of D value when the tubes or,cans are incubated directly.”

TABLE

25-6 : Survival Data on Direct Incubation

Duration of heat- | Numbcr of tubes ing (correctcd)(min) per serics ie

apne

iE

—

Positive tubes -

10 pe

40

10

10

45

10

9

50

10

5

55

10

2

60

10

°

}

740 ~~ Analysis of Fruit and Vegetable Products

Initially each tube contained 1,000 spores in 2 ml. Each positive tube is assumed to contain one surviving spore, so that the number of positive tubes

is equal to the number of surviving spores out of all spores (1,000 x 10) initially present.

log 10,000—log

9

4—0.95

ee ene ge er Ot ETS log 10,000—log 5

D = log

ae.

10,000 — log

KT

4—0.7 —0.

eer

Phantom TDT Curve or Thermal Resistance Curve

On a semi-log paper, plot the D values on the logarithmic scale and: the “temperature on the linear scale. Draw a straight line of the best fit (Fig. 25.5). The straight line gives the relationship between log D and temperature. This

Temperature

°F

Fig. 25.5 Thermal resistance curve (also called phantom thermal death time curve).

straight line represents the “Phantom” thermal death time which according to

Ball® has direction, but no position. Schmidt and Olson®* named it as chermal resistance curve as they thought that it represents the real measure of thermal resistance. It can be characterized by the D value at some reference tempera-

ture (usually 250° F). The slope measured as the number of °F required for the curve to pass over one log cycle is equal to x of the TDT curve. The z value of the thermal resistance curve, therefore, represents the temperature necessary to bring about a ten-fold change in D value and has essentially the same value under constant set of conditions whether determined from a TDT curve or from thermal resistance curve.

Determination of Thermal. Process Time

741

The F values given in the literature are often based on different initial number of cells. Hence, they are not comparable with one another. On the con-

trary, the D values are independent of initial numbers and hence can be compared irrespective of the initial number of cells. Since the death rate of microorganisms is assumed to be logarithmic, itis

theoretically impossible to obtain complete destruction, It is necessary, therefore, to accept for F value at a given temperature a heating time which will result in a defi..ite proportion of survivors. This is usually one surviving

spore in 1,00,000 (5 D) for FS 1518 and PA 3679, and one in 1 Xx 10! (12D) for C/. botulinum spores. This is explained further in the subsequent paragtaphs. F Value for Process Calculations In the heat processing of any foods, it is necessary to have some measure. of the effectiveness of any process in killing microorganisms in the food. Pio-

neering studies by Bigelow and Esty’® and Ball** *° in the 1920’s led to the adoption of 1 min at 250° F as the unit of process value. The symbol F now stands for the sterilizing value orthe lethality of the process. Thus, if it is said that the process has the value of F=3, it is meant that the process is equivalent in the sterilizing value of 3 min at 250° F assuming instant heating to this temperature at the start and instant cooling to a sub-lethal temperature at.the end. The word equivalent in the previous sentence means that if a

batch of cans was given this hypothetical process, the degree of sterility should be the same as that attained by the actual process that has been valued. Irrespective of the can size, process time. and temperature, the material should get the same degree of sterility. The plain symbol F is generally accepted as referring to 250° F as the reference temperature. The symbol F, is used in America to indicate that it is based on the z value of 18. Symbols such as F,,,, Fy99> Fy g9 and F,55 are used to denote the pasteurizing processes for acid packs. A fully defined F symbol should have

two adscripts, the z value and the reference temperature, e.g., F,43.The effect of temperature increases as the x value diminishes considerably at temperatures lower than250°'F Esty and Meyer!” showed that a preparation containing 6 x 10'° spores of the most heat-resistant strain available of C/. botulinum was killed by heat treatment at 250° F for 2.78 min (2.45 min according to the corrected value of

Townsend ef a/.*). While the number of spores used by Esty and Meyer"? in their TDT tion. It is _ ducing 1 x probability

runs was 6 x 10", their TDT's were based on complete destrucnow customary to assume that their TDT curve resulted in re-

10™ spores to one spore, or that F=12 D. In such a case the of survival of any C/. botulinum present will be no greater than 1

in 10!*, These maximum values obtained by Esty and Meyer" are sometimes referred to as classical values.

742

Analysis of Fruit and Vegetable Products

The classical values were obtained by Esty and Meyer'® by heating very large numbers of spores of different strains in neutral phosphate buffer and plotting the first destruction time following the longest survival time obtained at each temperature for the TDT curve. In ‘DT determinations with sporcs of Cl. botulinum, lower survival values are usually obtained than those accepted as the maximum. To interpret TDT data for C/. botulinum sporcs in a product (where y= 18) in terms of any F value for process calculations, it is necessary to convert the data obtained to the classical value. For this purpose, apply the following formula to get the classical resistance in the product.*»® : ! F(i.e., classical

Classical resistance of Resistance of Cl.botulinum Cl. botulinum in phos- X obtained in the product in

DEVAN of

Cl. botulinum : ity tie Pesta

phate buffer (= 2.45)

TDT run

~~ Resistance of Cl. botulinum obtained in phosphate buffer in TDT run

The same procedure may also be applied for calculation in terms of D, since D= oe

where, U ie 2.45 min, a=1X10” spores, and b=/spore.

Classical Di in phosphate buffer

= ae

=

0.204

To obtain the classical D}§ for C/. botulinum spores apply the following formula : Classical

D2 in _

the test product

heated in a product

0.204 x Dj8 obtained in the test product

Dys Obtained in the test run using phosphate buffer

From a study of much unpublished data in which F was determined by the conventional procedure and D calculated by the probability method, Schmidt*® has stated that the following formula gives the best interconversion of D value to F value or vice versa. D=

log, + 2

or

F =

D (log,

+

2)

Determination of Heat Resistance of Non-spore-forming Bacteria, Yeasts and Moulds

There is no standard procedure but methods similar to those described for

spores can be used. Murdock et a/.37 made use of Lactobacilli, Leuconostoc and

yeasts. Bacteria : Select some spoiled cans of the product which might have under-

gone spoilage due to underprocessing. Open

the cans under sterile condi-

Determination of Thermal Process Time

743

‘tions (see page 676) and examine microscopically. Grow the organisms on nutrient agar. Sometimes, they do not grow on nutrient agar in which case prepare a medium consisting of 10 g of tryptone, 3 g of ycast extract, 4 g of dextrose, 3 g of dipotassium phosphate (K,HPO,), 17 g of agar, 200 ml of filtered juice or centrifuged serum of the test product and 800 ml of distilled water. Adjust the pH to 5.5 and sterilize at 15 psig for 20 min. Murdock e¢ al.” made use of orange serum for growing Lactobacillus sp.,Lesconostoc and yeasts. In the case of mixcd cultures, isolate the organisms by plating and incubating at 30° or 37° C for 1 to 3 days. For preparing stock suspension, grow on slants and harvest as in the case of spores. The organisms may also be grown in the broth (same as before but do not add agar), Centrifuge the broth under aseptic conditions and wash the residue twice with sterile saline. Suspend the organisms in sterile saline and store in refrigerator until required for use. Count the concentration of cells in the stock suspension by serial dilution and plating technique. Prepare the medium for determination of TDT by any one of the following methods. If the test material is a juicy fruit, extract the juice and filter. 1f it is a pulpy fruit, blend (with water, if necessary) and centrifuge. Adjust the total soluble solids in the filtrate or centrifugate to the same strength as that of the.finished product by adding sugar and similarly the pH by adding acid. Sterilize at 15 psig for 20 min and cool to room temperature. To the sterilc juice, inoculate a known quantity of the stock suspension, shake thoroughly, distribute uniformly (2 ml) into TDT tubes and seal. Determine the heat resistance using a water bath at different temperatures. Because the TDT of the vegetative cells responsible for spoilage of acid foods is immeasurably small at 250° F, reference temperature lower than 250° is necessary for these organisms. Hence when pasteurization treatments are considered, a reference temperature of 150° F (65.5° C) could conveniently be used and the TDT at this temperature is designated as F.

Heat the tubes for various time intervals at each temperature (130—150° F). . Use four tubes per time interval. After heating, cool immediatcly by plunging _ in ice cold water. Transfer the contents of the tubes to sterile plates, and then add 15-20 ml of sterile medium (same as that used for cultivating the organism)

ot inoculate the medium contained in tubes. Incubate at 30° C and 37° C and examine for the presence or absence of growth. The destruction point is the shortest period of time showing no growth on any of the replicate plates or tubes. The survival time is the next shorter period of exposure or the longest period showing any survival. Note the lag in the come-up time using thermocouples in two reference tubes containing the medium and included in each ,

heating batch. Apply correction for the heating lag as described earlier or by adding 42°/ of the come-up time to the actual heating time to compensate for the lag.*® Heat resistance of spoilage organisms in orange juice is given in. Table 25-7.

Analysis or Fruit and Vegetable Products

744

TABLE 25-7 : TDT Data for Leuconostoc, Lactobacilli and Yeasts in Single Strength

Orange Juice”

Survival time

Destruction time

(CF) ee a a ee 130 340,000 Leuconostoc

(min)

(min)

24.1

34.1

135

4.2

6.2

140

°0.8

1.1

Organism

Lactobacilli

Yeasts

Concentration per nal

10,00,000

8,60,000

‘Tempe~ ratute

135

39.1

140

6.1

8.1

145

an!

Zot

' 140

17.1

24.1

145 150

— 0.3

_

2.1

; scp

IIT

0.04

7

0.28

7

0.35

©

0.4

(Reprinted with permission of the Institute of Food Technologists, USA, Copyright ©)

Yeast® : The difference in resistance between yeast spores and cells in the vegetative state is not as great as it is for bacteria. Strict adherence to various

environmental conditions is necessary before yeast will sporulatc.*® One of these is the need for an optimum amount of oxygen, which is absent in properly exhausted cans and thus becomes a limiting factor in the sporulation of yeasts. Some fruits, as in the case of cherries, may have natural inhibitors.® Cells in the vegetative state are most heat-resistant in the maximum stationary

phase.*® Standardize the growth conditions and note the time required for the cells to enter the stationary phase. The growth rate may be observed by (i) measuring the optical density of the growth medium at 600 nm at given intervals after inoculation of a standard strength culture, or by (ii) determination of the total number of cells at given intervals after inoculation by

counting. Prepare the medium for determination of TDT as in the case of bacteria.

Harvest the yeast cells from the growth medium in the maximum stationary

phase by centrifugation, wash twice with sterile 0.9% saline solution and suspend in fresh solution. Count the number of viable cells in the suspension after serial dilution by the plate count procedure using ‘“‘bacto plate count” agar. Determine the TDT as in the case of bacteria.

Determination of Thermal Process Time

745

Determination of Thermal Inactivation and Decimal Reduction.

Times of Enzymes*™ >
?-®4 The probabilities of survival throughout the container are summed up and the most probable surviving number in the container determined. Based on this concept, a mathematical system for evaluating process time has been deve-

loped by Stumbo.®®

The procedure is based on the hypothesis that in order

to evaluate the capacity of a heat process to reduce the number of bacteria in a container of food, consideration must be given to heat treatments received by all points throughout the container rather than that received by any one point. These methods are given below.

The General Method This method; also known as the Graphical Method, was developed by Bigelow ef a/.*° It is essentially a graphical procedure for integrating the lethal effects of various time-temperature relationships existing at the cold point during heating and cooling of a product. Each temperature represented by a point on the heat penetration curve is considered to have sterilizing or lethal value. The theory behind the method is that the rate of destruction of an organism per minute at any given temperature (T’) in a process is the reciprocal of the time in minutes (/) required to destroy the organism at that temperature. The requirements are: (i) the TDT of the most heat-resistant spoilage organism likely to be encountered must be known at all temperatures attained during the process, and (ii) the heat penetration data. The TDT is converted to lethal rates for the various heating temperatures. The lethal rate for a temperature is the reciprocal of the TDT. The process calculation-is explained by taking the example given by Schultz

and Olson.”° Enter the data from the TDT curve and the heat penetration curve

as given in Table 25-16. Enter column 1 (time) and 2 (temperature) systemati-

Determination of Thermal'Process Time

TABLE 25-16: Data Used in Plotting Lethality Curve

Time

Tempera-

TDT

(min)

ture

(min)

(CF)

°

82

2

Lethal

rate

(1/TDT)

—

3

217

670

0.0015

4

230

129

0.0078

6

233

88

0.0114

8

233

88

0.0114

Il

228

165

0.0061

14

232

100

17

237

“53

0.0189

20

240

36

0.0278

24

242

3

0.0357

29

245

19

0.0526

32

246

16.7

0.0599

35

247

14.8

6.0676

40

248

13.0

0.0769

45

248.5

{2.4

0.0806

47

248.5

12.4

0.0806

47-5

247

14.8

0.0676

129.0

0.0078

48.0 *

49.0

| 230 /

217

670.0

5

0.0100

'

0.0015

(Reprinted with permission of the American Can Co., USA.)

76'7

768

Analysis of Fruit and Vegetable Products

cally from the heat penetration curve. From the TDT curve, note the TDT corresponding to the temperature in column 2 and enter in column 3. Find the lethal rates by taking the reciprocals of the TDTs in column 3, and enter 0.10

pro lca a aefad Eel os ES

82 3

a

°

aate Le Pasir, PCE SP TL s| TEL telat Shed LT Ain co) 30 PROCESS TIME IN MINUTES

iFig. 25. 15 Lethality curve plotted from columns 1 and 4 of Table 25-16 (Courtesy :American Can Co.)

in column 4. Then plot the data in columns 1 and 4 ona coordinate paper (Fig. 25.15) and join the points by a smooth curve to form a lethality curve. Because the product of lethal rate and time is equal to lethality, the area below the curve may be expressed directly in units of lethality. A unit sterilization area is defined as the area on the lethality curve which just represents complete sterilization. To determine what process time must be employed to give unit lethality, the cooling position of any given lethality curve may be _ shifted to the right or left so as to give an area equal to 1. The number of squares in such unit sterilization area depends on how the coordinates are labelled. In Fig. 25.15, each side of the square represents the lethality of 0.01 and a time of 5 min so that one small square area represents a lethality of 0.05. An area equal to 20 squares is equal to unit sterilization area. If the area under the lethality curve is less than 20 squares, the process is inadequate and if more than 20 squares, it is greater than needed. The OCBADEFO under the Icthality curve representing 47-min process is found to be equal to 38.4 squares which is 192%, of the unit sterilization areca. This means that the process is considerably longer than necessary. To find the time required for unit sterilization-under the lethality curve, draw cooling curves similar to the original cooling curve several places along the heating curve. Two cooling curves are drawn (Fig. 25.15) starting-at 25 and 35 min. The areas under. these curves (OCFO and OCBEFO) are 42% and 97% of the unit sterilization area. By graphical interpolation, it will be found that by processing for 35.5 min, complete sterilizationis obtained. This is a trial and error procedure and, for this reason, the method is sometimes referred

Determination of Thermal Process Time

769

to as the graphical trialand error method.’ Alternately, carry out three or more heat penetration runs for different periods, plot the lethality curve and find the area under the Icthality curve in each case. By plotting the lethality against process time in min, find the actual time required. If the TDT at onc temperature is known, the TDT at any other temperature may be found using the following expression. log” fea

log ne 4

log 1Ti eae log 10 =

or

/ =I

250L—o7-

zg

#

250 —T

&

g

1, therefore log -— = —

antilog

vit — x

where, == I’ = x = ?/F = equal to 1 F/¢°=

temperature whose T'DT is required time in minutes (/) required to destroy the organism at 250° F slope of the TDT curve in °F time required to destroy the organism at temperature (T) if F is Lethal raterat. T-

Example : TDT of C/. botulinum at 250° F is 2.78 min with a z vaiue of 18. Find

TDT

at 240° F.

Advantages and gp sepdat slod: The general method is simple, versatile and can be readily applied to complex heating and cooling curves.** It is also useful for determining the exact lethality of a particular process, including the come-up time and the cooling time.® It is especially valuable when the heat

penetration curve on semi-log paper cannot be represented by one or two straight lines (see “Formula Method”). The disadvantages are that it is long and laborious and not well suited for routine calculation.5® The TDT at every temperature must be calculated. The method cannot be used readily for calculating lethalities based on initial temperature, retort temperature or con: tainer size other than those used for the particular heat penetration test,” although a formula developed by Schultz and Olson®® is available for the

purpose. Calculation of process time using special coordinate paper :Schultz and Olson®# have described a method for the construction of special coordinate or lethalrate paper which reduces the effort required for calculation and decreases the chances of misplotting. Method for construction of lethal-rate paper for a z value of 18 as described by the authors is given in Fig. 25.16.

Analysis of Fruit and Vegetable Products

770

FAHRENHEIT DEGREES

i] A

fo)

1o

20 PROCESS

30 40 TIME-IN MINUTES

Fig. 25.16. Lethality cutve plotted on lethal-rate paper directly from heat penetration data. (Courtesy: American Can Cg.)

Draw on a sheet of paper, two horizontal lines a unit distance apart, Consider the bottom line as the zero line and mark the top line as 250° F or the maximum temperature likely to be obtained in the processing range employed. For every degrec of temperature in the processing range, calculate the L value see page 772\ (or find from Table 25-17 or 25-18), and draw a horizontal line whose distance should be L units above the zcro line. Number these lines not with the values of L, but with the corresponding tempcratures. Draw vertical lines in the linear scale for the time. It will be seen from the lethality curve that if the highest temperature reached in the heat penetration test of the product is 227° F, the curve would occupy a very narrow band at the bottom and would be too small to be measured conveniently. This difficulty can be easily overcome by giving‘to the top line 232° F which is 18° F less than 250° F, a value as close as possible above the

maximum temperature observed during heat penetration. Adjustment of the top value to be used should be done in units of x value used. For example, if the x value used is 18, the top line instead of being numbered as 250, could be numbered as 268, 232, 214, etc. A different lethal rate paper must be cons-

tructed for each different z value employed. To calculate the F value of the process, mark the points corresponding to the time-temperature points of the heat penetration data on the lethal rate paper. Draw a smooth

curve

connecting

these points. Measure the area under the

curve by any convenient means, as described under “Improved General Method.” Calculate the F value of the process from the area under the curve by using the equation:

,

sie

preci

10" x d

Determination

of Thermal Process Time

771

vee == number of minutes represented by one inch on the time scale, A =» area under the curve in square inches, » = the number of changes in X units that must be made to change the number of the top line from 250 to the desired number (# is positive when the top line is numbered less than 250 and negative when it is numbered more than 250), and d = number of inches from the bottom line (zero line) to the top line. The above method of finding the area under the curve and calculation of the F value gives the sterilizing value of the process. If it is desired to determine the processing time having a given F value, draw cooling curves at different points described earlier. Calculate the areas under these curves and find the F values from the above formula. By graphical interpolation, find the process time equivalent to the desired F value.

Conversion of retort and initial temperatures : If an experimental heat penetration curve has been determined for a particular retort temperature and it is desired to find corresponding can temperatures that should be obtained if a different retort temperature is used, these may be calculated from the follow-

ing equations :°° i. When the initial temperature remains the same, but the retort temperature is changed :

cr, — rt, — where, RT =

original ‘retort

2a =

RT

— IT

temperature,

er ~ cr RT, =

new retort temperature,

IT= initial temperature, CT = a can temperature of the original set, and CT, = anew can temperature corresponding to CT. If CT, is plotted against CT, the graph will be a straight line since aie is a linear Emetion of CT. Hence, it is necessary to use the formula for determining only two temperatures and join the points with a straight line. From this, other temperatures may be taken. ii. When the retort temperature remains the same, but the initial temperature is changed: CI, =

Kr=

RT — IT,

Rar

(RT —

CT)

It has been found convenient to use a value of CT of about 200° F in order to determine the other point.

If it is desired to change both the retort temperature and the initial temperature, the two formulae can be applicd successively in either order. These equations have been found to be valid for conduction as well as convection heating products. The Improved General Method

Since the general method of Bigelow ef a/.°° is laborious and not well suited for routine calculations, Ball®= and Schultz and Olson® introduced further improvements which resulted in what is now called the! “Improved General Me-

thod.” Ball°5 made several improvements, the chief of which is the construc-

772 ~~‘ Analysis of Fruit and Vegetable Products COOLING COMMENCED RETORT

TEMPERATURE

240°F

240

3 ry ° (°F) Temperature 160

140

Time (min)

Fig. 25.17. Heat penetration curve of a 303 x 406 can of solid food and the corresponding L value vs time curve. Areas representing Fo values of 1 are also shown.

(Courtesy: P.W. Board)

tion of hypothetical TDT

curve passing through the point one minute

at

250° F (F = 1). The area under the lethality curve (Fig. 25.17) in terms of the

unit sterilizing area will then give the sterilizing value (F value) of the process directly. The symbol F’ was introduced by Ball** to designate the equivalent in minutes at 250° F of the combined lethality of all time-temperature relationships at the point of slowest heating represented by heating and cooling curves for a product during process. For instance, if the entire contents of a

can could be heated instantly to 230° F, held at that temperature for 36 min, and then instantly cooled, the process would be equal to 2.78 min at 250° F * and has an F value of 2.78 min. A second improvement contributed by Ball®® consisted of calculating the lethal rate (L) from the equation :

Tred Ah eee log * 250 —

T

where,L= lethal rate time at 250° F that is equivalent

in sterilizing value

to 1 min at some other temperature, T = any lethal temperature in °F,and X = the number of °F required for the slope of the TDT curve to pass one log cycle.

'

To illustrate these two significant contributions of Ball®4’? described.

above, it will be seen from the TDT curve (Fig. 25.2) that the TDT for C7. botulinum at 250° Fiis 2.78 min while at 240° F it is 10 min. Therefore, 1 min at 240° F is equivalent to 0.278 min at 250° F. L values for a range of tempera-

ture when z = 18 are given in Table 25-17 and for values of z other than 18 in Table 25-18.

Determination of Thermal Process Time

TABLE 25-17 : Lethal Rates when t(°F)

oo

'

Pi iE a

2

3

4

773

z= 18 _

5

a cdE-Alerts

190 209

0.0005 0.0017

0.000§ 0.0019

0.0006 0.0022

210

0.0060

0.0068

0.0077

22Q

0.0216

0.0244

0.0278

Mi

jd

3

.O

0.0007 0.0025

0.0008 0.0028

0.0009 0.0032

0.0088

0.0100

0.0114

0.0316

0.0359

0.0408

4

oS

0936 0129 0464

0.0041 0.0147 0.0528

26

225

0.0408

0.0415

0.0419

0.0424

0.0430

0.0435

0441

0.0447

0.0464

'0.0470

0.0476

0.0482

6.0489

0.0495

O§OL

0.0508

227 228 229

0.0528 0.0600 0.0681

0.0534 0.0607 0.0690

0.0541 0.0615 0.0699

0.0548 0.0623 0.0708

0.0555 0.0631 0.0717

0.0562 0.0639 0.0726

0579 0647 -0736

0.0577 0.0656 0.0745

0836 0950

0.0847 0.0962

1080 1228

0.1093 0.1243

1395

0.1413

1585

0.1605

1801

0.1824

230

0.0774

0.0784

0.0794

0.0805

0.0815

0.0825

0.0880 ©.1000

0.0891 6.1013

0.0903 0.1026

9.0914 0.1040

0.0926 0.1052

0.0938 0.1066

233. 234

0.1137 0.1292

O.11§I 0.1308

0.1166 0.1325

0.1181 0.1342

0.1196 0.1359

0.1212 0.1377

235 236 237

0.1468 0.1668 0.1895

0.1487 0.1690 0.1920

0.1506 0.1712 0.1945

0.1525 0.1733 0.1970

0.1545 0.1756 0.1995

0.1565 0.1778 0.2021

238 0.2155 239 0.2448

0.2182 0.2480

0.2211 0.2512

0.2239 0.2545

0.2268 0.2577

0.2297 0.2610

240 .0.2782 241 0.3162 242 0.3594 243 0.4084 244 0.4642

0.2818 0.3203 0.3640 0.4137 0.4701

0.2855 0.3244 0.3687 0.4190 0.4702

0.2892 0.3286 0.3725 0.4244 0.4822

0.2929 0.3329 0.3783 0.4298 0.4885

0.2966 0.3371 0.3831 0.4354 0.4948

2047 0.2073 2327 0.2356 CTO GUOS LO ©9900 000000 .2643 0.2678 3004 3414

0.0053

0167

0.0189

0600

0.0681

OB0 999900 Si9)00000

9.3043 0.3458

3881

0.3930

-4410 OF .OF +0) So 6 .§012

0.4467 0.5077

-3697 0.5769 -6474 0.6557

245

0.5274

0.5343

0.5412

0.5481

0.5552

0.5623

246 247

0.5995 0.6814

0.6073 0.6910

0.6151 ©.6990

0.6230 0.7079

0.6310 0.7171

0.6392 0.7264

248° 249

0.7743 0.8800

0.7843 0.8913

0.7943 0.9028

0.8046 0.9145

0.8151 0.9262

0.8254 0.9383

-7357 8362 9501

9.7452 0.8468 0.9625

250

1,0000

1.0131

1.0262

1.0393

1.0§21

1.0662

.0803

1.0931

|

0.0015

0046

-7

226

231 232

.0012

/

(Reprinted with permission of the P.W. Board.)

3981 4525 Men Xo oy jaa Xo) Ya ©e 5141

0.4033 0.4582 0.5208

5857

0.5920

6642 7548

0.6726 0.7645

774

Analysis of Fruit and Vegetable Products

TABLE 25-18 : Lethal Rates for Various

Temperatures and Values of

Se

Tonecmtare eS Ge Us ee on:

So

EL MEN IN TS Come-up time Corricted zero

°

s

s

8

r)

g

$

TIME IN MINUTES

Fig. 25.18. Straight line heating curve

Come-up time x 0.58 = 10 X 6.58 = 5.8 min I= RT —IT=240°—140°= 100° F jl=240 —115 =125°R

J= jl/1 = 125/100 = 1.25 f,=36.9—8.4=28.5

min.

Determination of Thermal Process Time

779

on the logarithmic scale. Plot the temperatures as retort temperature minus can temperature. Rather than making this subtraction each time to denote the temperature difference on the log scale, turn the log paper upside down and

start numbering the temperature divisions from the top. Plot the actual temperatures from top to bottom. Denote to the top line a temperature 1° F less than the retort temperature. The temperature at the bottom of the first log cycle will be 10 degrees below retort temperature, and the bottom of the second log cycle will be 100 degrees below. Plot the time divisions on the linear scale, starting with zero time (steam on time) at the left. Plot the temperatures for the corresponding times, starting with the initial temperature. Draw a straight line through the points, ignoring the lag period, and attempt to position the line not more than 1° F from any point above 210° F for low acid products and 140° F for acid products. A comparatively large variation in the position of-the temperature points may be noted when they approach the retort temperature. However, the logarithmic scale in this area is large enough: to accommodate the points within -|- 1° F. Plot the cooling curve on a separate sheet. In this case, do not turn the semilog paper upside down. Mark the bottom line to represent 1° F above the cooling water temperature. In order to have a clear understanding of the formulac developed, and the concepts on which the development of mathematical methods are based, the meaning and significance of the following terms are important. Heat penetration factors I and f,: The heating curve plotted on a semi-log

paper is defined by the factors /I and f,. The factorjwas introduced by Ball*4°*® to locate the intersection of the extension of the straight line portion of the heating curve and the vertical line representing the beginning of the process, when no time is considered in bringing the retort to holding, or processing temperature (see Fig. 25.18).- Calculation of 7 is made from the expression :

_

Retort temp. — Theoretical initial temp.

J ~~

Retort temp. — Actual initial temp.

In the above expression, the theoretical initial temperature is found by ex-

tending the straightline portion of the heating curve until it intersects a vertical line representing the beginning of the process adjusted to include the lethal value contributed by the come-up period of the retort. The coming-up

time of the retort does not have the heating value of the holding temperature, but will have heating effect which will be more than zero minutes. Conventionally, the heating value of the come-up time of the retort is taken as 0.42 of the come-up time. Be Corrected

zero _

of process

Retort come-up

x 0.58

time

This means that after steam is let in, if 10 min are required for the retort to come up to the holding temperature, the corrected zero or the begirining of the process is (10 x 0.58) =5.8 min from the time steam is turned on.

780

Analysis of Fruit and Vegetable Products

Actual initial temperature is the temperature of the food at the time steam begins to enter the retort. The temperature difference between the retort temperature (RT) and the actual initial temperature (IT) is designated as J. Multiplication product of jandI or jI designates the point of intersection of the vertical line representing the corrected 0 or the beginning of the process by the straight line por-

tion of the heating curve. ; ; The factor j has a similar application with reference to the cooling curve when it is multiplied by the quantity 7, where,m is the difference between the

maximum

temperature

attained by the food at the cold point during the

heating of the can and the temperature of cooling water. ‘The slope of the heating curve is representcd by /),. It is equal to number of minutes on the time scale required for the straight line portion of the heating curve to traverse through one log cycle. It is easily ascertained by plotting the heat penctration data on semi-log paper. The slope of the cooling curve

is represented by f.. In the General Method, each temperature extant during the thermal process--

ing of a food is assigned a lethal rate value,while in the “Improved General Method,” it is designated by L value. Any method which sums up the lethal rate values or L values representing all temperatures existing during the process, multiplied by the times during which the respective lethal temperatures were operative will express the total lethal value of a process. The General Method accomplishes this graphically while the Formula Method*™ accomplishes the summation mathematically. Heating and cooling curves drawn on semi-log paper represent the centre temperatures of a can during thermal processing. During heating at the cold point, the can attains a maximum temperature which is slightly lower than the operating retort temperature. This difference in degrees Fahrenheit between the retort tempcrature and the maximum temperature attained in the can at the cold point is called g. Immediately after processing, the steam is turned off and the can is cooled by plunging in cold water. Since g is the difference between the retort temperature and the maximum tcmperature attaincd, the cooling water is » +g degrees below retort temperature.

Ball** developed three equations to describe the theoretical curves representing centre temperature of food during process, vjz.,(i) an equation of the heating curve (logarithmic), (ii) an equation of the first part of the cooling curve (hyperbolic), and (iii) an equation of the cooling curve (logarithmic). These equations were integrated. The limits of integration were taken as g° F and 80° F (since a temperature which is 80° F lower than the processing temperature will have very little lethality). The formula derived was:

F=AXLXC where, F = sterilizing value of the process expressed as minutes at the reference temperature in °F,

Determination

of Thermal Process Time

781

fn = time in minutes required for the heat penetration curve plotted on logarithmic paper to traverse one log cycle, I, = lethality corresponding to the maximum temperature at the cold point of the can, and

C = a value obtained from the tables.

The tabulated value of C was calculated from the equation

C= (Ch + Cn + Ca)/m where, Cy, Con and Cy are the contributions to C from the heating, the initial

cooling and the final cooling phases respectively; and p is In (10). Steele et a/.® have given the formulae used by Ball and Olson for calculating Cy, Ca and Cy.

Since the value of C is a function of g, m and x, Ball™ tabulated the necessary values of variables g, # and x. From these ¢ : g curves were constructed for

different values of y and m + g. To evolve the processing time, f, is obtained from the heating curve. A value of g, corresponding to a certain value of ¢ on the TDT curve and to a certain value of Con the ¢: g curves, is ob‘tained. The value of g obtained referred to the heating curve gives the length of process necessary. While using the above formula in calculating process times, Ball** found that when considering a single value of ~ + g and a single value of ¢, any given value of the ratio ts (where,U is the timein minutes required to destroy

an organism at retort temperature,) has a value of g corresponding to it.

On the basis of these findings, Ball®* has presented graphs in whichtn — 7 Values were plotted against g values giving oe: g curves for the different ais of (m + g) and x (Fig. 25.19). (In the figure, the term afaeinstead o biais used).

Steele et al.©° using computer found that C:g tabulated values evolved by Ball and Olson?# by the mathematical technique may be upto 26% higher. This means that

most F values calculated from C: g table are over estimates, and reduce the safety factors associated with thermal processing. The f,/U:g values are equal to

fa/U = exp (g/z')/C where,z’ = z/ in which pu is In (10)

~

Steele et al. have published revised f,/ U:g values derived from C:g values. These are given in Table 25-20.

Ueki

2B;

where ys is the TDT at retort temperature, F is the time in minutes required to

destroy the organism at 250° F and F; is the time required to destroy a given

782

Analysis of Fruit and Vegetable Products

organism at retort temperature (RT) when F is equal to one. This is calculated from the expression: 250 — RT F; =log™

z

F; values for different retort temperatures and 04

values are given in Table 25-21.

06086!

m+9*!30°

(recess iemoaretre FEE

a

SA:

— =

ae vit

ea

| |

wad

a)

ZB 7M

ate

apa |

AA is _A

Af |

VACA

ZY

Y

id =

Wer

Fig. 25.19. (Reprinted from

C. O. Ball

&

Hill Book Company, New

Relation between f/u and g, m, z. F. C. W.

Olson,

Sterilization in Food Teistalogy, McGraw

York, 1957, p- 339, with

permission

of the publishers.)

When these values are substituted in the equation of the heating following equation is obtained:

curve, the

By = fy, (logjl— log g) where, Bg = process time in minutes from the corrected zero time to the end of the heating period. The lethal heat conferred during cooling is

accounted for by the equation of the heating curve through the relationship of & to the ratio of f,/U.

—

For processes where thezy =h yalue is. below 0.5 and the g value is below 0.1, Stumbo’ has given the ee gives quite close approximation.

ag

sik which according

By = U + f, (log jl— 0.85)

to him

783

Determination of Thermal Process Time

TABLE 25-20: g Values for m + g = 130 z=22

4.000 4.500 5.000 5.500 6.000 6.500 7.000 7.500 8.000 9.000 10.000 12.500 15.000 17.500 20.000 25.000 30.000 35.000 40.000 45.000 50.000 60.000 70.000 80.000 90.000 100.000 150.000 200.000 250.000 300.000 350.000 400.000 500.000

0.0727 0.1108 0.1576 0.2127 0.2753 0.3446 0.4199 0.5004 0.5854 0.6743 0.7665 1.2605 1.7800 2.3008 2.8114 3.3062 3.7828 4.2408 4.6803 5.1020 5.5069 5.8960 6.2702 6.9776 7.6362 8.2519 8.8298 9.3744 9.8890 10.3769 10.8407 11.7050 12.4967 14.2281 15.6935 16.9655 18.0896 20.0113 21.6187 23.0017 24.2165 25.3000 26.2784 27.9905 29.4559 30.7374 31.8765 32.9020 36.8990 39.7747 42.0218 43.8648 45.4258 46.7775 49.0351

0.0667 0.1016 0.1446

0.1951 0.2524 0.3160 0.3851 0.4589

0.5369 0.6185 0.7030 1.1561 1.6324

2.1101 2.5784

3.0321 3.4692 3.8891

4.2922

4.6789

5.0502

5.4069

5.7500 6.3987 7.0025 7.5671 8.0969

8.5961 9.0679 9.5153 9.9405

10.7328 11.4585 13.0457 14.3889 15.5548 16.5852 18.3467

19.8201 21.0881 22.2017 23.1953 24.0925 25.6630 27.0076 28.1839 29.2299 30.1719

33.8482 36.4996 38.5767 40.2849 41.7356 42.9964

45.1081

z= 20

0.0607 0.0548 0.0926 0.0836 0.1317 0.1189 0.1777 0.1605 0.2076 0.2299 0.2878 0.2599 0.3168 0.3507 0.4180 0.3775 0.4416 0.4890 0.5087 0.5633 0.5783 0.6403 1.0529 0.9509 1.4867 1.3427 1.9217 1.7356 2.3481 2.1207 2.4938 2.7613 2.8533 3.1594 3.5418 3.1987 3.5301 3.9088 4.2610 3.8481 4.1534 4.5990 4.4467 4.9239 4.7288 5.2363 5.2622 5.8269 6.3767 5.7587 6.2228 6.8907 6.6584 7.3731 7.8276 7.0688 7.4566 8.2572 8.6644 7.8243 8.1739 9.0515 8.8251 9.7728 10.4336 9.4215 11.8783 10.7259 13.1010 11.8296 14.1622 12.7876 15.1000 13.6340 16.7032 15.0811 18.0442 16.2914 19.1981 17.3328 20.2117 18.2475 21.1159 19.0635 21.9326 19.8004 23.3621 21.0904 24.5861 22.1949 25.6570 23.1614 26.6095 24.0209 27.4673 “24.7952 30.8174 27.8190 33.2363 30.0031 35.1336 31.7172 36.6961 33.1296 38.0250 34.3315 39.1820 35.3783 41.1236 37.1368

z=18

0.0491 0.0747 0.1063: 0.1435 0.1856 0.2324 0.2832 0.3375 0.3948 0.4548 0.5170 0.8502 1.2004 1.5516 1.8959 2.2295 2.5509 2.8596 3.1559 3.4402 3.7132 3.9754

z=16

z= 14

z=12

0.0433 . 0.0376 0.0320 0.0660 0.0573 0.0488 0.0939 0.0816 0.0694 0.1266 0.1100 0.0936 0.1639 0.1424 0.1212 0.2052 0.1783 0.1517 0.2500 0.2172 0.1848 0.2980 0.2589 0.2203 0.3486 0.3029 0.2577 0.4015 0.3489 0.2968 0.4564 0.3966 0.3374 0.7505 0.6521 0.5548 1.0598 0.9208 0.7834 1.3699 1.1902 1.0126 1.6738 1.4543 1.2373 1.9683 1.7101 1.4550 ~ 2.2520 1.9567 1.6648 2.5246 2.1935 1.8663 2.7861 2.4207 2.0597 3.0371 2.6388 2.2452 3.2781 2.8482 2.4234 3.5096 3.0493 2.5945 2.7591 4.2267. 3.7322 3.2428 4.7043 4.1531 3.6085 3.0702 5.1481 45449 3.9489 3.3599 5.5630 49112 4.2671 3.6307 5.9524 5.2549 4.5658 3.8848 6.3192 5.5788 4.8471 4.1243 6.6659 5.8848 5.1130 4.3506 6.9947 6.1750 5.3652 4.5650 7.3071 6.4508 5.6048 4.7690 7.8891 6.9647 6.0513 5.1489 8.4223 7.4353 6.4601 5.4908 9.5879 8.4643 7.3542 6.2576 10.5745 9.3350 8.1107 6.9013 11.4305 10.0906 8.7671 7.4598 12.1870 10.7582 9.3472 7.9534 13.4801 11.8996 10.3387 8.7971 14.5615 12.8540 11.1679 9.5027 15.4920 13.6751 11.8812 10.1097 16.3093 14.3963 12.5078 10.6428 17.0383 15.0397 13.0666 11.1184 17.6967 15.6206 13.5713 11.5478 18.8491 16.6375 14.4545 12.2993 19.8357 17.5080 15.2107 12.9427 20.6989 18.2696 15.8722 13.5055 21.4667 18.9469 16.4605 14.0061 22.1583 19.5570 16.9904 14.4569 24.8590 21.9394 19.0593 16.2171 26.8099 23.6601 20.5534 17.4881 28.3410 25.0105 21.7259 18.4855 29.6028 26.1232 22.6920 19.3073 30.6767 27.0704 23.5143 20.0067 31.6128 27.8950 24.2306 -20.6162 33.1845 29.2830 25.4344 21.6401

z=10

0.0265 0.0403 0.0574 0.0774 0.1002 0.1254 0.1528 0.1821 0.2131 0.2454 0.2790 0.4587 0.6477 0.8373 1.0231 1.2031 1.3765 1.5431 1.7030 1.8567 2.0039 2.1454 2.2816 2.5389 2.7782 3.0021 3.2123 3.4102 3.5974 3.7747 3.9434 4.2576 4.5453 5.1744 5.7068 6.1687 6.5769 7.2747 7.8582 8,3602 8.8011 ott 9.5496

10.1712 10.7033 11.1688 11.5828 11.9556 13.4114 14.4627 15.2875 15.9672 16.5456 17.0495 17.8961

z=6

z=8

0.0210 0.0320 0.0455 0.0614 0.0794 0.0995 0.1212 0.1444 0.1690 0.1947 0.2213

0.3639. 0.5138 0.6641 0.8115 0.9543 1.0920 1.2242 1.3510 1.4727

1.5896 1.7019 1.8098 2.0140 2.2040 2.3816 2.5483 2.7054 2.8538 2.9945 3.1283 3.3776 3.6059 4.1051 4.5275 4.8940 5.2180 LTA, 6.2348 6.6332 6.9831 7.2953 75771 8.0705 8.4928 8.8622 9.1908 9.4868 10.6423 11.4767 12.1314 12.6709 13.1300 13.5299 14.2019

0.0156 0.0238 0.0338 0.0456 0.0590 0.0739 0.0900 0.1073 0.1256 0.1446 0.1644 0.2703, 0.3817 0.4934 0.6029 0.7091 0.8113 0.9095 1.0038 1.0942 1.1810 1.2645 1.3447 1.4964 1.6376 1.7696 1.8935 2.0102 2.1205 2.2251 2.3245

2.5098

2.6795 3.0504 3.3645 3.6369 3.8777 4.2893 4.6336 49298 5.1899 5.4220 5.6316 5.9984 6.3124 6.5871 6.8314 7.0515 7.9108 8.5313 9.0182 9.4195 9.7609 10.0583 10.5583

Reprinted from Steele et al®° with the permission of Institute of Food Technologists ©

Analysis of Fruit and Vegetable Products

784

TABLE 25-20: g Values for m+g= fe/U

7=2%6

2222

z=24

2220

22182216

a

z=14

180 z=12

z= 10

ee

z=8

z=6

as

500

.0710

.0652

.0595

.0539

.0482

0426

0370

0316

0261

.0208

0154

550

0.1083

.0995

.0907

.0820

.0735

.0650

.0565

.0481

0399

0316

.0236

.1045 .1168 .1291 .1415 .1410 1574 .1741 .1908 2037. = 1825. 2253 .2470 ~=««.3092.—«2820.=2'«s«2552 = .2284.—Ss 3769 3438 3110 2783 .4489 4095 3705 3317.

.0924 1246 «1613S 2019 2460 .2932

0685 .0804 0924 .1084 s«1195. «1403'S ~—«=«1757'—s«1497) 2140 .1823 2551 2173

0567 0765 0990S“ Ss«1239:

1539 @0 .2078 650 .2689 700 750.3365. 800 4100 850 4887

900 950 1.000

5718 .6587. .7487

.5253 .6050 .6879

1.250 1.500 1.750 2.000 2.250 2.500 2.750

1.2310 1.7384 2.2470 2.7454 3.2283 3.6936 .4.1405

1.1310 1.5970 2.0643 2.5221 2.9658 3.3934 3.8040

3,000

4792 5519 6276

.0335 0450 0452 .0607 0786 = .0585 «0984 = 0732

.1510 .1799

1199 1430

2543. 2929 3329

2106) 2425. .2756

.1672 ~— 1245 .1926. .1433 2190 .1629

4335 4991 5675

3880 4471 .5081

«=3431. 3952 4492

1.0317 — 9332 1.4567 1.3177 1.8828 1.7031 2.3008 2.0810 2.7056 2.4471 3.0953 2.7998 3.4702 3.1386

8355 1.1795 1.5248 1.8631 2.1910 2.5067 2.8102

.7387 1.0429 1.3479 1.6472 1.9368 2.2162 2.4844

6426 _ 5475 9072 7731 1.1728 9992 1.4330 1.2209 1.6852 1.4357 1.9281 1.6428 2.1615 1.8415

4533 6400 8273 1.0109 1.1888 1.3601 15247

3601 5085 6572 8030 9444 1.0805 1.2112

2985) :3439 3908

.0893 ~—-.1064

.2679 .3783 4890 5976 7027 .8040 9014

4.5695

4.1980

3.8296

3.4638

3.1012

2.7417

2.3854

2.0323

1.6827

1.3368

.9948

3.250 4.9812 3.500 - 5.3762 3.750 5.7559 4.000 6.1208 4.500 68110

4.5763 4.9393 5.2880° 5.6235 62575

4.1746 4.5056 4.8238 6.1298 5.7082

3.7760 4.0754 4.3633 4.6399 5.1632

3.3806 3.6488 3.9064 4.1543 4.6228

2.9887 3.2259 3.4536 3.6727 4.0869

2.6003 2.8066 3.0048 3.1955 3.5558

2.2155 2.3913 2.5601 2.7225 3.0297

1.8343 1.9800 2.1198 2.2543 2.5085

14572 1.5729 1.6840 1.7909 1.9929

1.0844 1.1705 1.2532 1.3327 1.4831

5.000

7.4536

6.8477

6.2466

5.6502

5.0590

4.4725

3.8913

3.3155

2.7452

2.1809

1.6230

5.500 6.000

8.0540 8.6176

7.3996 7.9173

6.7499 7.2224

6.1056 65329

5.4665 5.8491

4.8329 5.1713

4.2049 4.4992

3.5827 3.8336

2.9665 3.1742

2.3568 2.5218

1.7539 1.8767

6.500 7.000 7.500

9.1486 9.6502 10.1259

8.4052 8.8661 9.3033

7.6673 8.0880 8.4867

6.9354 7.3160 7.6766

6.2096 65503 6.8731

5.4899 5.7911 6.0766

4.7766 5.0387 5.2871

4.0698 4.2931 4.5049

3.3699 3.5548 3.7301

2.6773 2.8242 2.9635

1.9924 2.1018 2.2055

8.000 9.000 10.000 12.500

10.5780 11.4203 12.1917 13.8781

9.7187 10.4927 11.2016 12.7508

8.8658 9.5719 10.2181 11.6329

8.0195 86582 9.2432 10.5224

7.1802 7.7522 8.2757 9.4211

6.3481 68537 7.3171 8.3298

5.5233 5.9633 6.3663 7.2478

4.7061 5.0811 5.4247 6.1753

3.896% 4.2074 4.4920 5.1138

3.0959 3.3427 3.5689 4.0627

2.3041 2.4877 2.6559 3.0236

15.000 17.500

15.3049 16.5429

14.0625 15.1997

12.8285 13.8670

11.6044 12.5435

10.3902 11.2310

9.1865 9.9300

7.9927 8.6399

68104 7.3623

5.6400 6.0967

44811 4.8438

3.3352 3.6052

20.000 25.000 30.000 35.000 40.000 45.000

17.6366 19.5047 21.0667 22.4112 23.5908 24.6425

16.2049 17.9237 19.3607 20.5958 21.6811 22.6485

14.7846 16.3525 17.6628 18.7908 19.7810 20.6650

13.3736 14.7919 15.9774 16.9978 17.8947 18.6943

11.9737 13.2443 14.3060 15.2201 16.0223 16.7388

10.5865 11.7095 12.6491 13.4566 14.1664 14.7992

9.2120 10.1886 11.0063 11.7094 12.3268 12.8778

7.8491 8.6821 9.3787 9.9780 10.5040 10.9738

6.4999 7.1901 7.7666 8.2629 8.6992

5.1648 5.7127 6.1714 6.5658 6.9122

3.8440 4.2522 4.5937 4.8873 5.1455

9.0883

7.2218

5.3753

50.000

25.5917

23.5228

21.4621

19.4155

17.3850

15.3713

13.3753

11.3975

9.4392

7.5007

5.5835

60.000

27.2533

25.0513

22.8580

20.6789

18.5162

16.3718

14.2459

12.1394

10.0537

7.9894

5.9473

70.000 80.000

28.6738 29.9160

26.3593 27.5033

24.0530 25.0975

21.7604 22.7067

19.4843 20.3313

17.2277 17.9774

14.9913 15.6429

12.7751 13.3309

10.5801 11.0404

8.4073 8.7733

6.2587 6.5312/

90.000 100.000 150,000 200.000 250.000

31.0201 32.0131 35.8826 38.6646 40.8396

28.5205 29.4362 33.0071 35.5803 37.5953

26.0274 26.8631 30.1302 32.4856 34.3321

23.5474 24.3053 27.2626 29.3966 31.0722

21.0856 21.7635 24.4131 26.3261 27.8266

18.6435 19.2439 21.5863 23.2783 24.6052

16.2231 16.7448 18.7838 20.2564 21.4122

13.8248 14.2700 16.0077 17.2631 18.2471

11.4499 11.8186 13.2581 14.2976 15.1138

300.000 42.6235 350.000 44.1358 400.000 45.4480

9.0990 9.3918 10.5364 11.3631 12.0119

6.7735 6,9920 7.8442 8.4601 8.9433

39,2527 -35.8537 40.6597 37.1458 41.8822 38.2714

32.4509 33.6240 34.6457

29.0629 30.1155 31.0321

25.6993 26.6300 27.4397

22.3634 23.1735 23.8793

19.0587 19.7493 20.3508

15.7858 16.3582 16.8559

500.000

12.5464 13.0007 13.3968

9.3413 9.6798 9.9750

43.9326

36.3632

32.5716

28.8029

25.0654

21.4615

17.6940

14.0631

10.4712

47.6382

40.1584

——

Reprinted from Steele et a/.© with the permission of Institute of Food Technologists ©

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785 of Thermal Process Time Determination

786

Analysis of Fruit and Vegetable Products

To compare the process time evolved by the formula method with the general method or the improved general method, add corrected zero time i.e., 58% of the come-up time to the process time calculated by formula method. Examples 1 and 2 illustrate the use of the formula method.

When the heating is by convection, the semi-log plots of heat penetration data do not always fall on a straight line. Hence, excluding the initial lag period, it is advisable to apply the analysis of least squares described earlier for plotting the TIT, TDT or thermal resistance curves (see page 747) to plot heat penetration data, and for finding f, and f..

In evolving the formula method, Ball assumed f, to be equal to f., and /, to be equal to 1.41, which may not be the case38. Stumbo and his coworkers’ found fn/U:g to be virtually independent of the process parameters such as can size, initial temperature of the product, processing temperature, cooling water temperature and / of the heating curve (/,), but to be greatly dependent on the j of the cooling curve (j-). Hence, they revised the earlier tables of Ball? and presented new tables of f,/U:g for different values of 7, and z. In evolving the process time of foods in which the heat penetration into the cans is by convection, great caution is necessary. In the calculation of process time of canned tropical fruits processed in boiling water, and in which the heat penetration into the canned product was by convection, various methods were examined. Of them, process time calculated by Ball's formula using f, and f, found by applying the analysis of least squares, and g corresponding to actual j, from fn/U:g tables of Purohit and Stumbo’ gave values very near to the values found by the graphical method.

Products which Exhibit Broken Heating Curve Products such as thick soups, mixed vegetables, syrup packed sweet potatoes, cream style corn, etc. exhibit broken heating curves. From initial convection heating, a definite shift to conduction heating is observed during the process. For want of a precise method of evaluation, all such products are treated as if they are heated by convection. The heating curves of such products, after the initial lag, may be represented by two straight lines. The formula method should be employed with great caution for evaluating the products in which the heating curve shows only one break. The heat penetration data should show that the break in the curve occurs consistently at the same place. Further, the heat penetration data for one set of conditions should not be used for another. A typical broken heating curve is illustrated in Fig. 25.20.

Determination of By : When the heating curve is broken, the process time is calculated by the equation : Bp

=f,

log jl af f, ie

log Lip

—f,

log She

Determination

of Thermal

Process Time

787

where, f,= time required for the second straight line portion of the heating curve to traverse one log cycle and can be determined from heat penetration curve, £;,, = Difference between retort and can temperatures (i.e.,g at which the curve suffers a change in slope). If the x,, (time ftom the beginning of the process to the point of break in the heating curve) is noted, g,, may be calculated by using the equation: log 2,, =

logjI — Sh

249

faba? Be :G23 bed 2] RE Le NL ea PAS Pa AGUS mG bmi bieiebilesiiy cdoo aLEE Peale = Pee tb VST

X rf eee i

rune (iia

(Logarithmic Scate)

TEMPERATURE DEGREES IN F 170

| i ial ee ew

150

ee S|

Sw

jess

410

{2 a

BeBe aii a

OT PS ee Seeih UES eeSe

a i ae 2 2 ae

EES EEE ALh BSE

Corrected zero of process

0

4

20

30

40

50

TIME IN MINUTES (Linear Scale)

Fig. 25.20. Broken Heating Curve

Sa =

58.2

On

=

49.2

Lbpn

=

250°

=

242.3-

=7.7°

Sh = 13.6 — 4.1 = 9.5 min.

= RT — IT = 250° — 155° = 95:0F Jf Ber,

BNP

OS

JI = 250° — 130° = 120°F Retort come-up

time X 0.58 = 6 X 0.58 = 3.5 min.

788

Analysis of Fruit and Vegetable Products

8h> is g at the end of the heating curve. Calculate fx/Un2 and using this value,

using g tables (Table 25-20). fr/Un2 may be calculated

find g from fh/U:.

the following expression: SrlUno

==

To solve the above expression, r,, (a proportionality factor corresponding

to g value), F; and f,/U,,, have to be found. Find rz, corresponding to gz, from Fig. 25.21. Find F; corresponding to retort temperature using the expression: 250 —- RT

B, =) logtt moat

or from Table 25-21.

Find f;,/Uz, value to the corresponding gy, value (Table 25-20). From the calculated f,/U,, value, find the corresponding g,,. value from Table 25-20.

Calculations involved are shown in Problem 4. Determination of F : To find F when the heating curve is broken, the following formula may be made use of : a2

F

Fy

_m Tbh

(fn{Ung) r,

(f, ~~ fy)

Fj filUen)

To find f,/Uj,, calculate g,, using the expression :

om ea ee et

,

bh

and find fr/U», from Table 25-20.

To find f,/U,,, calculate Shy using the expression :

Us = Thy OS on — Ba dpcumae om at toma 8 fag froggy 2

;

and find fr/Un2 from Table 25-20.

r,, = A proportionality factor corresponding to g value at which the curve suffers a break in the slope. Find r value corresponding from Fig. 25.21.

Example 5 illustrates the calculations involved.

to the g,, value

Determination of Thermal Process Time

Fig. 25.21 : ton—

789

log gj, relationship

(Courtesy: American Can Co.)

Correction for Lethality During Cooling From the heat penetration curve ¢Fig. 25.22), it will be seen that in the cooling phase > a significant amount of lethal heat is being extended on the surviving organisms. From the Fig. 25.22 > it will be seen that the slope of the

cooling curve (f,) is almost equal to the slope of the heating curve (f,). The

lethality during the cooling phase has been taken into account in the formulae in the case of a product which does not exhibit broby assuming that f, a

ken heating curve (Fig. 25.18) and to

hr in the case of broken heating curve

(Fig. 25.20). This assumption does not introduce significant error. However,

in the case of products with broken heating curves, which are initially heated

790

Analysis of Fruit and Vegetable Products

247

Bsn teaSs Sees |}

=e

168

Gees Fs -_

oe

ee

u 238-4

re

ceases FeelB70

88

lmao (BREN 5 0 REG2 © sea

=

an

££

—s

|S

(ay, 4 5 BES

rrp

tip) &: |

n

eH

=

Bee

Q

tye ph (ied SSE] Su!

70°

09l48R oF

(a oO

a

EE EES oO

Heating

20 30

Time

in Min.

Cooling

Fig. 25.22. Heating and cooling curves. (These curves have to be plotted separately on

different graph papers. See page 778 for procedure)

by rapid convection and then by slow convection, initial cooling may occur by fast convection in which case, the slope of the cooling curve would be closer to f, than to f,. Under such conditions, a correction would be required to the cooling phase. An empirical method for making corrections for the cooling curve

depends

on a correction to the log g,, value depending on the value of Se For constant values of ‘B, the value of log g,, is decreased by 0.007 for each 0.1 de-

viation from unity of the ratio fe‘ The value of log g,, may thus be corrected

f

by adding or subtracting the term 0.07 (1 — 7) When calculating processing

2 time (Bg) it is subtracted and when calculating sterilizing value (F), it is added. These are illustrated in the calculation of problems

4 and 5.

Conversion of Process Time According to Can Size °

If the heating rate f, of a product in one size of can is known, the heating rate of the same product in other sizes of cans may be calculated. The / value is assumed to be invariable with the change in the size of the can.

Determination of Thermal Process Time

791

Simple Heating Curve Calculation of Processing Time (Bz)

Problem

1

Problem

1.

Data

2.

RT (Retort temperature)

230° F

240° F

3.

IT (Initial temperature)

160° F

160° F

4. J

2

1.25

5» Sh

60

40

6.

12.1

3

18

18

130

180

F

Fe

8.

mt+e

9.

Calculation

“to.

2

I = RT — IT

250—160 = go

240—160 = 80

11.

jl

2 X go = 180

1.25 X 80 = 100

12.

log jl

2.253

2

13. Fj (from Table 25-21)

“4

3-594

14.

U=FxF;

12,1X1=12.1

3X 3.$94=10. 782

15.

Srl U

6o/12.1=4.96

40/10. 78= 3.71

16.

g corresponding ‘to f,/U (from Table 25-20)

5.1126

3.8652

17. log g

0.7086

18. -Bp =f, (log jl — log g)

60 (2.2553 — 0.7086)

0.5872 40 (2 — 0.5872)

= 92.8 min.

_ = 56.5 min

Products in which heating is by conduction : The heating rate f, is a function of can size and shape, and thermal diffusivity of the product. Can factor

“tee

Thermal diffusivity

Thermal diffusivity is constant for a specific product, iA=

Can factor A —————._.—_——

Can factor

Thermal diffusivity A

p B= an d fi

Thermal diffusivity B

where,.A and B are two different can sizes. Since thermal diffusivity of can A is equal to that of can B,

San Can factor A

Therefore, f,B

B

———_____—__—_.

ala Can factor B

= f, A X conduction factor (B/A) -

792

Analysis of Fruit and Vegetable Products

Simple Heating Curve Calculation of Sterilizing Value (F) Problem 3 1.

Data

me

de

240° F

gen

160° F

Hise Ff]

1.25

5+ Sh

m8

Gs

&

18

7

Bp

56.5f

8

m+e

180

oy Ff

To be calculated

to.

Calculation

1.

I= RT — IT

240 — 160 = 80

12 nuh

1.25, X \8O.== \1090

13.

log jI

-

14.

log g = log jl — =

Gin

4

= he 40 = 0.5875 3.8681

16. f,/U corresponding to g.

.

(from Table 25-20)

17.

Fy (from Table 25-21)

=

tn

(falU) Fi

Ee

3.7128

3.594

«437128 X 3-594 set aie

ee

Determination of Thermal Process Time

793

Broken Heating Curve Calculation of Processing Time (Bz) _————————— a

Problem 4 $$

240° F 140° F

1.2 12.2 50 13.65

10 18° F 180° F

SOS ae eem+ Z Calculation 10.

I= RT —

IT

II.

jl

12.

log gy, = logjI

240 —

100 X1.2=120 13.65

— bh

13. bh

140 = 100

2.0792

Sh

—-——— Taz

= _0.9603

9.1264

14. f,/Uy, corresponding to g,, of 9.1264

11.8568

(from Table 25-20) I§-

0.74

rpp cotresponding to log gy, of 0.96 (from Fig. 25-21)

F; corresponding to 240° F and z = 18 (from Table 25-21).

Sal Ung= 2 16.

fo

Exe 4

®

—

3-594 50

10 X 3.594 +

Sal

0.74 (50 —

12.2)

= 1.3055

11.8568

0.9119

Sh_ cotresponding to Sn{ Une Of 1.3055

(from Table 25-20) =

Bg==f, logjl+ (fa Sh) 108 gon

12.2 X log 1204+. (50 —

18.

12.2)log

9.1264 — 5° X log 9.9119 =,63.67

— fz log gns

min

=

When f, = fh, correction for

108 Zh, = log 8h,—0.07 (: Be *)

log 0.9119

=

— 0.07 G =

12.2 ) 50

0.093

2

19.

Bp = 12.2 X log 120 E: (50 —

12.2) log 9.1264

—50

(— 0.093)

=

663 min.

Nore : When f, == /2, calculate Bp as in column 17. When it is equal tof, apply correc-

tion for log g,, as shown in column 18, and calculate Bp as in column 19.

794

~~ Analysis of Fruit and Vegetable Products Broken Heating Curve Calculation of Sterilizing Value (F) Problem 5

Data

1.

RT

2, at 3.

ee

240° F

-

140°F

.

12.2

J

1.2

4:

Sh

5-

ha

aS

pi)

a0

13.65

6.

Xbh

7

2

8

m+ez

Ric

180

9.

Bg

a

64

F

aie

To be calculated

ae

240 — 140 = 100

as

1.2 X 100 = 120

10.

18

Calculation wu.

12.

J=RT — IT jl ;

x

13.

log gp, = log jl — a

14.

£bh

13.65

ae

Sn

15. fnlUs, corresponding to gp Of 9.124

log 120 — and SE 0.9603

12.2

A

9.1264

=

11.8568

(from Table 25-20)

Phen

_

Srlogjl+ (fe — Sn) log 8p, — Bp

2

Ss

12.2 X 2.0792+(50 — 12.2)0.9603 on

=

16. Bh, 1]. fh/Up2 corresponding to gh ot 0.8981 (from Table 25-20) 18. Mn Corresponding to log gyn of 0.96 (from Fig. 25.21)

19,

=) So F=—-*—_

20.

When f, = fh, correction for

(fal Una) Fy

- 0.04669

**

0.8981

An

1.2955

Sc

(fa — Sh) ~ ron —“~*_“*

Ons 5°

Fi Srl Usa)

1.2955 X 3.594

;

log Sh, is log gq,+ 0.07 (: Ee 5

Ta »

6 @h2 is 1.014

22.

fr/ Une corresponding to gh value

Me

=

:

ig

=

of 1.0145 (from Table 25-20) 2

Fe

hh

(falUne) Fs

_

Tbh fs — Sn)

Fifn/Usa)

— 64

jo

0.74(50—12.2) a

3.594.K 11.8568

= 1110/03

12.2

:

-0.04669-+0.07( eure

"0.00623

1.38 Dy

50

1.38X3-594

9.74 (50 - 12.2)_ane

3.594 X 11.8568

=

Note : When f/, =/,, apply correcticn to log gjq'as in column 20 and calculate F as in column 23.

795 Determination of Thermal Process Time

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Determination of Thermal Process Time

797

BO

798

Analysis of Fruit and Vegetable Products

Conduction factors for different sizes of cans are given in Table 25-22. Procedure for using the table is given below.

Whenf, of can size 401 x 411 is 40 min, find the f, of can size 300 x 407. From Table 25-22,conduction factor B/A for can size 300 X 407 = 0.592. Therefore, f, = 0.592 x 40 = 23.68 min.

Products in which heating is by convection ; Generally, in homogencous products, the viscosities of which are not considerably different from that of water, heating rate is proportional to the surface/volume of the container which has been incorporated into a can factor by Schultz and Olson.™ The can factor may be further simplified into convection factors which are listed in Table 25-23. The procedure for using the table is same as described above for conduction heating.

The equation involved for determining the heating rate in glass jars when the same in cans is as follows:™ fa jar = fy can X factor for jar/can. The factors are given in Table 25-24.

The Nomogram

Method

The Nomogram Method developed by Olson and Stevens® is merely a gtaphical solution of the Formula Method developed by Ball.** Different nomogtams are required for canned products in which the heating is represented by a single straight line heating curve and by a broken or two- slope heating curve. These are given in Figs. 25.23 and 25.24 respectively. Careless operation and faulty constitution of the nomogram may give rise to erruneOus results.

A few examples are given to illustrate the calculation of problems by the Nomogram Method.

Calculation of Process Time Simple Straight Line Heating Curve Problem 6: The following data are given RT =257 IT = 160° j =2

Find Bz

tr FE.

F F

= 0 = 12.1

Proceed as given below: Step 1. On Fig. 25.23, connect Fy (12.1) on scale 1 to RT (250° F) on scale 4: get a point on scale 3.

Determination of Thermal Process Time

799

: Co.) Can America (Courte

a

-

N

e

om’ . \ x yi

—S

SR Sex

SS

HAY SS

\

CS

q

‘

SS

CEES ~>

AN

x Ore’

oS)

\\

heating nomogr Proces curve.

3 /

Fig. 25.23.

800

Analysis of Fruit and Vegetable Products

|

: mogram—b heating (Courtesy Co.) American Can curve.

Fig. 25.24. Process no:

Determination of Thermal Process Time

801

Step 2. Connect point on scale 3 to f;, (60) on scale 2 and extend to eet a new point on scale 4. Step 3. Follow parallel to the ladder lines between scales 4 and 5 from the new point on scale 4 to point on scale 5. Mark this point. Step 4. Connect 7 (2.0) on scale 5 to RT — IT (250 — siG0ic2 90) on scale 7 to get a point on scale 6. Step 5. Connect point on scale 6 to marked point on scale 5 and get a new point on scale 7. Step 6. Connect this new point on scale 7 with f, (60) on scale 8 and get By Which is 90.5 minutes. Note : There are two scales 8, one marked 8A ata the other 8B. The one

to use depends on which of the ladder arrangements between scales 4 and 5 was used. The A lines start on-the lower two-thirds of scale 4 and the B lines start on the upper third. Which ladder to use depends on the condition of the

problem and whichever is used, the corresponding scale 8 must also be ‘used. There are a few simple tricks that can be used to make problems workable on the nomogram which at the first glance might appear to be out of range. Bg on scale 9 of Figure 25.23 only includes the range 0 to 120 min, and process times longer than this are not uncommon. A little study will show that a line

passing through a given point on scale 7 intersects scales 8 and 9 so that the ratio of Bz to f, is constant. Therefore, the same ratio can-be maintained when both B, and f, are multiplied or divided by the same number. If By is 180 min, a line should be drawn between 180/2 = 90 and /,/2 which will give the identical point on scale 7 that would have been found if the line had been drawn

between the real values of Bz and /f,. _ A line drawn through a point on line’3 at any angle will intersect points on scales 1 and 4 so that for every increase in the value of RT by 18, the value

of Fo is increased tenfold. This means that if the retort temperature is 270° F,

the value on the scale can be taken as 252° F providing the value of F, is divided by 10.

Broken Heating -Curve Problem 7

F, = 10.0 Sy,

= 122

ti,

= 50

J

=12

4, = 13.65 RT = 240.0

©

802

Analysis of Fruit and Vegetable Products

Find By Proceed as given below : Step 1. On Fig. 25.24, connect xph (13.65) on scale 1 to fy (12.2) on scale 4 and extend to get a point on scale 5. Step 2. Connect / (1.2) on scale 7 to RT — IT (240 — 140 = 100) on scale 5 to get a point on scale 6. Step 3. Connect point on scale 6 to marked point on scale 5 and get g,, (9.4) on scale 7. Step 4. Follow the ladder from g,, 9.4 to point on scale 8. Step 5. Connect this point on scale 8 to f, — f, (50 -- 12.2 = 37.8) on

scale 11 and get U, (= 2.75) on scale 9 and mark. Connect F, (10) on Rete 6 with RT (240) on scale 8 and get a point on scale 7. Step 7. Connect this point on scale 7 with RT (250) on scale 8 and extend it to scale 6 to get U, (= 35.5). Step 8. Get U, by adding mentally U, from Step 7 to U, from Step 5. In this example,U, = 35.5 (see Step 7) and U, = 2.75 (see Step 5). Step 6.

Therefore,U, = 35.5 + 2.75 = 38.25.

Step 9.

Connect U, (38.25) on « scale 6 with f, (50) on scale 10 and get a new

point on Har ys Step 10. Follow the ladder from this point and get a point on scale 6. Step 11. Connect this point on scale 6 with z,, = 9.2 (from Step 3) on scale 7 and extend to get a point on scale 5.

Step 12. Connect this point onscale 5 with f,= 50.on scale 3 and extend to

Step 13.

scale 2 to get Ba —-:.2,, (= 50).

To get Bg mentally add Bg — x,, from Step 12 to x,,. In this example

By — x, (from Step #2)= 50. x, (given in the data)= 13.65. Therefore,B, = 50 + 13.65= 63.65 min.

Calculation of Sterilizing Value Simple Straight Line Heating Curve :

Problem 8

fy jf RT IT

= 12.0 =15 = 240° F = 170° F

'F, = To be found

Determination of Thermal Process Time

803

Proceed as follows :

Step 1. In Fig. 25.23, connect Bg (30) on scale 9 with ff, (12) on scale 8 and extend to scale 7 to get-a point. Step 2. Connect RT — IT (240 — 170 = 70) on scale 7, and / (1.5) on scale 5 and get a point on scale 6.

Step. 3. Connect this point got on scale 6 with the point marked in Step 1 on scale 7 and extend to scale5 to get a point. With the figures chosen, if it is found that the point does not come

of 5,.scale 8A chosen using scale 8B.

within the

scale

is wrong. Repeat the steps from Step 1

Step 4. From this point on scale 5, follow the ladder lines to scale 4 and get |

a point.

.

Step 5. Connect this point on scale 4 with f;, (12.0) on scale 2 and get a point: on scale 3, Step 6. Connect the point on scale 3 with RT (240) on scale 4, extend to scale

1 and read F,. (F, found should be within a few tenths of the figure

found by Formula Method). In this example, F, is 3.82. Broken Heating Curve:

Problem 9 Data given

Spe = 122 ft, = 50 Ys A2 a yis=o19765

IT = 140° F Bz = 63 min F, = To be found

RT = 240° F Step 1. In Fig. 25.24, connect xn (13.65) on scale 1 to fy (12:2) on scale 4 and extend to scale 5 to get a point. Step, 2. Connect j (1.2) on scale 7 to RT — IT (240 — 140 = 100) on scale 5 and get a point on scale 6. Step 3. Connect this point on scale 6 to marked point on scale 5 and extend to scale 7 to get g,, (9.2). Step 4.

Follow the ladder line from g,, on scale 7 to. a point on scale 8.

Step 5. Connect scale 11, Step 6. Connect on scale

this point on scale 8 to f, — f, (50 — 12.2 = 37.8) on and get U, (2.7) on scale 9. By— x, (63 — 13.65 = 49.35) on scale 2 to f, (50) 3 and extend to scale 5 to get a point.

;

804

Analysis of Fruit and Vegetable Products

Step 7.

Connect this new point on scale 5 to g,, (9.2 from Step 3) on scale

7 and get a new point on scale 6, Step 8. From this new point on scale 6, follow the ladder line to a new point on scale 7.

Step 9.

Connect this point on scale 7 to f, (50) on scale 10 and extend to scale 6 to get U, (40).

Step 10. Subtract mentally U, (2.3 from Step 5) from

U, (40 from Step 9)

to get U, (37.3). ‘Step 11. Connect U, (37.3) on scale 6 to 240 on scale 7.

on scale 8 and get a point

Step 12. Connect this point on scale 7 to RT (240° F) on scale 8 and extend to scale 6 to get F, (10.2). Other Methods of Process Calculation

The classical methods of process evaluation described in the previous pages are based on the destruction of spores at the cold point in the can. Stumbo®™ pointed out that the number of survivors is proportional to the spores present initially. He visualized the contents of the can to consist of a series of concentric shells ranging from one, the size of the container itself to one, the

size of a thin pencil lead at the can axis. With uniform distribution of bacteria, the volume of material and the number of spores subjected to the conditions at the slowest heating point of the can are very small and the number progressively increases from the central cylinder towards the periphery. Although the spores near the periphery receive more severe heat treatment than the centre, the chances of survival would be greater because of the larger initial

_number of spores. Therefore, the F value required at the surface of each of these cylinders increases with its area, which is indirectly dependent on its distance from the can axis. He, therefore, proposed that processes should be based on calculations of the chance of survival of a spore in the region of the can for which this is greatest. Ball®™ criticised this concept on the grounds that the probability of survival is loosely defined and should strictly be taken as the ratio of the number of survivors to the initial number of organisms in the same volume, location, etc. In the process of heating, since the cold point receives

less heat than any other, the probability of survival at this point is greater than elsewhere.

Stumbo,”™* Gillespy®’ and Hicks*! went a stage furthet and suggested that the most logical procedure is to add up, or integratc, the chances of survival over the whole volume of can. Each of these workers first determined the survival ratios for particular points or small regions in the can and then integrated

them over the whole volume. Although the algebra employed by these three workers differed,

application. % «

the three procedures

give essentially the same

results in

Determination of Thermal Process Time

805

In the methods developed by Stumbo, Hicks and Gillespy, an iso-j region that encloses somewhat one-tenth or less of the total can volume is of greater concern and the survival outside this iso-j region is negligible. However, very significant amounts of the heat-vulnerable quality factors may be lost in the outermost regions of container. Jen et al. have modified the formula of Stumbo for the.. determination of sterility to calculate the retention of the quality factors. They

assumed the slopes of the heating and the cooling curves to be equal in developing their formula, but they are generally different. Downes and Hayakawa have modified the formula of Jen et a/.® to take the difference between.the slopesof the heating and the cooling curves into consideration. In the count reduction system proposed by Yawager,*’ the cans inoculated with known concentrations of microorganisms are taken out from the processing line at

different intervals, and subcultured to determine the number of the surviving bacteria. The data so obtained are used to calculate the lethality of the process.This procedure does not require heat penetration data. The same principle can be. employed to determine the concentration of residual enzyme and chemical degradation taking place during the process. This method is specially suitable for evolving thermal! process by flame sterilization where it is difficult to get a reliable heat penetration data®. To determine the process time for conduction-heating products, in addition to the graphical and the formula methods described above, in recent. years, the

following methods have been proposed:— i. Method of Teixeira et a/. based on the finite difference method. ii. Method of Herndon e¢ al.”° where population distribution of slope indices

had been used in place of the traditional individual or mean single value of slope indices in process calculation. iii. Method of Flambert and Deltour’! in which the sterilization value is calculated by the numerical integration process using time-temperature relationship obtained by exact solution method of heat conduction. iv. Experimental formula of Hayakawa’? based on dimensionless parameters. v. Method of Lenz and Lund’} based on Fourier number. vi. Method of Steele and Board’4 based on sterilizing. ratios instead of temperature differences used commonly in thermal process calculation. Based on the formula method of Ball, computers have been used to calculate the

process time.””” Bimbert et ai’* connected the can thermocouple directly to an analogue computer which gave directly the sterilizing value of the process. Teixeira et al.?? developed a computer programme on the basis of the formula developed by Stumbo to calculate the F value and nutrient retention in conduction heating foods. Most of the research work has been devoted to the study of conduction-heating foods in which the heat penetration is slow. The temperature of the product at different points in the containers is different, and it is difficult to predict mathematically the temperature pattern of the canned food. Comparatively little

806

Analysis of Fruit and Vegetable Products

work has been done in the case of convection-heating products, as the temperature, and hence, the lethality is assumed to be uniform throughout the can. Procedures based on calculation of the lethality at single point are ideal for convection heating products as the temperature remains essentially uniform at all the points in the can due to the movement of the convection currents. Flash and High Temperature Short Time Processes” |

_ When high temperature (270° - 290° F) processes are used, .it is possible to reduce the holding time to a few seconds. The alignment chart (Fig. 25.25)

provides a means of computing the sterilization value of thermal processes for liquid and semi-liquid food products in the temperature range 260°—300°F. The equation on the alignment chart expresses the relationship. The factor z represents the “F to which the process temperature must be increased to compensate for a 90% reduction of holding time, while maintaining the same

sterilization value,

and

is usually

taken

as 18 for preliminary

~

x

Fe

cal-

400 300 260 :

0:16 0.15 0.14 o.13

Ww

ae & 20 > Zt.

9 -%

Or: 6

0.10

-

za

5 =

0.0 M 0.06 0.05

= $s 8 WwW

oc

on

z a

(7

Fig. 26.3: Tough string tester for French (green or wax) beans. (Source: FAO/WHO Codex Alimentarius Methods of Analysis for Processed Fruits and Vegetables (CAC/RM 36/39-1970))

retain the pod material for weighing. Fasten the clamp assembly to one end of the string. Grasp the other end of the string with the fingers (a cloth may be used to aid in holding the string) and lift gently. If the string supports the 250 g assembly for at least 5 sec, consider the bean unit as containing tough string. If the string breaks in less than 5 sec, retest the broken parts that are 13 mm or longer to determine if such portions are tough. Weigh the bean units which contain tough strings. CALCULATION % w/w pods containing tough strings

_ Pods containing a ee

tough strings (g) eee oe STO Test sample (g)

References

1. Official Methods of Analysis, Association of Official Analytical Chemists, PO Box 540, Benjamin Franklin Station, Washington 20044 DC, 10th edn., 1965.

2. CAC/RM 39-1970.

Total Solids, Insoluble Solids and Soluble Solids Total Solids

Total solids are considered as an alternate method to minimum drained weight in canned mature processed peas.

Examination of Canned Products

833

PROCEDURE

Prepare the sample as described earlier. In a flat bottom metal dish having a

tught-fitting lid, take diatomaceous earth like Supercel, Kieselguhr or Celite at the rate of 15 mg/sq cm, and dry at 110° C for 30 min. Cool in a desiccator and weigh. To each dish, add sufficient sample to give a dry residue of 9-30 mg/sq cm. Weigh as rapidly as possible to avoid moisture loss. Mix with filter aid, and distribute uniformly over the bottom of the dish, diluting with water, if necessary, to facilitate distribution. Reduce the moisture content to approximately 50% by drying on a water bath or hot air oven at 70° C. When the samples are apparently dry, complete the drying in a vacuum oven for 2 hr at 70° + 1° C (see Chapter 1). Cool the dried sample in a desiccator, weigh as soon as possible after the sample reaches room temperature, and calculate the per cent total solids content. Convert the percentage of total solids found by the above method to total solids on a percentage of water capacity as follows:

Total solids as a percentage of water capacity

%. Total solids X net contents Water capacity

Insoluble Solids Wash 20 g of the blended solids repeatedly with hot water, centrifuge after each addition of water and pour the clear supernatant on to a Buchner funnel containing previously dried (for 2 hr at 100° C) and weighed filter paper. Use a second paper, if necessary, if the first paper becomes clogged. After 4 or 5 washings, transfer the remaining insoluble matter to the filter paper, dry in uncovered dish for 2 hr at 100° C, cover, cool in a desiccator and weigh.

Soluble Solids Find by calculation as given below: % Soluble solids = % Total solids - % Insoluble solids Reference

Official Methods of Analysis,

12th edn., Association of Official Analytical Chemists, Washington

DC,p. 597 (1975).

Alcohol Insoluble Solids in Canned Peas and Corn

The quality of canned peas is determined by the maturity at the time of harvest. The maturity is related to the alcohol-insoluble solids (AIS) which consist mainly of starch, hemicellulose, fibre and proteins. Tender green peas have lower AIS values than mature ones. The generally accepted limits in U.S.A. for smooth-seeded and wrinkle-seeded varieties are 23.5 and 21% respectively. Taste tests have indicated that the AIS range for first quality size graded canned peas is 11-16%. In Grade C canned peas, the AIS of early type of peas are not more than 23.5% and of sweet type of peas not more than 21%.

834

= Analysis of Fruit and Vegetable Products

PROCEDURE

The following procedure is based on A.O.A.C. method’. After determining the drained weight, transfer the peas to a white enamelled pan and remove any foreign material. Wash the peas with water equal to double the volume of original sample. Drain the water from the peas by sieving through a screen. Grind the peas in a blender to a smooth homogeneous paste. Weigh 20 g of ground material into a 600 ml beaker. Add 300 ml of 80% alcohol, stir, cover _ the beaker, and bring to boil. Simmer slowly for 30 min. Fit into a Buchner funnel, filter paper of appropriate size, which has been previously dried and weighed. Before using for filtration, dry the filter paper in a flat-bottomed dish for 2hr at 100°C. After drying, cover with a tight fitting cover, cool in desiccator, and weigh at once. Apply suction and transfer the contents of the beaker to the Buchner funnel taking care to avoid running over the edge of the paper. Suck dry, and wash the residue on the filter paper with 80% alcohol until the washings are clear and colourless. Transfer the paper and the alcohol-insoluble solids to the same dish as that used for drying the filter paper. Dry uncovered for 2 hr at 100° C. Place the cover on the dish, cool in a desiccator, and weight at once. From this weight deduct the weight of the dish, cover and paper. CALCULATION

Alcohol-insoluble

solids% =

et

ee

Wt of sample taken for estimation

AIS content has also been successfully applied for determining the quality of canned corn. In Grade C cream style corn, the weight of AIS of the washed and drained material should not exceed 27% by weight of such material. In

Grade C canned whole kernel corn, the limiting line for AIS is 27% of the drained weight. Reference

1. Official Methods of Analysis, uth ed., Association of Official Analytical Chemists, Washington D.C., U.S.A.. (1970).

Determination of Calcium in Canned Vegetables Codex

method

(CAC/RM

38-1970)

based on AOAC

method

determination in vegetables and tomato products.

for

(Applicable to canned lima beans, potatoes and tomatoes) The procedt'ye involves complexometric titration of the calcium in the product after ashing and passage through ion exchange column with high phosphate capacity.

.

Examination of Canned

Products

835

REAGENTS

Prepare the reagents using glass-distilled or deionized water. 1. Potassium hydroxide—potassium cyanide solution: Dissolve 280 g KOH and 66 g KCN in 1 litre water. 2. Calcium carbonate: AR grade, dried for 2 hr at 285° C.

3. Hydroxynaphthol blue—calcium indicator:

Available from Mallinckrodt

Chemical Works. Store in dark. Use fresh supply of this indicator after one year. 4: Ascorbic acid 5. 5% HCl: Mix 3 volumes conc HCI with 22 volumes H,O.

6. 10% HCl 7. 5% w/v Sodium carbonate 8. 0.01 M Disodium dihydrogen ethylenediamine tetraacetate (EDTA) standard solution: Dissolve 3.72 g of EDTA (at least 99% purity) in water and make up to 1,000 ml in a volumetric flask. Weigh accurately enough CaCo, to givea titre value of about 40 ml with 0.01 M EDTA and transfer to 400 ml beaker. Add 50 ml

deep blue end point, using magnetic stirrer. Add last few ml of EDTA solution dropwise. Molarity of EDTA : mg CaCO3 solution EDTA (ml) X 100.09 APPARATUS

1. Titration

stand:

Fluorescent

illuminated,

such

as Titra-Lite

Precision

Scientific Co. or equivalent. . 2. lon exchange column: Approximately 20 X 600 mm, fitted with coarse porosity sintered glass disc and Teflon stopcock. Place 30-40 g moist Amberlite

IR-4B resin (anion exchange resin with high phosphate capacity) in 600-ml beaker, and exhaust with three 250-ml portions 5% NagCO3 or NaOH. Wash with

water until free of excess of alkali. Treat the resin with three 250-ml portions of 5% HCL, mixing thoroughly after each treatment. Rinse with water until the colour is removed and transfer with water to the column. Column is ready for use after draining water to the top of resin column. (Exchange capacity for phosphate is approximately 1,500 mg; therefore a number of aliquots can be passed through the column before regeneration is necessary. Rinse the column with approximately 250 ml of water before each use until the eluate is colourless). PREPARATION OF SAMPLE

A. Liquid from canned whole tomatoes: Drain the liquid from tomatoes, centrifuge and pass through fast paper. Weigh 100 g of filtrate into platinum or porcelain dish. Evaporate to dryness on a water bath. Ash at 500-525° C until apparently free of carbon (grey to brown). Cool, add 20 ml of water, stir with a glass rod, and add 10 ml conc HCl cautiously under watch glass. Rinse the watch

836

Analysis of Fruit and Vegetable Products

glass into dish, and evaporate to dryness on steam bath. Add 50 ml of 10% HCl, heat on steam bath for 15 min, and filter through paper for quantitative analysis into 200-ml volumetric flask. Wash the paper and the dish thoroughly with hot water.

Cool the filtrate, dilute to mark, and mix.

B. Canned vegetables: Thoroughly comminute the entire contents of the can

(representative portion from cans of diameter 8cm or larger in high speed blender). Weigh 50 g of sample (100 g if there is no declaration of added Ca) into platinum or porcelain dish. Evaporate to dryness, ash and tteat as for liquid from canned whole tomatoes. DETERMINATION

Transfer 50 ml or 100 ml aliquot of prepared sample to 250-ml beaker, and adjust to pH *. 5 with dropwise addition of 10% KOH solution using pH meter and magnetic sti: : er. Pass the sample through the resin column (column is in chloride form), adjust the flow rate to 2-3 ml/min, and collect the effluent in 400-ml beaker.

Wash the column thoroughly with 100 ml of water in two 50-ml portions. Pass first 50 ml through the colu:in at the same rate as samples. Pass second portion through at 6-7 ml/min. Finally pass enough water freely through the column to make a total volume of 250-300 ml. Mix thoroughly and adjust the pH to 12.5— 13.0 (using pH meter and magnetic stirrer) with KOH-KCN solution (approximately 10 ml). Add 100 mg of ascorbic acid and 200-300 mg hydroxynaphthol blue indicator. Titrate immediately with 0.01 M EDTA solution through pink to deep blue end point, using magnetic stirrer. CALCULATION

For 50 ml aliquot, % w/w Ca =

Titre value X 0.4008 X 4 X 100 Sample (mg)

For 100 ml aliquot,% w/w Ca =

Titre value X 0.4008 * 2 X 100 Sample (mg)

Results are expressed as per cent w/w Ca of the final product or of the packing medium, as appropriate. References

t. Anon, J. Assoc Offic, Anal. Chem., 49, 211 (1966); 51, 494 (1968)

2. CAC/RM 38-1970.

Mineral Impurities (FAO/WHO CAC/RM 49-1972

Method)

PRINCIPLE

Sand and other inorganic materials are separated from plant tissue by a process

of floatation and sedimentation. The sand and earthy particles being heavier, sink

Examination

to the bottom of the receptacle which

of Canned

Products

837

is collected, incinerated, weighed and

reported as “mineral impurities”. Technically, it can be considered as “waterinsoluble inorganic residue” and will include not only silica but also other matter such as particles of limestone. REAGENT

15% (w/v) NaCl solution. PROCEDURE

Blend the entire contents when the capacity is 500 g or less in a blender. When the capacity is more than 500 g, blend the contents, and quickly remove 500 g. Transfer the blended material to a 2-litre beaker taking care to include any sand that might settle out. Nearly fill the beaker with water and mix by swirling using a stirring rod,if needed. Allow to stand for 10 min, and decant the supernatant material and water into a second 2-litre beaker. Refill the first beaker with water,

mix and swirl, and again allow to stand for 10 min. Fill the second beaker with water, mix, swirl, and allow to stand for 10 min. At the end of 10 min, decant the

supernatant from beaker No. 2 to beaker No. 3, and from beaker No. 1 to beaker No. 2. Repeat the sequence carefully by decanting the supernatant from beaker No. 3

to sink, until all fruit tissue is removed from the sample. Finally, collect the residue from the beakers 1 and 2 into beaker No. 3. Remove

seeds or fruit tissue that settle out by treating the residue in beaker No.3 with hot 15% w/v NaCl solution. Remove NaCl by washing with hot water. Test the washings with AgNOs to ensure complete removal of NaCl. Transfer the residue remaining in the beaker to a funnel fitted with ashless filter paper. Use small portions of water to ensure transfer of all the residue. Discard filtrate. Transfer the filter paper to a weighed crucible. Dry in a hot air oven or over Bunsen burner, and ignite in a muffle furnace for about 1 hr at 600 °C. Cool in a desiccator and weigh. Subtract the weight of the crucible from the weight

of crucible plus incinerated residue. CALCULATION

Mineral impurities _ mg of incinerated residue (mg/kg)

~-g of test sample taken

1.000 ae

In canned strawberries, according to Codex Standards (CAC

mineral impurities should not exceed 300 mg/kg.

12-1972), the

CHAPTER 27

Frozen Fruits and

Vegetables Standards

No specifications have been laid down under the Fruit Products Order or Prevention of Food Adulteration Act of the Government of India for frozen fruit and vegetable products. The FAO/WHO Codex Alimentarius Commission have issued recommended international standards for quick frozen peas (CAC/RS 411970), strawberries (CAC/RS 52-1971), raspberries (CAC/RS 69-1974), peaches (CAC/RS 75-1976), bilberries (CAC/RS 76-1976), spinach (CAC/RS 77-1976), blueberries (CAC/RS 103-1978) and leek (CAC/RS 104-1978). Of these, the recommended specifications for peas!and peaches? are given below briefly as illustration. Quick Frozen Peas

Quick frozen peas are the product prepared from fresh, clean, sound,whole immature seed of peas which have been washed, sufficiently blanched to ensure adequate stability of colour and flavour,and conform to the characteristics of the species Pisum sativum L. Freezing should be carried out in such a way that the range of temperature of maximum crystallization is passed quickly. The quick freezing process shall not be regarded as complete unless and until the product temperature has reached -18° C (0° F) at the thermal centre after stabilization. The product shall be maintained at low temperature.

If peas are size graded, they shall conform specifications for the size names:

to one of the two following

Specifications A for sizing Size designation Small Medium Large

Round hole upto upto over

sieve size in mm 8.75 10.2 10.2

Size designation Extra small Very small Small Medium

Specifications B for sizing Round hole upto upto upto upto

sieve size in mm 7.5 8.2 8.75 10.2

Large

over 10.2

Frozen Fruits and Vegetables

839

Tolerance for sizes: If size graded, the product shall contain not less than 80%

either by number or weight of peas of the declared size or smaller sizes. It shall

contain no peas of sizes larger than the next two larger sizes nor more than 20% either by number or mass of peas of the next two larger sizes, if such there be. Not more than one quarter of these peas,whether by number or mass, shall belong to the larger of the next two sizes.

Analytical Characteristics: The alcohol insoluble solids content for peas and garden peas must not exceed 23% and 19% respectively. Definition of Defects and Tolerance Limits 1. Blond peas: Peas which are yellow or white but edible, i.e. not sour or

rotten —2%

2. Blemished peas: Peas which are slightly stained or spotted -5% 3. Seriously blemished peas: Hard, shrivelled, spotted, discoloured, worm-eaten

or blemished to the extent of seriously affecting the eating quality —1% 4. Pea fragments: Crushed, partially broken or individual cotyledon but not intact peas—12% 5. Extraneous vegetable material: Presence of vine or leaf or pod material from the pea plant or other vegetable material such as poppyheads or thistles—0.5% but not more than 12 cm? in area. Any sample unit from a sample taken in accordance with the sampling plans for prepackaged foods, 1969, shall be regarded as “defective” when any of the above defects are present in more than twice the amount of the specified tolerance for the

individual defects, or if the total of defects 1 to 4 inclusive exceeds 15% w/w. A lot is considered acceptable when the number of such “defectives” as specified in the above paragraph does not exceed the acceptance number, C of the Sampling

Plans for Prepackaged Foods (see Chapter 34). The cooking time for quick frozen peas may vary within the range of 3-5 min depending upon variety, maturity and size of the peas.

Frozen Peaches

Peaches may be either freestone or clingstone. The colour of the ripe flesh depending on the variety may be designated as white (or yellow-white), yellow (pale yellow to light orange), red (orange red to red) or green. They may be presented as whole (unpitted), halves, quarters, sliced, pieces or diced (cube-like parts having a maximum length of 15 mm on one edge). Peaches prepared with dry sugar shall contain not more than 35% w/w and not less than 18% w/w TSS at 20° C. When prepared with syrup, TSS shall be not more than 30% w/w and not less than 15% w/w at 20° C. Any sample unit that falls outside the limits of TSS range specified shall be regarded as “defective” provided it does not exceed the limits of the range by more than 5% w/w soluble solids.

840

Analysis of Fruit and Vegetable Products

Quality Factors General Requirements Quick frozen peaches shall be (a) clean and practically free from foreign material; (b) free from foreign flavour and odour; (c) of similar varietal characteristics; and (d) of good, reasonably uniform colour characteristics of the

varietal type. With respect to visual or other defects subject to a tolerance shall be: practically free from (a) dark discolouration or green areas (except for green in green types), (b) blemished units, (c) stalks (stems), or portions thereof, or other extraneous vegetable matter (EVM), (d) fibrous units, (e) pit fragments free from whole pits (stones) except in whole style; reasonably free from (i) overripe, mushy or

disintegrated fruit, and (ii) peel; and practically intact units for the style, and may be materially altered in shape due to excess trimming or mechanical damage. Definition of ‘defective’ for Quality Factors

Tolerance for defects are given in Table 27-1. Any sample unit taken in accordance with the sampling plans for prepackaged foods (see Chapter 34) and which is adjusted to a standard sample unit size for applying the tolerances relating to visual defects shall be regarded as defective for the respective characteristics as follows: (a) any sample unit that fails to meet the general requirements of quality factors. (b) any sample unit that fails the Total Allowable Points for Defect Categories—Minor, Major or Serious; or which fails the Total Allowable Points

for the combined

total of the respective categories given in Table 27-1.

Lot Acceptance for Quality Factors A lot is considered acceptable when the number of “defectives” as defined in the above paragraph does not exceed the acceptance number (C) for the appropriate sample size as specified in the sampling plans for prepackaged foods (see Chapter 34), provided that the number of whole pits (stones) does not exceed the tolerance on a sample average basis.

Food Additives Ascorbic acid Citric acid

750 mg/kg Limited by good manufacturing practice

Parameters of Freezing’ Deep-Frozen Foods Foods subjected to freezing process in accordance with good commercial Practice to reduce the average or equalization temperature to—18° C (0° F) or lower, and then stored at -18° C (0° F) or lower are called frozen foods.

yrep A194 -yauad

qu

zUP

Sues eyi

I

ody |

snosqiy

wed,3¢

ajousud

[OL squIog

2w

(sauois)

SIFEMOY

siuauisesy ypeq aoaid

1 weg

yseq yun

‘adi3saaQ Aysnur weg yun

jeoruerpaus aseusepYeq yun

SSOOXyUWI] pUe

J29q

‘Yy ug

3

J

‘a

“p

gu?

ysayj

Peg 1-S'0 gu?

~eg y

uoNLIZaqUISIG JO

Use|

WAG Jeu20

susais ‘S¥[BIS D>

qe

usaig

asaid yseq

“e

quawamnseau

I

I

-

-

>

s

. From Fig. 27.4 *: Reciprocal of the stable period 4: Quality deterioration per day x number of days.

Find the stable period corresponding to temperatures entered in column 2 from the time-temperature —tolerance curve or by calculation using Qjo in case of °C or gio in case of ° F,and enter in column 4. Find the quality deterioration per day which is the reciprocal of stable period, and enter in column 5. Multiply the data in column 5 with the number of days in column 3 to get the quality loss for the time at each temperature and enter in column 6. The total of entries in column 6 gives the total quality loss from the factory to the consumer.

The deterioration per day may be plotted against days on a graph paper and the area under the curve represents the quality loss. Divide the area under the curve by an area equal to one which would give total quality loss. The value of 0.8650 (see Table 27—3).represents the total quality loss from the time of manufacture until it reaches the consumer. Although the value is lower than 1.0, yet there is no detectable deterioration in the quality.

When the total quality loss is equal to 1, it is indicative ot the first detectable deterioration. The storage period corresponding to the first detectable difference by a taste panel is referred to as “the high quality life” in respect of a product. In order to make the term “first detectable difference” more comparable, the

International Institute of Refrigeration recommends the use of the term with statistical analysis of the results. . Another question of importance to the frozen food industry is the relation

between the first detectable difference and the practical storage life. As wide variations exist in the time required for the first detectable difference between different products, no given factor can generally be used. Based on the data available at present, the International Institute of Refrigeration suggests using a

Frozen Fruits and Vegetables

865

factor of 2 when the producers would be on the safe side in assuming that their products are still of high quality. Processing methods and packages used (i.e., use of less water vapour and oxygen permeable plastic films) have a significant effect like lowering temperature of storage. These have to be considered. Time-temperature-tolerance investigation can only be used as a guideline of the influence of changes in the manufacturing and distribution on frozen foods. For a particular product, this knowledge may then be combined with accelerated storage tests for calculation of storage life. Table 27-4 gives the values for ‘high quality life’ showing the primary limiting factor. Table 27-5 shows the commercial practice.

Mineral Impurities Determine as in the case of canned fruit and vegetable (see Chapter 26) except for preparing the sample as described below: Fruit Products: When the unit weight of the products is 500 g or less, use the entire

contents (fruit + syrup). Blend the material in a blender or macerator and transfer TABLE 27-4: High Quality Life at 0°F (-18° C) for Various Frozen Fruits and Vegetables in Commercially Good Quality Retail Packages.

Beans, green (French)

Quality

Temp.

Temperature

High quality

tested

quotient

range”

life at 0°F®

gio

Ae

(days)

Flavour

3.2

0-25

Colour

3.6

0-25

100

Cauliflower Peas

Flavour Flavour

3.4 3.4

-10-12 0-25

365 320

Spinach

Colour Fiavour Colour

4.2 3.4 555

0-25 -10-20 0-20

210 140 350

Boysenberries (‘n atural)

Flavour

Colour Flavour Colour Flavour Cloud

Boysenberries in syrup Orange juice concentrate

:

300

3.1

10-30

390

2.8 5.1 3.6

10-30 10-30 10-30

650 650 310 750 275

Peaches

Colour

7.8

10-25

365

Raspberries in syrup

Flavour Colour Flavour Colour

6.6 6.6 5.6 5.6

10-30 10-30 0-30 0-30

730 900 365 365

Strawberries — sugared

—

*; For averaging gio;

>. By triangle test.of change *; Values range from 3.5-5.0. Source: Van Arsdel, W.B., Food Processing, 22 (12), 40. (1961).

866°

Analysis of Fruit and Vegetable Products

tt

TABLE 27-5: Expected Storage Life of Frozen Fruits and Vegetables* eee. ne ee ke es Expected

Commodities

Temp.

Storage life

cx

(months)

ge

Fruits

-18 -18

Apricots in sugar Cherries (sour) in sugar

12 12

Cherries (sweet) in sugar

-18

8-10

Peaches in sugar

-18

8-10

-24

Peaches in. sugar and ascorbic acid Raspberries without sugar

12-14

-18

12

-24

18

-18

12

-24

18

Raspberries in sugat

-18

18

Strawberries in sugar Other frozen fruits

~24 -18 -22 to -18

24 12 12

-20

9-12

4

Fruit juices

citrus or others, single strength or concentrate Vegetables Asparagus

-18

8-10

Beans, snap Beans, Lima

-18 -18

8-10 12

Broccoli

-18

12

Brussels sprouts

-18

8-12

Carrots

-18

Cauliflower

-18

12-15 10-12

Corn on the cob Cucumber (sliced)

-18 =18

8-12 5

-24 -29

8 12

Mushroom

-18

8-10

Peas

-18

8-12

Potatoes (French fried)

-18

6

Potatoes (scalloped)

-18

1

Spinach

~18

10-12

-22 to -18

12

'

;

Other frozen vegetables

Note: Relative humidity should be as high as possible. Because of large deviations in the quality of the raw material and packaging, the expected storage life may vary within a still wider range than indicated in this table. 4

to a beaker using small quantities of water to assure complete transfer of the material. When the unit weight is more than 500 g, mix the contents of the entire container thoroughly and quickly remove a representative 500 g portion. Blend and transfer to a beaker. Vegetable Products: Take 250 g of the sample, comminute in a blender with small amount of water to facilitate maceration of the material.

Frozen Fruits and Vegetables

867

Maximum permissible limits for mineral impurities (sand, grit and silt) according to FAO/WHO Codex Alimentarius International Standards are as follows: Bilberries

On whole product 0.05%

Spinach Strawberries

0.10% 0.10%

References

1. Codex Alimentarius Commission: Recommended International Standard for Quick Frozen Peas. CAC/RS 41-1970.FAO/WHO Food Standards Programme, FAO, Rome. : 2. Codex Alimentarius Commission: Recommended International Standard for Quick Frozen Peaches. CAC/RS 75-1976.FAO/WHO Food Standards Programme, FAO, Rome. Ww. International: Institute of Refrigeration, Recommendations for the Processing and Handling of Frozen Foods, 1964.Internationa! (17e), France.

4. Association

Institute of-Refrigeration, 177, Boulevard Maleshrbes, Paris

of Official Analytical Chemists, Offsctal Methods

of Analysis,

12th edn:, 1975,

Association of Analytical Chemists, Washington, D.C.

5. Codex Alimentarius Commission: Recommended International Standard Procedures for Thawing of Quick Frozen Fruits and Vegetables and Cooking of Quick Frozen Vegetables for Examination Purposes, CAC/RM 32/33-1970. FAO/WHO Food Standards Programme, FAO, Rome. 6. Codex Alimentarius Commission, FAO/WHO Codex Alimentarius Methods of Analysis for Quick Frozen Fruits and Vegetables, First Series CAC/RM 34-1970; 43-1971; 54-1974. FAO/WHO Food Standards Programme, FAO, Rome.

7. Thompson, R.R., Ind. Eng. Chem., Anal. Edn.,.14, 585 (1942). 8. Cobey, HS. jr., and Manning, G.R., Quick Frozen Foods, 15 (10), 54 (1953). 9, Masure, M.P. and H. Campbell, Frust Prod. J., 23, 369 (1944).

10. Anon, J. Assoc. Offic. Agric. Chem., 30, 76 (1947). 11. Joslyn, M.A., J. Assoc. Offic. Agric. Chem., 40, 338 (1957).

12. Lee, F.A., D. DeFelice, and R.R. Jenkins, Ind. Eng. Chem., Anal. Edn., 14, 240 (1942). 13. Lee, F.A., Whitcombe, J., and Hening, J.C. Food Technol., 8, 126 (1954). 14. Varseveld, G.W., J. Assoc. Offic. Anal. Chem., 57, 701 (1974).

15. The Almanac of the Canning, Freezing and Preserving Industries, Edward E. Judge & Sons, Inc., 79

Bond St., Westminister. 16. Arighi, A.L.,M.A. Joslyn, and G.L. Marsh, Ind. Eng. Chem., 28, 595. (1936). 17. Gutterman, B.M., Lovejoy, R.D. and Beacham, L.M., J. Assoc. Offic. Agric. Chem., 34, 231 (1951).

18. Gutterman, B.M., J. Assoc. Offic. Agric. Chem., 39, 282 (1956).

19. Van Arsdel, W.B., M.J. Copley and R.L. Olson (Eds..) Quality and Stability of Frozen Foods; TimeTemperature-Tolerance and its Significance, 1969, Wiley-Interscience, New York.

CHAPTER 28

Fruit Juices, Concentrates and Beverages . Propucrs

included under this group are canned or bottled juices, pulps,

nectars, concentrated

fruit juices and beverages. Beverages include squashes,

cordials, barley waters, crushes and syrups which are preserved using chemical preservatives and meant for use after dilution; ready-to-serve beverages containing juice and preserved by physical methods or carbonation; and carbonated beverages with no added juice. Standards for Fruit Juices, Nectars, Concentrates and Beverages

The Fruit Products Order specifications for fruit juices, mectars, concentrates and beverages are given at the end of this book. The joint FAO/WHO Codex Alimentarius Commission (CAC) has drawn up intetnational standards for

a number of these products in. series of publications, references to which are made in Table 28-1. The numerical values with respect to composition and quality | factors are summarized in the table. The variations and explanations needed only are discussed below. The standards laid down are with respect to the products preserved exclusively by physical means but do not include ionizing radiation. Orange juice refers-to the product prepared using sound ripe orange (Cstrus sinensis L. Osbeck) juice. In orange juice concentrate, the soluble solids contributed by the addition of mandarin juice should not exceed 10% of the weight of total soluble solids in the finished concentrate. Concentrate prepared by physical removal of water may include (i) the addition of juice or concentrate or water for maintaining the essential composition and quality factors, and (ii) the addition of natural volatile components where they had

been previously removed. Apple and grape juices, and their concentrates

may be turbid or clear, but the

grape juice concentrate should be substantially free of crystals of salts of tartaric acid. The concentrates of apple and grape juices may be clarified with the aid of clarifying and filtering agents. The juices may be prepared by reconstituting the concentrate or by addit:on of concentrate to the juice. Sucrose, dextrose and dried glucose syrup, and in some

cases fructose (to orange juice concentrate), may be added in concentrations not exceeding the limits indicated in Table 28-1. The products are to be prepared according to the Code of Hygienic Practices for Canned Fruit and Vegetable Products (CAC/RCP 2-1969) and Quick Frozen

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Fruit Juices, Concentrates and Beverages 869

870

Analysis of Fruit and Vegetable Products

Fruits, Vegetables and their Juices (Ref. ALINFORM 71/13, Appendix IV). The products so prepared, when tested by appropriate methods of sampling and examination, should be free from microorganisms capable of growth under normal conditions of storage; and should not contain any substance(s) originating from microorganisms in amounts which may be toxic. The limits of mineral contaminants permitted are summarized/in Table 28-2. When added sugar, wher¢ permitted, is more than 15 g/kg, the words “X added” should be mentioned on the label. If the ratio of TSS to acid (as anhydrous citric acid) is more than 15 to 1 in orange juice and 12 to 1 in grapefruit juice, the word “sweetened” may be used instead of “X added” on the label. The minimum fill of the container should not be less than 90% v/v of the waterholding capacity of the container. ; The label should bear a complete list of ingredients including the presence of juice in the descending order of proportion, except that added water need not be declared. In case the juice is made from concentrate, the fact of reconstitution should be declared on the label, e.g., “Orange juice made from concentrate’,

“Reconstituted juice” or as “Orange juice made from concentrated orange juice”. The label should have the address of the manufacturer, packer, distributor,

exporter or vendor, country of origin, and information whether refrigeration is needed for keeping, and, if necessary, thawing the product. If the product is reprocessed in a second country, that country shall be considered as the country of origin for purposes of processing. The label should not have any picture of fruit. The net contents should be declared in metric system, US or British units as required by the country in which the product is sold. In case of bulk packs, the above information shall either be declared on the label or given in the accompanying documents.

Sampling Draw the samples according to the sampling plan for AQL 6.5. (see Chapter 34). Mix the sample thoroughly by shaking. Filter through cotton or Whatman No.4 paper. Pour the carbonated beverage from one beaker to another several times to expel CO, before commencing analysis. To sample juices or pulps from casks or barrels, mix the contents thoroughly either by rolling, rocking or stirring using the sampler. Collect the sample in a glass container with a sterile cork. Use a wide bore glass tube for sampling juices, and a long handled aluminium or stainless steel ladle for pulps and concentrates. Wash the sampler with water, then with a germicidal solution like a strong solution of sodium acid sulphite, and finally with clean water. Before removing the bung, clean the area surrounding the bung hole with sodium bisulphite solution. After removing the bung, mix the material with the sampler before drawing the sample. Draw a large sample (500 ml) by moving the sampler in different directions in the cask. Transfer to the glass container, cork, mix thoroughly, and

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Analysis of Fruit and Vegetable Products

draw from it subsidiary samples into small containers. Store the main container in good condition to serve as reference sample, if need be. FRUIT JUICES Total solids

Determine by drying in a vacuum oven at 70° C.

Total Soluble Solids Soluble solids may be determined by means of Brix hydrometers which measure the specific gravity or by a refractometer which measures refractive index. Brix is a measure of soluble solids only in the case of pure sucrose solutions. Generally, fruit juices contain more sugar than any other soluble constituent, and hence, Brix \provides a useful guide of soluble solids or sugar content. Soluble solids other than \sucrose do not affect the specific gravity and the refractive index to the same extent as sucrose, and the effect on refractive index is not the same as the effect on specific gravity. Hence, Brix readings found using a hydrometer and refractometer differ. For single strength juices, a hydrometer or refractometer may be used.

Measurement of soluble solids in Juices and Syrups using Hydrometer

The hydrometers commonly used in canneries for the testing of syrups are the Brix or Balling, Baume, Specific Gravity, and Twaddle. Brix or Balling

hydrometer gives directly the percentage of sugar by weight in the syrup. It is always necessary to make a temperature correction (Table 15-1), since the hydrometers are usually calibrated at 20° C (68° F). Each instrument used by canners usually covers a range of only 10° Brix, e.g.,10 to 20, 20 to 30, 30 to

40, 40 to 50, and 50 to 60° Brix, respectively, and are graduated in 1/10° divisions. Brix is defined as per cent sucrose measured by a Brix hydrometer. Brixe and Baume

The Brix hydrometer is the same as the Balling. It gives the per cent of sugar (sucrose) by weight Baume hydrometer, to 70°. The original heavier than water,

at the temperature indicated on the instrument. In the still used in some canneries, the divisions range from 0 Baume hydrometer scale is graduated so that for liquids 0° is the point to which the hydrometer sinks in wi ter,

and 10%, the point to which

it sinks in a 10% solution

of NaCl;

for liquids

lighter than water, 0° is the point to which the hydrometer sinks in the 1097, solution of NaCl and 10°, the point to which it sinks in water, both liquids being at. 17.52%.

The continental Baume hydrometer has the “rational” scale proposed by Lunge, in which 0° is the point to which the hydrometer sinks in water, and 66° the point to which it sinks in Hy SO, of sp. gr. 1.842, both liquids being ats,

Fruit Juices, Concentrates and Beverages

873

Baume “Rational” Scale : Sp. gr. at 45° C (eompated to water at 15° C=1) a

144.3 — “Baume 144.3

\

The American Baume hydrometer scale, adopted by the Manufacturing Chemists’ Association of the USA, is calculated from the following formulae: \

For liquids heavier than water at 15.55° C (60° F) :

Baume reading = 145 —

.

145 Sp. gr.

145

P- St. =

445 — Baume reading

For liquids lighter than water:

140 Baume reading = Ces oe 130 p- gt. sig 140 SPA Siomeagay ae Baume reading Tu'addle Hydrometer Generally used in England, the Twaddle hydrometer has a scale from 0-to 200°, corresponding to change in specific gravity from 1 to 2, the degrees representing constant increases. Water at 4° C is considered to have a specific gravity of 1000 units. Hence, an increase in specific gravity of 5 units corresponds to an increase of 1° TW’. One degree Twaddle corresponds to 1.005

specific gravity. Testing of Hydrometers Since continued use of hydrometers in hot syrups aflects their accuracy, they should be checked frequently by more accurate instruments. Hydrometers with long stems having greater distance between divisions are more easily read than hydrometers with short stems. It is desirable for the canner to have duplicate or triplicate sets of accurate hydrometers. Temperature Corrections Hydrometers are calibrated for use at a “standard” temperature. The calibration temperature is painted on the stem of the hydrometer. The test must

be made at that temperature, or suitable correction must be made. Usually the syrups are at much higher temperatures than those at which the instruments are calibrated. At temperatures above the standard, the observed reading will be too low, because the higher temperature expands the volume and corres-pondingly decreases the Brix degree. To avoid this, corrections given in Table 28-3 should be applied to the observed readings.

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Fig. 28.2: Capillary Viscometer (Reprinted from Official Methods of Analysis (12th edition) 1975, with the permission of Official Analytical Chemists,

Arlington, VA 22209)

Fruit Juices, Concentrates

and Beverages

897

CALIBRATION

Add water to the tube at 24 + 2° C. Allow to flow till a steady state is reached. Stop the flow by placing finger over the end of the tube. Fill the tube until it

overflows and level off using a spatula or by sighting across the top of the tube. Remove the finger and start the timer. Mark the level reached by the top meniscus at the end of 13.0 +0.2 sec. Etch the calibration line on the outside of the reservoir. DETERMINATION

Clean the apparatus, dry and maintain at 24 + 0.5° C. Adjust the sample to 24+ 0.5° C. Add the sample to the tube and allow to flow until a steady state is reached. Stop the flow by placing a finger over the end of the capillary tube. Fill the tube almost full avoiding air bubbles. If air bubbles are present, remove by stirring with a glass rod or thermometer. Check the temperature at this point. Fill completely and level off as before. Remove the finger, start the timer immediately, and note .

the time required to the nearest 0.1 see for the top of the meniscus to reach the calibration line. Take 2 or more readings. Mix the sample before each determination, and rinse the viscometer with water between each reading of viscous samples.

According to the standards of identity

for fruit nectars as a means of

distinguishing between fruit nectars and other fruit juice products, the time required for the nectar to flow through the tube at 24° C must be not less than 30 seconds (see Table 28-1, foot note i). The viscosity may also be determined using a suitable size of Ostwald viscometer at a specified temperature. Determine the time required for flow of distilled water and the sample. The relative viscosity can be calculated from the following expression}: a Dit. Nw ..Dw X% tw

where, D = density t =time of flow 5 = sample

w = water

The value of » for water at 15° C is 0.0134 in cgs units. One unit is the poise. To follow the enzyme clarification of juices and for quality control, what is generally required is 1/Nw.References

1. Lamb, F.C., and L.D. Lewis, J. Assoc. Offic. Agric. Chem., 42, 411 (1959). 2. Official Methods of Analysis, Assoc. of Official Analytical Chemists, Washington DC., 12th edn. 1975, 22.009. 3. Shacklady, J., in Food Industries Manual,

Leonard Hill, London, 20th edn., p. 248, 1969. 4

898

Analysis of Fruit and Vegetable Products

SQUASHES, CORDIALS AND CRUSHES

‘Read the label carefully and note the omissions. Mark the level of the container with a marking pencil. Remove the closure and note the odour of the contents. Pay particular attention to yeasty or turpentine odours. Test for the presence of yeasts as detailed under microbiological examination of beverages. Turpentine odour is indicative of the presence of oxidized essential oil and is often indicative of age. The presence of an oily ring near or slightly above the liquid level is due to separation of the flavouring oil used in the squash. The formation of such a ring may occur rapidly or slowly depending on the amount of oil present and on the method used to incorporate it in the squash. Note whether the beverageis clear or cloudy. These beverages are allowed to contain chemical preservatives such as sulphur dioxide and benzoic acid either alone or in combination. Artificial sweetening agents are not permitted in India. The chemical examination of these beverages includes determination of refractometer solids, acidity, pH,

reducing and total sugars, preservative(s) and

saccharin.

Preservatives and Artificial Sweetening Agents Benzoic acid and sulphur dioxide are the only two preservatives permitted in fruit juice beverages. Benzoic acid can be estimated by extracting with chloroform

(see page 310)-or ether. SO, should preferably be determined by the distillation procedures although direct titration method is generally made use of for control purposes (see. page 306 ). The only synthetic sweetening agent permitted in artificial carbonated beverages is saccharin but none in any fruit juice beverages. Their presence could be detected by methods given on page 303. Methods for the quantitative estimation of total SO2, benzoic acid and saccharin

are given below: Total sulphur dioxide in Fruit Juices by Direct Titration To 50 ml of juice, add 25 ml of 1 N NaOH and allow the mixture to stand for 10 min. Add 10 ml of dilute H2SO, (1 + 3) and titrate with 0.05 N iodine solution

using starch as indicator. One ml of 0.05 N iodine is equal to 0.0016 g of SOx. Calculate the SO2 content using the expression. SOz ppm

_ Titre value X 0.0016 X 10° ® lowndo Woaup tT bal Soi

Detection of saccharin

The schematic procedures for sweetening agents and preservatives are given in Fig. 12-3. Saccharin can be extracted by‘diluting the beverage with water (1+ 4), making strongly acidic with HCI, and extracting with ether. Remove the solvent from the ether extract, and taste the residue which would be intensely sweet, or test using

Fruit Juices, Concentrates

and Beverages

899

resorcinol. To the residue, add a few mg (50 mg) of resorcinol and 10 ml of H2SO,. Heat gently until a dark green colour is formed. Cool carefully, add water (10 ml),

and excess of 5 N sodium hydroxide solution. Saccharin gives fluorescent green colour. Benzotc acid and saccharin

The procedure is based on selective extraction of benzoic acid at pH 4.0, and then saccharin by making the liquid strongly acidic. PROCEDURE

Benzoic acid: To 50 ml of the sample ina separating funnel, add 25 ml of pH 4 buffer solution containing 10% sodium carbonate and 6.5% citric acid. Add 25 ml of diethyl ether, shake, allow to separate, and draw off the aqueous phase to another separating funnel. Make two further extractions of the aqueous phase similarly with 25 ml portions of ether. Wash each ether extract with the same 5 ml portion of water by transferring buffer solution from one funnel to another. Combine the ether extracts, filter, and carefully evaporate the ether over a water bath. Remove the last 5 ml of the solvent at room temperature using an air blower. Dissolve the residue in 5 ml of 60% acetone, and titrate with 0.05 N sodium

hydroxide using phenol red as indicator. Calculate the benzoic acid content using the relationship that 1 ml of 0.05 N NaOH = 0.0061 g benzoic acid. Saccharin: To the aqueous phase containing the sample in the separating funnel, add the water used for washing, and 10 ml of conc HCl. Extract the saccharin three times with 25 ml portions of diethyl ether. Wash the combined ether extracts with three 5 ml portions of distilled water. Do not discard the wash water. Combine the washings and extract with 10 ml of ether. Mix this ether extract with the main ether extract, filter, and wash the filter paper with more

ether. Evaporate the ether on a water bath. Dissolve the residue in 5 ml of acetone, and evaporate to dryness. Add 4 ml of distilled water, warm to dissolve, cool to room temperature, and titrate with 0.05 NN aOH solution using bromothymol blue as indicator. Calculate the saccharin content using the relationship that 1 ml of 0.05 N NaOH = 0.00916 g of saccharin. Estimation of Saccharin in Beverages Containing

al Oils

For the estimation of saccharin in beverages containing esscuitial oils, it is advisable to remove the oil present. The procedure consists of acidifying the beverage with HCl, repeated extraction with ethy] ether and shaking the combined ether extracts with dilute ammonia at least twice. The lower aqueous phase is run into a fresh separating funnel, acidified and extracted with diethyl ether. The saccharin can then be extracted and estimated in the ether extract as in the

previous method, or by the AOAC

nesslerization method given below:

900

Analysis of Fruit and Vegetable Products,

REAGENTS

1. Ammonia-free water: To a large volume of water, add a little dilute H2SO,

and distil. Start collecting the water when the distillate gives no yellow colour with Nessler reagent.

2. Nessler reagent: Dissolve 100 g of mercuric iodide (Hglz) and 70 g of potassium iodide (KI) in a small quantity of ammonia-free water, and add this mixture slowly, stirring to a cold solution of 160 g of NaOH in 500 ml of ammoniafree water. Store in Pyrex or Corning glassware. The solution is stable for 1 year.

3. Standard ammonium chloride solution: Make up 0.2921 g of NH,CI to 1 litre with ammonia-free water. 1 ml = | mg of saccharin. Dilute 200 ml of this to 1 litre. The resulting solution would contain 200 ppm saccharin. PROCEDURE

In the procedure described below, use only ammonia-free distilled water. To 50 ml of the sample in a separating funnel, add 2 ml of HCl, and extract twice with 50 ml ether. Combine the ether extracts, filter through cotton, and wash with 5 ml of water containing 1 drop of HCl. Separate the ether layer, and evaporate to, dryness on a water bath. To the residue, add 5 ml of water and 6 ml of HCl, and

evaporate on a hot plate with constant stirring to approximately 1 ml. To the residual 1 ml, add 5 mlof water and 6 ml of HCI, and evaporate as before to 1 ml.

Dilute to 50 ml with water. Make up 2 ml of this solution to 25 ml with water. Add 1 ml of Nessler reagent and compare with ammonium chloride standards. Find the saccharin content using the following relationship: 0.2921 g NH,ClI = 1 g of saccharin (C7HsNO3S) which is an iasoluble form = 1.317 g of sodium salt of saccharin (C7H4N NaO3S. 2H20)

Fruit juices may be clarified using (i) pectic enzymes, (ii) tannin and gelatin, (iii) using a combination of the methods (i) and (ii), and (iv) by centrifuging and filtering. The first three methods give a relatively clear juice which is

passed through a filter press using diatomaceous earth to ensure the removal of all small particles.

Enzyme Clarification of Fruit Juices Pectin degrading enzymes are usually used in the clarification of fruit juices. There is a possibility of obtaining an increased colour yield from grapes if the enzyme is mixed with and allowed sufficient time to react with the milled

Fruit Juices, Concentrates and Beverages

901

fruit. When used with juices, enzymes work only slowly at temperatures below 10° C and are largely destroyed when exposed to temperatures above 40°C. If large quantities of juice are being treated, the amount of enzyme needed should be determined. A simple preliminary test is described below for the

purpose.

Mix equal quantities of strained juice and absolute alcohol in a standard test tube. Allow the contents to stand for 20 min or longer. Inspect the pectin layer formed on top of the liquor and measure its depth. A rough and ready guide to the quantity of pectin in the juice is afforded by the depth of the pectin layer.

2-+

mm

--

Slight pectin content

5-25 mm

—

Medium pectin content

30-50 mm

—

Considerable pectin content

The quantity of pectic enzyme to be added for each of these three grades of juice can be calculated as under :

Slight pectin content in juice add 0.02 to 0.045% (w/w)

Medium pectin content in juice add 0.05 to 0.075% (w/v) Considerable

pectin content in juice add 0.08 to 0.15% (w/v)

The quantity given above is only an approximate indication and may vary with the source. A more exact preliminary test is as follows : To each of the six cylinders, add 100 ml of juice. Dissolve 10 g of single strength enzyme in one litre of water to provide a 1% stock solution. Pipette 2.5, 5, 7.5, 10, 12.5 and 15 ml of the enzyme solution to each: cylinder. Mix the

samples well and examine after 1-2 hr by visual observation ora filtration test. If no sample shows adequate clarification, attempt gelatin clarification.

For bulk enzyme action, suspend the required amount of enzyme in a little

of the juice which has been raised to 30-40° C and maintain at that temperature for one hour. Mix this volume of enzyme suspension into a large quantity of

juice and adequately incorporate in the bulk juice for clarification. At 10° C, time required for clarification may be 8-10 hr or more.

Tannin-Gelatin Clarification! The laboratory method

for standardization of clarification of apple juice

using tannin-gelatin flocculation is given below. This method can be applied to other juices with suitable modifications.

902

Analysis of Fruit and Vegetable Products

Prepare stock solutions of tannin and gelatin as given below: Tannin : Dissolve 9.5 g in 1000 ml of water. Gelatin : Weigh 21.25 g of gelatin. Make into a soft mass with water (600-800 ml). Heat to boiling, cool, and make up to 1000 ml.

cold

To preserve these solutions for more than a few days, replace 200 ml of water with 200 ml of 95% alcohol.

Use trial amounts of these solutions per 40 0z of juice as indicated in Table

28-11. Add the tannin first and stir the juice thoroughly. Then add gelatin in a thin stream with vigorous stirring. Select the least amount of gelatin giving the desirable clarification. Usually only the first four or five sets are necessary, but if satisfactory clarification is not obtained with any of these, try the complete series. After these trials have been made, add the equivalents

given under the

columns “Amounts per 100 gallons” in Table 28-11 to the large batch to be clarified. Prepare both solutions in water in the same manner as that used for the stock solutions. Only enough water should be used to dissolve the tannic acid completely. The gelatin solution should not be too thick. For example, if 3 oz of gelatin is required per 100 gallons of juice, dissolve in 1 to 1.5 litres of hot water. The gelatin may also be dissolved in hot juice from the pasteurizer. A heavy flocculent precipitate will appear in a few minutes and will settle completely overnight.

TABLE 28-11: Trial Amounts Trial amounts

of Tannin

and Gelatin Solutions Amounts

Test

Tannin

Gelatin

‘

number

solution

solution

Fannin

per 100

gallons

: Gelatin

ml

ml

oz

oz

I

12

5.0

155

1.5

i2 3

12 12

6.6 8.3

EAS AS

2.0 2.5

4

12

3.0

12

10.0 11.6

1.5

5

1.3i5

5 5

6

12

13.3

1.5

4.0

7 8

12 12

15.0 16.6

ins Da5

4-5 5.0

9 10

2: 12

18.3 20.0

Ess. TS.

5-5 6.0

Reference

1.

Charley, V.L.S., Recent Advances in Fruit Juice Production, Commonwealth Bureau of Horti-

culture and Plantation Crops, Commonwealth Agricultural Bureaux, London, p. 62 (1950).

Fruit Juices, Concentrates and Beverages

903

Carbonation of Beverages Carbonation is the process of mixing sufficient CO, with water or beverage so that when served, the product gives off the gas in fine bubbles and has the characteristic pungent taste suitable to the beverage carbonated. Carbonation adds to the life of a beverage and contributes in some measure to its tang. Fruit juice beverages are generally bottled with CO, content varying from 1 to 8 g per litre. Though this concentration is much lower than that required for complete inhibition of microbial activity (14.6 g/litre), it is significant in supplementing the lethal effect of acidity on pathogenic bacteria. Another advantage of carbonation is the removal of air which results not only in anaerobic condition,

but also reduces

the oxidation

of ascorbic acid.

Carbon dioxide volume : At atmospheric pressure, the amount of CO, dissolved by water (w:v) depends solely on the temperature. Solubility is greater at lower temperature than at higher temperature. The amount of CO, dissolved by water at a given temperature is proportional to the pressure the

former exerts on the latter (Table 28-12). Carbonation, therefore, depends on pressure and temperature. Unit of measurement: The unit of measurement accepted by the industry as standard is the ‘volume’. This is defined as the amount millilitres that a given volume of water will absorb at atmospheric i.e., at 760 mm of Hg at a temperature of 15.5° C (60° F). These. are

beverage of gas in pressure, arbitrary

points set by agreement. This condition registers as zero on the gauge commonly used to measure the volume of CO, absorbed in carbonated beverages. Thus at 15.5° C, beverage water will absorb one volume of carbon dioxide

represented as zero on the gauge. When the pressure is increased to 15 lbs (actually 14.7 lbs) by applying a pressure of one additional atmosphere, the water will absorb two volumes of the gas, and for each additional 15 lbs or

atmosphere of pressure an additional volume of duction of the temperature will permit the water of CO,. When the temperature is reduced to 0” C will be absorbed. There will consequently be

CO, will be absorbed. Reto dissolve greater amounts (32° F’), 1.7 volumes of CO, an additional ‘absorption a

1.7 volumes of CO,. Thus, if a bottle is filled at 32° F under 30 lbs of pressure,

the

volume

intermediate

of CO,

dissolved in the water

pressures and temperatures,

will be 1.7 x 3 = 5.1. For

intermediate

volumes

of the gas

will be absorbed (Table 28-12). Solubility of CO, in sugar solutions : As the concentration of sugar increases, the solubility in terms of volume of gas decreases, but this is not very highly significant for practical purposes (1% sugar solution at 60° F dissolves 0.995 volume of CO,, while 13% sugar solution dissolves 0.902 volume).

Analysis of Fruit and Vegetable Products

904

TABLE

28-12 : Solubility of Carbon Dioxide in Water Recalculated into Degrees Fahrenheit and Pounds per Square Inch

Pounds

Volumes of CO, gas dissolved by one volume of water at

per sq.

Temperature (“F)

inch

Pressure 35 36 tate ei 15 3.46 3.19

40 2.93

6o 55 Se fa SL el 2.50 2.20 2.02

3-15 3.61

2.92 3-35

4.06

Te

75

80

85

1.86

1.71

1.58

1.44

1.37

1.84

1.69

1.58

1.48

2.69

2.48

2.29

2.10

1.93

1.80

1.70

3-77

3-03

2.80

2.58

2.37

2.18

2.03

1.91

4-52

4-19

3.69

3.11

2.86

2.63

2.42

2.26

2 515

5-39

2:49 Zotz

2.34

25 30

5-21

4.81

3-92 4-41

5-35

4.91

5-89 6.43 6.95

4-97

4.61

4-05

3-37 3.71

3.42

3-15

2.89

2.67

5.88

5-43

5-93

4-43

4.06

3-74

3.44

3.16

2.91

6.36

5-45

4.80

4-40

4-05

7.48

6.86

5-89 6.34

5-17

4-74

8.02

7-35

6.79

5.87 6.29

5-53

5.08

4-37 4.68

9.09

8.33

7-7°

7-13

6.27

5-76

10.17

9.31

8.61

7.98

7.00

12.18 11.25

10.30

9.52

8.82

13.34 12.33 11.29 10.43

9.66

9 II.02

27)

3-04 531

3-42

4-27

.80

go

2.57

3-73

4.58

100

Jo

CI57) 2.00

4:04

j 40 6 45 6 5° 7-53 55 8 8

65

2.36

20

35

48

3.142

3-73 4.02

3.69

4.31

5-30

4.89

6.43

5-92

5.46

7-74

7.11

6.54

6.04

8.4

7:79

7.18

6.62

S010

2.56

12.0402. 77

3.40

3-17

2.99

3-95

3.64

3.39

3.20

4:49

4.14

3.86

3.63

5.02

4.62

4.31

4.06

5-55

5-12 5.60

4-77 5.22

4:49

6.08

4.91

Source : A, Seidell, Solubilities of Inorganic and Metal Organic Components, 4th edn,D. Van

Nostrand Co., 1958. (Reprinted with the permission Society, Copyright ©)

of American Chemical

Degree of carbonation : The level of carbonation required varies according

to the type of fruit juice (Table 28-13) and type of flavour. Sample calculation for carbonation ; The carbonation pressure required to carbonate a beverage at 40° F so as to maintain a gas volume of 4 is calculated as given below: Solubility of CO, at 60°F =: 1 volume Solubility of CO, at 32° F = 1.7 volumes (1.7 —1) x (60 Solubility of CO, — 40)

at 40°F Gas volume

the beverage

_

eee oeO (C required in

4

It is, therefore, necessary to raise the volume by 2.5 (1.6.4 — atmospheric solubility of 1.5 volumes of CQ, at 40° F.

The carbonation pressure required =

1.5) over the

poy en JDP Se eens

1.5

Fruit Juices, Concentrates and Beverages

905

The accurate gauge pressure to be maintained during carbonation to get he desired volume of CO, in the beverage may simply he ascertained from Table 28-12. TABLE

28-13: Composition of Carbonated Beverages

Beverage

Sugar

Gas

(°Brix)

\ volume

Citric

(%)

a

Lime

Onl

Lime

=

acid

pH

4.0

0.14

3 02

11.10

357

0.23

2.90

Lemon

11.18

332

O.12

3.07

Lemon and lime

11.04

Byers

0.18

3.01

Orange

13.40

203

0.19

3.39

Grape

14.50

1.3

0.12

3.10

|

Measurement of Carbon Dioxide in Bottled Beverages APPARATUS

Use a device consisting of a gauge with a hollow spike having holes on the side with a slot in the neck to introduce the bottle. PROCEDURE

Introduce the bottle into the slot provided in the neck of the tester. Secure the bottle in place by tightening with a threaded stem. Insert the pressure gauge until the needle point touches the crown cork. Close the sniff valve on the gauge stem until the néedle point of the pressure gauge is forced through the crown cork. Note the reading on the gauge. Open the sniff valve and allow the pressure to escape until the first bubble rises in the liquid. Shut the sniff valve and shake the bottle until the gauge reaches maximum pressure. Note the pressure on the gauge. Sometimes, the final pressure noted may be lower than the pressure noted before sniffing the head pressure in the bottle. Usually it is the same as the final pressure when the sample is cold (40-50° F). Remove the gas volume testcr and crown cork from the bottle and note the tempera-

ture with the help of a thermometer. Refer to Table 28-14 for the volumes of carbon dioxide dissolved.

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Analysis of Fruit and Vegetable Products

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Fruit Juices, Concentrates and Beverages 907

908

Analysis of Fruit and Vegetable Products

References 1. 2.

Jacobs, M. B., The Chemisiry and Technology of Food and Food Products, 2nd edn., Vol. 3, Interscience Publishers, Inc., New York, p. 2365 (1951). Publishing Corpn., New Von Loesecke, H. W., Outlines of Food Technology, Reinhold York, p. 382 (1948).

Caffeine

Caffeine (CsH,)N,Oz) is added to some of the carbonated beverages particularly of the cola type. The amount of caffeine in such beverages should not,

however,

exceed 200 ppm.

PRINCIPLE

The method of determining caffeine is based on its extraction from the beverage using chloroform and measurement of its absorbence in the ultraviolet region at 276.5 nm. Alternatively, the chloroform can be distilled off and the nitrogen in the caffeine determined by the usual micto-Kjeldahl digestion and distillation procedure. REAGENTS

1. Reducing solution : Dissolve 5 g of sodium sulphite (Na,SO3) and 5 g of potassium thiocyanate (KCNS) in water and dilute to 100 ml. 2. Dilute phosphoric acid (H,PO,) solution : To 15 ml of acid, add 70 ml of water. 3. 1.5% Potassium permanganate solution: dilute to 100 ml.

Dissolve 1.5 g in water and

4. Sodium hydroxide solution : Dissolve 25 g of NaOH in 75 ml of water. 5. Caffeine standard solution : Weigh accurately 100 mg of caffeine (recrystallize using chloroform,if necessary), dissolve in chloroform, transfer to a 100-ml volumetric flask and dilute to mark (1 ml = 1 mg of caffeine). Dilute 10 ml of this solution to 100 ml with chloroform (1 ml == 0.1 mg). PROCEDURE

To remove CO,, pour the effervescence ceases. funnel and add 5 ml of mixture stand for exactly

carbonated beverage from one beaker to another till Pipette 10 ml of the sample into a 150-ml separating potassium permanganate solution. After letting the 5 min, add 10 ml of reducing solution, 1 ml of phos-

phoric acid solution, and 1 ml of NaOH solution. Mix after addition of each solution. Add 50 ml of chloroform and shake for 1 min. Allow the layers to separate and drain off the chloroform layer through a 7-cm rapid filter paper into a 100-ml glass-stoppered volumetric flask. Add 2 to 3 ml of chloroform to the separating funnel and drain off as before. Wash paper with 2 to 3 ml

Fruit Juices, Concentrates and Beverages

of chloroform. Re-extract the solution with 40 ml of chloroform

909

and drain

off as before. Wash the stem of the funnel and the paper with a few ml of chloroform and dilute to mark. Measure the absorbence of this solution at

276.5

nm

in a spectrophotometer.

Note

the caffeine

content

from the

standard curve. STANDARD

CuRVE

Into separate volumetric flasks, pipette 1, 2.5, 5, 10, 15 and 20 ml of caffeine

standard solution containing 0.1 mg/ml, and dilute to mark with chloroform to give 0.1, 0.25, 0.5, 1, 1.5

and

2 mg of caffeine per

100

ml.

Measure

absorbence at 276.5 nm. Plot absorbence against concentration. CALCULATION

Caffeine, mg/100 ml =

ALTERNATIVE

mg of caffeine per 100 ml x 100 oe 3S ml of sample taken

PROCEDURE

To 50 ml of the sample, add 7.5 ml of potassium permanganate, mix and allow to stand for 5 min. Then add 50 ml of reducing solution, 5 ml of phosphoric acid solution and 5 ml of NaOH solution, mixing after each addition. Extract five times with 25 ml portions of chloroform; drain and filter after each extraction as before. Distil off most of the chloroform in a water bath. Transfer the remaining 10 to 15 ml to a 100-ml Kjeldahl flask and evaporate the chloroform extract to dryness in a boiling water bath. Dissolve the residue in water. Add 10 ml of conc H,SO,, 2 g of sodium sulphate, and 0.2 g of copper sulphate. Digest for 45 min and then allow to cool. Transfer the digest to the distillation apparatus directly (without making up to volume), add NaOH and distil into boric acid solution as described under micro-Kjeldahl distillation procedure (see page 16). Titrate the ammonia with 0.02 N HCl, 1 ml of which is equal to 0.971 mg of anhydrous caffeine. References sb 2.

Wilson, J.B., J. Assoc. Offic. Agric. Chem., 41, 617 (1958). Harris, F.J.T., J. Sci. Food Agri., 4, 205 (1953):

FRUIT JUICE CONCENTRATES

Insoluble Solids and Pulp Content Insoluble solids which consist of pulp affect the appearance and ‘“‘mouth

feel” of a juice. They also affect the consistency and the tendericy of the free

910

Analysis of Fruit and Vegetable Products

liquid to separate from the juice. The ultimate quality of citrus juice concentrates on storage is governed by the pectinesterase activity, pectin and glucoside content. All these parameters vary with the insoluble solids content. The determination of pulp content may be carried out on the basis of total pulp or screened pulp. The total pulp content is determined by the use of a centrifuge. Screened pulp, which is the coarser material almost entirely provided by cut-back juice, is the pulp retained ona standard 20-mesh screen. The average value for centrifuge solids in Florida citrus concentrates is 11.66% and that for screened pulp is of the order of 1%. The free and suspended pulp content in grapefruit juice is from 10 16,.15°% and in frozen concentrate, 10%. According to U. S. Standards, pineapple juice requires not less than 5% and not more than 30% of finely divided insoluble solids. Screened Pulp

Apparatus: Use 20-mesh screen (12.7 cm diameter and 7.0 cm deep) made of

woven stainless wire. Sample size and the basis for reporting of resuJts are as follows: oduct arn

Sample size j

Results grams of 20-mesh pulp per

Bulk juice Chilled juice

1000 ml 946 ml (one quart)

1,000 ml Quart of juice

Retail concentrate

6 oz can of concentrate

6 oz can of concentrate

reconstituted to 710 ml of juice

Bulk concentrate

1000 ml of reconstituted 1000 ml of reconstituted juice

juice

PROCEDURE

Pour the entire sample through a 20-mesh screen and allow to drain for 2 min.

Shake the screen vigorously either manually or using a sieve shaker for 2 min until

pulp coalesces and is free on the screen surface. Wipe off the excess water from the bottom of the screen using cloth or tissue paper. Transfer the pulp to a tared dish, weigh accurately (+ 0.1 g), and report the results as stated earlier.

The screened pulp primarily consists of large pulp particles which are

contributed by juice sac material. The determination of suspended solids.

screened

juice may

be used

for the

Suspended Solids (Centrifuge Pulp)

The suspended solids of citrus juices consist of fine Pieces of juice sacs,

Fruit Juices, Concentrates

and Beverages

911

membrane, and other structural components. The suspended solids are also referred to as insoluble solids. centrifuge solids, bottom pulp or sinking pulp. APPARATUS

Laboratory centrifuge, 4 x 50 ml cups

PROCEDURE Measure 50 ml of the juice into each of two graduated 50-ml long-cone cen' trifuge tubes approximately 43 inch overall length. In the case of concentrated citrus juices, reconstitute the product and pass through a 204mesh sieve before centrifuging. Place in a suitable centrifuge and adjust the speed according to diameter, as indicated in Table 28-15.Centrifuge the juice for exactly 10 min (3 min in the case of canned pineapple juice). As used here, diameter means the overall distance between the bottoms of opposing centrifuge tubes in the operating position. After centrifuging, note the reading,at the top of the layer of pulp in the tube and multiply by 2 to give the percentage of free and suspended pulp.

The following procedure for determination of insoluble solids is based on the AOAC method :

Determine the total solids in the juice by oven drying. Filter another portion of juice and determine the total solids in the filtrate. Calculate the insoluble solids content, using the following expression : So

creole Saat

100 (Total solids % — Soluble solids in the filtrate %)

100 — Soluble solids in the filtrate % Folds of Concentration

Concentrates are described 4-, 5—, or .6- fold which indicates that the fresh juice is concentrated 4, 5 or 6 times. Conversely, one volume of concentrate can be

diluted to give 4, 5 or 6 volumes of juice. The degree (fold) of concentration is

usually assessed industrially from the specific gravity or refractive index. Specific gravity of the concentrates cannot be determined by the usual method as

it tends to occlude air bubbles. The method available is given below: Weigh 20-25 g of concentrate into a tared 150-ml conical flask fitted with a tightfitting rubber cork. Add a quantity of water equal to not less than 3 times the

weight of the concentrate, and reweigh. Mix thoroughly by swirling to minimise formation of air bubbles and allow to stand for 1 hr. Into a 50-ml specific gravity bottle which has. been standardised at 20° C, fill the diluted concentrate by swirling the conical flask at intervals to ensure that a proper proportion of cellular matter enters the specific gravity bottle. Adjust the temperature to 20° C and determine

the specific gravity as described earlier. Calculate the specific gravity of the original

concentrate as described subsequently.

912

= Analysis of Fruit and Vegetable Products TABLE 28-15: Relation Between Diameter and Approximate

Speed of Centrifuge Diameter

Approximate revolu-

(inch)

tions

per min

10

1609

103

1570

II

1534

114

1500

12

1468

12}

1438

13

I4Io

133

1384

ci

1359

143

1336

Ty

1313

154

1292

16

1271

163

1252

7

1234

173

1216

18

1199

18)

1182

‘9

1167

19} 20

|

1152 1137

Reference

1.

Standard Methods for the Analysis and Examination of Foodstuffs, sth edn.,Commonwealth Food Specifications Committee , Department of Primary Industry, Canberra, A. C. T. 2690, P-. 105 (1969).

Fruit Juices, Concentrates and Beverages

TABLE 28-16: Domke Table of Apparent Specific Gravity of Sucrose

913

Solutions at 20°C*

Calculated from tables of Kaiserliche Normal-Eichungs-Kommission and accepted by International Commission for Uniform Methods of Sugar Analysis. Degrees Brix or

per cent

0

a]

7

3

A

2)

6

oy

8

2d

0 1 2 3 4

1.0000 1.0039 1.0078 1.0117 1.0157

1.0004 1.0043 1.0082 1.0121 1.0161

1.0008 1.0047 1.0086 1.0125 1.0165

1.0012 1.0051 1.0090 1.0129 1.0169

1.0016 1.0055 1.0094 1.0133 1.0173

1.0019 1.0058 1.0098 1.0137 1.0177

1.0023 1.0062 1.0102 1.0141 1.0181

1.0027 1.0066 1.0106 1.0145 1.0185

1.0031 1.0070 1.0109 1.0149 1.0189

1.0035 1.0074 1.0113 1.0153 1.0193

5 6 7 8 s

1.0197 1.0237 1.0277 1.0318 1.0359

1.0201 1.0241 1.0281 1.0322 1.0363

1.0205 1.0245 1.0285 1.0326 1.0367

1.0209 1.0249 1.0289 1.0330 1.0371

1.0213 1.0253 1.0294 1.0334 1.0375

1.0217 1.0257 1.0298 1.0338 1.0380

1.0221 1.0261 1.0302 1.0343 1.0384

1.0225 1.0265 1.0306 1.0347 1.0388

1.0229 1.0269 1.0310 1.0351 1.0392

1.0233 1.0273 1.0314 1.0355 1.0396

10

1.0400

1.0404

1.0409

1.0413

1.0417

1.0421

1.0425

1.0429

1.0433

1.0438

11 12 13 14

1.0442 1.0484 1.0526 1.0568

1.0446 1.0488 1.0530 1.0573

1.0450 1.0492 1.0534 1.0577

1.0454 1.0496 1.0539 1.0581

1.0459 1.0501 1.0543 1.0585

1.0463 1.0505 1.0547 1.0589

1.0467 1.0509 1.0551 1.0594

1.0471 1.0513 -1.0556 1.0598

1.0475 1.0517 1.0560 1.0603

1.0480 1.0522 1.0564 1.0607

15 16 17 18 19

1.0611 1.0654 1.0698 1.0741 1.0785

1.0615 1.0659 1.0702 1.0746 1.0790

1.0620 1.0663 1.0706 1.0750 1.0794

1.0624 1.0667 1.0711 1.0755 1.0799

1.0628 1.0672 1.0715 1.0759 1.0803

1.0633 1.0676 1.0719 1.0763 1.0807

1.0637 1.0680 1.0724 1.0768 1.0812

1.0641 1.0685 1.0728 1.0772 1.0816

1.0646 1.0689 1.0733 1.0777 1.0821

1.0650 1.0693 1.0737 1.0781 1.0825

20 21 22 23 24

1.0830 +. 1.0874 1.0919 1.0965 1.1010

1.0834 1.0879 1.0924 1.0969 1.1015

1.0839 1.0883 1.0928 1.0974 1.1020

1.0843 1.0888 1.0933 1.0978 1.1024

1.0848 1.0892 1.0937 1.0983 1.1029

1.0852 1.0897 1.0942 1.0987 1.1033

1.0856 1.0901 1.0946 1.0992 1.1038

1.0861 1.0905 1.0951 1.0997 1.1043

1.0865 1.0910 1.0956 1.1001 1.1047

1.0870 1.0915 1.0960 1.1006 1.1052

25 26 27 28 ‘29

1.1056 1.1061 1.1066 UT1OS ieISHOT ad tee) 1.1149 1.1154 1.1159 1.1196 1.1201 1.1206 1.1244 1.1248 1.1253

1.1070 tz 1.1163 1.1210 1.1258

1.1075 Ltt 1.1168 1.1215 1.1263

1.1079 TIT 26) 1.1173 1.1220 1.1267

1.1084 SST 1.1178 1.1225 1.1272

1.1089 1 NIS5. 1.1182 1.1229 1.1277

1.1093 WU40) 1.1187 1.1234 1.1282

1.1098 1.1145 1.1192 1.1239 1.1287

30 31 32 33 34

1.1291 1.1339 1.1388 1.1436 1.1486

1.1296 1.1344 1.1393 1.1441 1.1490

1.1301 1.1349 1.1397 -1.1446 1.1495

1.1306 1.1354 1.1402 1.1451 1.1500

1.1311 1.1359 1.1407 1.1456 1.1505

1.1315 1.1363 1.1412 1.1461 1.1510

1.1320 1.1368 1.1417 1.1466 1.1515

1.1325 1.1373. 1.1422 1.1471 1.1520

1.1330 1.1378 1.1427 1.1476 1.1525

1.1334 1.1383 1.1432 1.1481 1.1530

35 36 37 38 39

1.1535 1.1585 1.1635 1.1685 1.1736

1.1540 1.1590 1.1640 1.1690 1.1741

1.1545 1.1595 1.1645 1.1696 1.1746

1.1550 1.1600 1.1650 1.1701 1.1752

1.1555 1.1605 1.1655 1.1706 1.1757

1.1560 1.1610 1.1660 1.1711 1.1762

1.1565 1.1615 ‘1.1665 1.1716 1.1767

1.1570 1.1575 1.1620 1.1625 1.1670 1.1675 1.1721 . 1.1726 1.1772, 1.1777

1.1580 1.1630 1.1680 1.1731 1.1782

by weight of sucrose

(Contd.)

Analysis of Fruit and Vegetable Products

914

TABLE 28-16 (Contd.-) eee E See D Eating, 6 WA Degrees Brix or

per cent

.0

i!

2

%

A

6

>

si

8

9

by weight of

sucrose ESE ne 40 41 42 43 _ 44

1.1787 1.1839 1.1891 1.1943 1.1996

1.1793 1.1844 1.1896 1.1949 1.2001

1.1798 1.1849 1.1901 1.1954 1.2007

1.1803 1.1855 1.1907 1.1959 1.2012

1.1808 1.1860 1.1912 1.1964 1.2017

1.1813 1.1865 1.1917 1.1970 1.2023

1.1818 1.1870 1.1922 1.1975 1.2028

1.1824 1.1875 1.1928 1.1980 1.2033

1.1829 1.1881 1.1933 1.1985 1.2039

1.1834 1.1886 1.1938 1.1991 1.2044

45 46 47 48 49

1.2049 1.2102. 1.2156 1.2211 1.2265

1.2054 1.2108 1.2162 1.2216 1.2271

1.2060 1.2113 1.2167 1.2222 1.2276

1.2065 1.2118 1.2173 1.2227 1.2282

1.2070 1.2124 1.2178 1.2232 1.2287

1.2076 1.2129 1.2184 1.2238 1.2293

1.2081 1.2135 1.2189 1.2243 1.2298

1.2087 1.2140 1.2194 1.2249 1.2304

1.2092 1.2146 1.2200 1.2254 1.2309

1.2097 1.2151 1.2205 1.2260 1.2315

50 51 52 53 54

1.2320 1.2376 1.2431 1.2487 1.2544

1.2326 1.2381 1.2437 1.2493 1.2550

1.2331 1.2387 1.2442 1.2499 1.2555

1.2337 1.2392 1.2448 1.2504 1.2561

1.2342 1.2398 1.2454 1.2510. 1.2567

1.2348 1.2403 1.2459 1.2516 12572

1.2353 1.2409 1.2465 1.2521 1.2578

1.2359 1.2415 1.2471 1.2527 1.2584

—-1.23641.2420 1.2476 1.2533 1.2589

1.2370 1.2426 1.2482 1.2538 1.2595

55 56 57° 58 59

1.2601 1.2658 1.2716 1.2774 1.2832

1.2606 1.2664 1.2721 1.2779 1.2838

1.2612 1.2670 1.2727 1.2785 1.2844

1.2618 1.2675 1.2733 1.2791 1.2850

1.2624 1.2681 1.2739 1.2797. 1.2856

1.2629 1.2687 1.2745 1.2803 1.2861

1.2635 1.2693 1.2750 1.2809 1.2867

1.2641 1.2698 1.2756 1.2815 1.2873

1.2647 1.2704 1.2762 1.2821 1.2879

1.2652 1.2710 1.2768 1.2826 1.2885

60 61 62 63 64

1.2891 1.2950 1.3010 1.3069 1.3130

1.2897 1.2956 1.3015 1.3075 1.3136

1.2903 1.2962 1.3021 1.3081 1.3142

1.2909 1.2968 1.3027 1.3087 1.3148

1.2914 1.2974 1.3033 1.3093 1.3154

1.2920 1.2980 1.3039 1.3100 1.3160

1.2926 1.2986 1.3045 1.3106 1.3166

1.2932 1.2992 1.3051 1.3112 1.3172

1.2938 1.2998 1.3057 1.3118 1.3178

1.2944 1.3004 1.3063 1.3124 1.3184

65 66 67 68 69

1:3190; 1.3252 1.3313 1.3375 1.3437

13197) 1.3258 1.3319 1.3381 1.3443

1.3203) 1.3264 1.3325 1.3387 1.3450

3209) 1.3270 1.3332 1.3394 1.3456

3215) 1.3276 1.3338 1.3400 1.3462

U32ZZ1 1.3282 1.3344 1.3406 1.3468

13227) 1.3288 1.3350 1.3412 1.3475

115233) 1.3295 1.3356 1.3418 1.3481

14239 1.3301 1.3363 1.3425 1.3487

1.3245 1.3307 1.3369 1.3431 1.3494

70 71 72 73 74

1.3500 1.3563 1.3626 1.3690 1.3754

1.3506 1.3569 1.3633 1.3696 1.3761

1.3512 1.3575 1.3639 1.3703 1.3767.

1.3519 1.3582 1.3645 1.3709 1.3774

1.3525 1.3588 1.3652 1.3716 1.3780

1.3531 1.3594 1.3658 1.3722 1.3786

1.3538 1.3601 1.3664 1.3729 1.3793

1.3544 1.3607 1.3671 1.3735 1.3799

1.3550 1.3614 1.3677. 1.3741 1.3806

1.3557 1:3620 1.3684 1.3748 1.3812

75 76 yh 78 79

1.3819 1.3884 1.3949 1.4015 14081

1.3825 1.3890 1.3955 1.4021 1.4087

1.3832 1.3897 1.3962 1.4028 1.4094

1.3838 1.3903 1.3969 1.4034 1.4101

1.3845 1.3910 1.3975 1.4041 14107

1.3851 1.3916 1.3982 1.4048 14114

1.3858 1.3923 1.3988 1.4054 1.4121

1.3864 1.3929 1.3995 1.4061 1.4127

1.3871 1.3936 1.4001 1.4067 1.4134

1.3877 1.3942 1.4008 1.4074 1.4140 (Contd.)

Fruit Juices, Concentrates .and Beverages TABLE

28-16

915

(Contd.)

80 81 82 83 84

1.4147 /1.4154 1.4214 1.4221 1.4281 1.4288 1.4349 1.4355 1.4417 1.4423

1.4160 14227 1.4295 1.4362 1.4430

1.4167 1.4234 1.4301 1.4369 1.4437

1.4174 1.4241 1.4308 1.4376 1.4444

1.4180 1.4247. 1.4315 1.4383 1.4451

1.4187 1.4254 1.4322 1.4389 14458

1.4194 1.4261 1.4328 1.4396 1.4464

1.4201 1.4207 1.4268 1.4274 1.4335 1.4342 1.4403 1.4410 1.4471 - 1.4478

85 86 87 88

1.4485 1.4554 1.4623 1.4692

1.4499 1.4567 1.4636 1.4706

1.4505 1.4574 1.4643 1.4713

1.4512 1.4581 1.4650 1.4720

1.4519 1.4588 1.4657 1.4727:

1.4526 1.4595 1.4664 1.4734

1.4533. 14602 1.4671 1.4741

1.4540 1.4609 1.4678 1.4748

1.4492 1.4560 1.4629 1.4699

1.4547 1.4616 1.4685 1.4755

Reprinted from Official Methods of Analysis, 9th edn., with the permission of Official Analytical Chemists,

Artinglton, VA

22209.

By referring to Domke’s Table (Table 28-16) of apparent specific gravity of sucrose solutions at 20° C, find the °Brix corresponding to the specific gravity of diluted juice. By interpolation express the Brix value in two decimal places. Let this Brix value found from the table be ‘b’. Calculate the Brix value of the original

concentrate using: p=

2%t+y

x where, B = Brix value of original concentrate

b = Brix value of diluted concentrate tound from Domke’s table. x = weight of concentrate in grams originally taken y = weight of water in grams added to the concentrate Find the specific gravity of original concentrate corresponding to Brix value ‘B’ from Domke’s Table. Calculate the fold of concentration as follows: Fol

Specific gravity of concentrate X Brix of concentrate Specific gravity of juice X Brix of juice

From the above expression, it is seen that °Brix and specific gravity are interrelated, and that the fold of concentrate is dependent not only on the specific gravity of concentrate, but also on the specific gravity of juice used in preparing the concentrate for which the USDA. assumes a value of 1.0743 at 20/20° C, ie., 11.75 °Brix. The linear relationship between refractive index and concentration in pure solutions is also found in mixed solutions, certain extreme cases of which are

exemplified by fruit juices and their concentrates. Basker? has described an accurate method for determining the specific gravity of concentrate which makes use of the linear relationship between the refractive index and the concentration (w/v).

The degree of concentration may be obtained from the specific gravity or refractive index using Table 28-17.

“sain Sn4jjzI>) JO UOTIBIJUZIUOTD JO 2039aq] ay? Jo NONeUTTISa ay2 SuTUIaDUO) sisTWaYD jo [aueg ay) WO; 130day JoIUOD sadnf yNIy sNIID—-foy 06¢ 1-18¢'I O8¢ 1-1 LET OLE T-19¢ T O09¢ T-1S¢1 OSE T-6EE 1 BEC 1-97E 1 C7E 1-FIE L CIE 1-l0¢E T 00¢ 1-687 T 887 1-9Z7'T CLO 1-¥9T I €9TT-1ST7'1 OSZ 1-6¢7 I 8E7 1-977 T S77 I-VIT I €17 1-107 1 0021-681 'T 881 T-9ZT'T SLI I-VOL' COL I-ISUT OST'1-G¢L'l

xopul

Auaeas

jo

$1 UOII¥IJUIIUOD

8 YL “AL ”L & YO Zt) Vite) 9 YS “”G YG ¢ YY “AY YY y YE ”AL YC g O8LY'T-9¢Ly'T CCLVI-169¥'T 069¥'1-1S9F'T OSOV'I-L19F'T O19P 1-99Sh'T cOSyI-1ZSh'I O@SV L-9LbV'T CLyY I-I¢yy'l O¢yy I-18¢h'1 O8cy I-I¢ey'l O The most abundant carotenoid of the tomato is lycopene, which comprises approximately 83% of the pigments present. Procedures for determining B-carotene and lycopene are given in Chapter 17.

Criterion for evaluating the red colour of tomatoes is the ratio between transmittance of light at 620 nm and 670 nm, and the criterion for green tomatoes

is the ratio between 520 nm and 545 nm.

Measurement of colour of tomato products including the juice is usually made by visual comparison. The Munsell system using the Maxwell spinning disc is used

in industry and by the Federal Graders in USA. A rapid method for determination

of colour consists of extracting the sample with benzene and measuring at 485 nm°.

Tomato

Products

973

Tomato products being opaque, the visible colour is determined from the light reflected from their surface. Procedures for measuring colour by reflectance are given in Chapter 17. The instruments generally used for measurement of reflectance colour in the tomato processing industry are the Hunter colour and colour difference meter, the Purdue colour ratio meter and Agtron colorimeter. Such instruments measure the relative redness and yellowness of samples in terms.

which can be used directly as a guide in classification or to estimate

tristimulus or Munsell factors. The reflectance values measured at 440, 550 and 620 nm, and expressed as a tatio of Rgz0/(Rsso0 - Rago), (where, R is the reflectance at the particular

wavelength) places the sample approximately in the order of visual ranking.’ The Macbeth-Munsell Disc Colorimeter, widely used in USA for designating grades for tomato products, consists of four coloured discs—red, yellow, black and grey. These are overlapped in such a way as to expose a desired percentage of each disc. The disc combination is then spun at 2,500 rpm in the instrument, and the colour of tomato product is visually compared with the standard under standard illumination condition of the instrument.

Grades A and B tomato products according to USD A specifications should have colour which is equal to or more red than the disc combination I, and Grade C product should have colour which is equal to or more red than the disc combination II as shown in Table 30-13.

TABLE

30-13: Munsell Colour Standards for Tomato Products

Disc Number

1. 5 R 2.6/13 (glossy finish)

2. 2.5 YR 5/12 (glossy finish)

Percentage of Grades A & B

disc exposed Grade C

I

Il

65

53

21

28

3. Black Ni

7

9%

4. Grey Na

7

oY

During ripening of tomatoes, with the changes in colour from green to red, chlorophyll decreases, while the total carotenoid and lycopene contents increase. If chlorophyll is present in the tomatoes used for processing, they are degraded during heat treatment. and/or storage. The use of red tomatoes having slight yellow patches yield ketchup equivalent to US Grade C, while red and red ripe tomatoes yield ketchup equivalent toGrades A and Bfor colour. Rather than lycopene content, chlorophyll content of fresh juice has been found to be a better index of quality of the resulting ketchup. Parameters of colour for fresh tomato juice for preparing ketchups are given in Table 30-14.

974

Analysis of Fruit and Vegetable Products

TABLE 30-14: Colour Standards for Fresh Tomato Juice Suitable for preparing Ketchup Conforming to US Standards Grades A and C Grade A

Particulars

Grade C

Chlorophyll, mg/100 g Redness index = Rg20/(Rsso—R4ao)

Max. Min.

0.027 10.0

Max. Min.

0.120 6.3

Tintometer reading: Red units Yellow units

Min. Max.

12.5

Min.

10.6

8.5

Max.

Dominant wavelength* nm

592

9.5

589.6

* Determined by reflectance colour measurement.

Measurement of Consistency of Tomato Pruducts

Viscosity or consistency is one of the important factors in determining the overall quality and acceptability of any tomato products. Consistency measurement may be made using Bostwick and Adam’s consistometers, Efflux tube viscometer, Stormer viscometer or Brookfield synchrolectric viscometer. See Chapter 18 for details. Mineral Impurities‘

The AOAC method (1975—3.005) which is the approved procedure of Codex Alimentarius Commission for determining mineral impurities is given below: Use concentrate, puree or paste equivalent to 250 g of 8% w/w soluble solids. Ash the sample at 500- 550°C ina platinum dish. Wet the ash with 5-10 ml of HCl, boil approximately for 2 min, evaporate to dryness on a water bath, and heat for another 3 hr. To the residue, add 5 ml HCl, boil for 2 min, add 50 ml of water heat on the water bath for a few minutes, and filter through filter paper. Do not discard the filtrate if metals are to be estimated. Sand: Wash the residue on the filter paper to the platinum dish, add 20 ml of saturated NagCO; solution, and boil for about 5 min. Add a few drops of 10% NaOH solution, and allow to settle. Decant the supernatant and filter through ignited and weighed Gooch crucible. To the residue in the dish, add 20 ml of saturated NagCOs solution, boil, decant and filter as before. Repeat the process once more. Transfer the residue in the dish to the Gooch crucible, wash with water, then with little dilute HCl (1+4), and finally with hot water until free from chlorides.

Save the combined filtrate. Dry the Gooch crucible, ignite at 5|00—550° C, cool and weigh. The difference in weight gives sand. Alkali-soluble SiOz: Acidify the combined alkaline filtrate obtained in the determination of sand with HCl, evaporate to dryness, and heat in a drying oven at 110-120° C for 2 hr. Moisten the residue with 5-10 ml HCl, boil for 2 min, add 50 ml of water, heat on a boiling water bath\for 15 min, and filter through an ashless

Tomato Products

filter paper or ignited and tared Gooch

975

crucible. Wash the residues with hot

water, ignite at 500-550°C, cool and weigh as SiO). Add the filtrate and washings collected in the determination of SiO, to the previous ones, and use for metal estimation. Find the sum of sand and alkali-

soluble SiO2, and express the results as mg/kg of mineral impurities on diluted product of 8% solids. Mineral Contaminants

See Chapter 6

Mould Count, Rot Count and Extraneous Contamination

See Chapter 24 References

1.

Official Methods of Analysis, Association of Official Analytical Chemists, Washington, DC, USA,

2.

Bigelow, W.D.,H.R.

11th edn, p. 559 (1970).

Smith & C.A. Greenleaf, Tomato Products, Pulp, Paste, Catsup, Chilli Sauce and Juice, Bull. 27-L, Revised, National Canners Association Research Laboratory, Washington, DC, USA (1950).

National Canners Association Research Laboratories, Laboratory Manual for Food Canners and Processors (The AVI Publishing Co. Inc., Westport), Vol. II, p. 277 (1968). 4. Official Methods of Analysis, Association of Official Analytical Chemists (12th edn.), pp.34 & 598 (1975). 5. Gould, W.A.,

Tomato

Production,

Company, Westport, Connecticut,

Processing and Quality Evaluation, The AVI

Publishing

USA, p. 228 (1974).

6. Kramer, A., & R.B. Guyer., Proc. Am. Soc. Hort. Sci., 51, 381 (1948). 7. Goose, P.G. & R. Binsted , Tomato Paste and other Tomato Products, Food Trade Press Ltd.,

London (1973).

& Pairat, N. & S. Ranganna, Indien Food Packer, XXXI (2), 42 (1977).

—

CHAPTER 31

Dehydrated Fruits and Vegetables Preparation of Sample (other than for enzyme test)

"TAKE a representative sample of at least 50 g and grind ina suitable grinder, so that at least 99 per cent passes through a 30-mesh sieve. Mix the material

remaining on the sieve with that passing through the sieve to constitute the prepared sample. Take care during grinding and handling to avoid uptake of moisture as dehydrated vegetables are, in most cases, hygroscopic. All operations should be carried out as expeditiously as possible. In the case of dehydrated onions, rapidly mix the bulk sample and pass a representative sub-sample of about 25 g through a coffee grinder. Rapidly mix the whole of the ground sample without sieving. Moisture

Dry in a metal dish (6-8 cm metal cover. Spread 4 to 8 g of the the bottom of the dish, cover and and the cover for 6 hr ina vacuum 50+ 10 mm of mercury. During

diameter) provided with a prepared sample as evenly as weigh. Remove the cover. oven at 70 + 1° C and at drying,

admit

tightly fitting possible over Dry the dish a pressure of air to the oven at the

rate of 2 bubbles per sec through a conc H,SO, bubbler. After 6-hr stop vacuum pump and introduce air at such a rate that the oven is pheric pressure within 5 min. Remove the dish, replace the cover, desiccator and weigh within 30 min. Report percentage loss of

of drying, at atmoscool in a weight as

moisture.

Enzyme Test

Catalase Reaction' Take a test tube of approximately 1 inch in diameter and 6 inch deep and fill to a depth of approximately 1 inch with small broken pieces of the dehydrated vegetable. Cover with water and allow to stand for 10 min. Add an equal volume of 3% hydrogen peroxide and mix the contents of the tube by shaking gently. The test is positive if there is an evolution of oxygen. Peroxidase Reaction

Take a small portion of the sample and reconstitute in cold water for 10-15 min. Pour off excess water. Add small pieces of the reconstituted sample to a

Dehydrated Fruits and Vegetables

977

depth of about 1 inch in a test tube 1 inch in diameter. Add 10 ml of water and 1 ml of a 1% guaiacol solution in 95% alcohol and 1 ml of a fresh 0.5% hydrogen peroxide solution [10 ml of “10 volume” (3%) hydrogen peroxide added to 50 ml of water]. Shake the test tube to mix and note the reaction as given below after 5 and 10 min. i Negative — No discolouration, or reddish brown specks, or a few larger spots especially on veins. Light positive— Light brown markings scattered throughout the tissue or heavy browning of a few pieces not‘ restricted to veins. Positive — More pronounced reddish brown colouration than in light positive. For dehydrated beetroot, in place of guaiacol solution, use a 0.5% solution of benzidine in 95% alcohol; a bluish black colouration indicates a positive test.

Check the effectiveness of the reagents by carrying out the test on fresh vegetables, and on the same vegetable after boiling for 5 min and cooling. The former should give a strong positive reaction and the latter, a negative reaction. Blemish Count?

Rehydrate 50 g representative sample of the dehydrated material. Visually count the number of surface blemishes. Multiply the number of blemishes by 2 and report as number of blemishes per 100 g of sample. Bulk Density? Bulk density indicates the weight of substance held in a unit volume and is of particular importance in the packaging of the product. For its determination, note the tare weight of a glass cylinder of ‘exactly 625 ml water capacity. Fill the sample from a hopper suspended 3 cm away. Strike off the excess of sample above the rim of the cylinder and note the weight again. Bulk density, lb/cu ft = Wt of sample in grams/10 Compacted bulk density may be determined by weighing after the cylinder has been repeatedly tapped and refilled.

Rehydtration Tests (Fruits and Vegetables) There is no standard method for measuring rehydration and it is, therefore, advisable that suitable procedures be developed in each plant so that daily evaluation of the quality of the product can be made. The technique of performing each test must be carefully standardized if comparable data are to be obtained. The following procedure for measuring rehydration is suggested by the US Department of Agriculture.? PROCEDURE

Weigh 2 to 10 g of the dry material. Place in 500-ml beakers, add 80 to 150 ml of distilled water, cover each with a watch glass, bring to a boil within 3 min on an electric heater, and boil for 5 min, The precise amount of water

978

Analysis of Fruit and Vegetable Products

will vary with the material, time and rate of boiling; excessive amounts of water should not be used. Remove from the heater and dump into a 7.5 cm Buchner funnel which is covered with a coarsely porous Whatman No. 4 filter paper. Apply gentle suction and drain with careful stirring for half to one min until the drip from the funnel has almost stopped. Do not dry by long suction. Remove from the funnel and weigh. Set the drained sample aside in a covered porcelain evaporating dish for quality tests. Repeat this test, and then rehydrate six,other 10-g samples, boiling two for 10 min, two for 20 min, and two

for 30 min. It will be necessary to use 20 to 30 ml more of water for the last two tests than for the shorter boiling, first test. Only small pieces will rehydrate in 5 min. CALCULATION

Calculate the results in terms of ‘rehydration ratio,” “coefficient of rehydration,” and “per cent of water in the rehydrated material,” as given below :

Rehydration ratio: If the weight of the dehydrated sample (a) used for the test is 10 g and the drained weight of the rehydrated sample (b) 80 g, then

Rehydration ratio

= a: b:: 10:80

Coefficient of rehydration : If the drained weight of 10 g of dried cabbage containing 5% moisture after rehydration is 95 g, and the fresh cabbage before

drying contained 93% moisture, Drained wt of deCoefficient of _ hydrated sample

100 —

rehydration

Amount of moisture present

— FWe of dried sample taken for rehydra- — tion

Moisture content of sample : before drying

in the dried sample taken for rehydration

| x 100

95 x (100 — 93) _ 0.7 ~ (10 — 0.5) x 100 Per cent water in the rehydrated material : The drained weight of the rehydrated sample being known, the per cent water content in the rehydrated material is given by Drained wt of rehydrated material

Dry matter content in the sample taken for rehydration Dreined wt of rehydrated material

ony

Following the values given under “coefficient of rehydration,” the moisture

content in the

rehydrated sample is see

x 100 = 90.0%

In making rehydration tests, it is suggested that the following conditions be met.3

Dehydrated Fruits and Vegetables

979

1. Work out atime and temperature sequence suitable! to the test material. 2. Determine the time of soaking and boiling that is compatible with optimum quality of each sample. 3. Always run a series of tests at various times and temperatures and evaluate the data on the rate of change in coefficient rather than on a single determination of a coefficient. 4. Start the test with at least enough water to submerge the pieces, but do not use so much water that excess amounts are present at the end of the test, especially when quality tests are being made on the samples. 5. Shake or stir, if necessary, to insure wetting of all pieces during the test. 6. Control the rate of heating so as to prevent rapid and variable losses of water while boiling. 7. Use unit heaters set up in such a way as to prevent rise in temperature from radiatedor convected heat. References 1. Standard Method for the Analysis and Examination of Foodstuffs. Commonwealth Food Specifications Committee, Department of Primary Industry, Canberra, 5th edn., p. 205, 1969. 2. Lees, R., The Laboratory Handbook of Methodsof Food Anzlysis, Leonard Hill Books, London, p. 93,

3. 4.

1968.

Anon, Vegetables and Fruit Dehydration, US Dept. Agr., Misc. Publ., p. 540, 1944., von Losecke, H.W., Drying and Dehydration of Foods, poration, New York, 2nd edn., p. 283, 1955.

Reinhold

Publishing

Cor-

CHAPTER 32

Pickles, Chutneys and Vinegar CHUTNEYS

AND PICKLES

CHUTNEYS and pickles constitute an important section of the fruit and vegetable processing industry in India. Fruits and vegetables form the most important constituent of these products. Onions, garlic, chillies and spices are added to improve the taste. Among chutneys, mango chutney occupies a prominent place. It is a product prepared by cooking the fruit in sugar syrup with salt, spices and vinegar or acetic acid to a thick consistency. There are many grades of chutneys—sweet, sliced, sweet Lucknow, Major Grey, Colonel Skinner,etc., prepared from sliced mango, and the manufacturing process controlled to preserve the shape and texture of slices; Bengal chutney which contains mango in the form of cubes instead of slices; and Kashmiri chutney prepared from mango pulp. In addition to mango, Major Grey contains candied peel; Colonel Skinner, raisins and currants; and Bengal

chutney, mustard. Sweet lime chutney is prepared from brine-cured limes to a limited extent.

Salt, oil, vinegar and sweet pickles are the important varieties commercially manufactured. Salt pickles contain fresh or brine-cured material preserved with 12 to 15% salt. Turmeric, chilli powder and spices are the other ingredients. In this group may be included pickle in lime juice in which the finished product should | contain not less than 12% salt and 1.2% acidity as anhydrous citric acid, Oil pickle is a salt pickle with fresh oil (groundnut, sesame or mustard) as an additional ingredient. These are generally prepared from mango, lime and lemon. Vinegar pickle is prepared either from fresh or brine-cured material from which the salt has been leached out, and preserved using vinegar or dilute acetic acid, and may or may not contain added sugar. Onion and mixed vegetable vinegar pickles are the ones commercially manufactured. In Europe, sweet pickle refers to the vinegar pickle containing higher amounts of sugar. Sweet pickle, as manufactured in India to a limited extent for home consumption but mainly for export to suit the Western palate, is a product prepared from fresh or brine-cured fruits or vegetables, and contains salt, sugar, acetic acid or vinegar, oil and ground garlic, ginger, turmeric, chilli (red, dry) and spices as ingredients; the whole mass is cooked.

The composition of pickles is given in Table 32-1.

Pickles, Chutneys and Vinegar

981

TABLE 32-1: Composition of Commercial and Laboratory Samples of Pickles Ent Sei ET, GE IIE OE SF) aS: DISS SUP. OS Salt pickle Oil pickle Sweet pickle Particulars ee Commercial Laboratory Commercial Laboratory Laboratory sample sample sample sample sample Range

Moisture Salt as NaCl Acidity as anhydrous citric acid Volatile acidity as acetic acid pH Ether extractives Sugars — Total

% 66.0 - 74.2 % 13.9 - 15.0 Jo

Range

66.13 17.61

49.6 - 73.0 13.5 - 17.24

53.74 18.74

39.48 8.81

1:79=.2.17

0.64

1.4 - 1.60

1.44

0.78

2.4 - 2.7 1.76- 3.29

3.15 3.02

% % %

2.45- 3.0 13.6 -39.6

3.05 20.40

1.79 2.30 16.40 26.25

Source: Sastry, M.V., S. Ranganna & G.S. Siddappa., Indian Food Packer, XV (5), 16 (1961); XV (6),

7 (1961).

Principles of Preservation of Chutneys and Pickles The concentration of salt which acts as a preservative in salt pickle, pickle in citrus juices and oil pickle should be not less than 12% but preferably maintained at 15%. In oil pickles, the salt content in the pickle mass minus the oil content

should be not less than 12%. Sufficient quantity of oil in the pickle to provide a thin layer at the top helps to create anaerobic conditions, and check the growth of aerobic microorganisms (aerobic bacteria, moulds and yeasts) which cause spoilage. In chutneys and vinegar pickles, the actual acetic acid content, and not the total acidity calculated as a percentage of the total volatile constituents, i.e.,

Total acetic acid activity X 100 (100 - total solids)

é

a value which may be called the “preservation index” is important.! The index should preferably be not less than 3.6% which generally helps to preserve the product. In such products, from the organoleptic point of view, the salt content should not exceed 3 to 4%. Hence, by increasing the sugar content, it is possible to reduce the acetic acid concentration required.

According to Burrell,? in vinegar-containing products like chutneys, pickles, mayonnaise, salad creams, sweet sauces, etc., which include sugar and/or salt solutions and oil emulsions, the nature of spoilage is common in all cases. The acidity (as acetic acid) — on water content — factor should be not less than 3.6%. The quantity of acetic acid required may be calculated using the expression:

982

Analysis of Fruit and Vegetable Products

Acetic acid in the whole product % =

3.6 X % moisture content

700

Bells and Etchells? have given the following formula where sugar is present in the pickles:

% Acetic acid in the whole product =

80 - % sugar in the whole product

50

This index provides for a slightly lower acetic acid content. In the indigenous sweet pickle which contains oil, although added sugar and salt have a preservative action, their concentration is not sufficient enough to preserve the product. Hence, the same expressions could be made use of for calculating the acetic acid content required. By determining the moisture content in the product mix before cooking in the oil media, the quantity of acetic acid required to be added may be calculated.

Quality Control Steps 1. Fruits and Vegetables—maturity, size, and freedom from damage or spoilage. 2. During Brining a. Salt and acid concentration with fermenting brine—Initial 8 to 11% but equilibrium salt content of 9% should be maintained. With nonfermenting brine,

salt content should be 15% with added 1% lactic acid. Add 2.0% lactic acid to 20% brine in the case of unfermented onions. Addition of 0.5% acetic acid to pricked cucumber brine reduces bloating. b. Check on brine concentration and topping of the brine. c. Adjustment of the brine concentration after completion of fermentation (3 to 8 weeks) to 15% and lactic acid concentration to 1%.

d. Prevention of growth of surface yeast (scum) with a layer of oil, ultra-violet

irradiation, or by adding 1,000 ppm of sorbic acid, if permitted. 3. During Preparation a. Vegetables—debrined,

acidified and/or sweetened, salt content, acidity,.

appearance, colour, texture, flavour, foreign matter, and refractometric solids

(when sugar is added). b. Packing liquor—acidity, refractometric solids if sweetened, colour, clarity, and flavour. SOx, if used. c. Oils—colour, flavour, refractive index, free fatty acids, peroxide value, Kries test, antioxidant, and tests for ensuring freedom from adulteration.

d. Containers—checking for dimensions, freedom from defects, weight and capacity. e, All raw materials—mineral residues (do as frequently as possible), pesticide residues particularly on fruits and vegetables, SOz, and any unpermitted preservatives. 4. Finished Product a. Carry out the cut-out examination of pickles in vinegar as detailed in Chapter . 26.

Pickles, Chutneys and Vinegar

983

b. Examine all products for net weight of contents, appearance, colour, odour, texture, flavour, defects and foreign matter, refractometer solids, total acidity, salt

content, and microbiological quality (mould, yeast and bacterial counts). c. Determine volatile acidity, as acetic acid, in chutneys, vinegar pickles andsweet pickles. d. Oil content in oil pickles. e. Mineral impurities. f. Extraneous matter. Determination Sampling, Examination and Preparation of the Sample Follow the procedure described for canned foods for sampling, determination of net weight, fill of the container, visual examination, determination of drained

weight, and refractometric solids. Blend the material to get a composite sample. Salt content

Determine by the procedure given in Chapter 9 (see page 204) or by the indirect (Volhard) method (see page 964).

Acidity—Total and Volatile Follow the procedure prescribed in the case of vinegar. Determine the total acidity in 25g of the composite sample. To another 25 g of the sample in an evaporating dish, add about 150 ml water and evaporate on a water bath. Determine the acidity which gives fixed acidity. The difference between the two values is the volatile acidity. Express the results as acetic acid. In the case of sweet pickle which contains oil, to 25 g of the composite sample, add 150 ml of water, stir vigorously and allow to settle. Transfer the supernatant to a 250 ml separating funnel, allow the water and oil layers to separate. Drain the aqueous layer to the same container which contained the sample. Wash the oil phase once or twice with 5 to 10 ml portions of water, and transfer the washings to the container which contained the sample. Determine the total acidity, fixed acidity, and by difference acetic acid content as before. Instead of evaporation and determining the volatile acidity by difference, the sample with water added (and oil separated in the case of sweet pickle) may be determined by steam distillation as in the case of vinegar. Repeated evaporation or continued distillation may have to be adopted to ensure accurate estimation of volatile acidity. Oil Content in Oil and Sweet Pickles

Dry a known weight of the composite sample in a hot air oven by following the procedure described for the determination of moisture (see page 3) and extract

984

Analysis of Fruit and Vegetable Products

the oil in a Soxhlet extraction apparatus using petroleum ether of 40-60° C (see page 10).

A more accurate method would be to extract, using chloroform-methanol . extraction procedure (see page 231). The oil extracted by this procedure could be used for further characterization and analysis.

Preservatives—See Chapter 12. Mould count, rot count, insect fragments and other Chapter 24. Mineral Acids—see page 988. Ash—Total and Acid-Insoluble—see page 8. Mineral contaminants—see Chapter 6. Mineral impurities—see Chapter 26.

foreign matter—see

References 1. Blanchfield, J.R., in Food Industries Manual (Ed.), Anthony Woollen, Leonard Hill, p. 422 (1969). 2. Burrell, J.R., Food Manuf., 4, 168 (1948). 3. Bell, T.A. & J.L. Etchells, Food Technol., 6 (12), 468 (1952).

VINEGAR Vinegars are of two types — brewed and artificial. Brewed vinegars are made from various sugary and starchy materials by alcoholic and subsequent acetic fermentations. Apples, grapes and pineapples are the fruits generally used for the purpose. Malt vinegar is derived wholly from malted barley, with or without the addition of the cereal grain, malted or otherwise, the starch of which is saccharified by the diastase of the malt. Distilled malt vinegar is prepared by distilling the malt vinegar. The product merely contains the volatile constituents of the vinegar from which it is derived. It is colourless and is generally used in the manufacture of pickled onions. Spirit vinegar (also called white vinegar, alcoho] vinegar, grain vinegar or distilled vinegar) unlike distilled malt vinegar, is the product made by acetous fermentation of a distilled alcoholic fluid which in turn is produced by fermentation. It is usually made by alcoholic fermentation of molasses and then distilled prior to acetic fermentation. Spiced vinegars are prepared by steeping the leaves or spices in an ordinary vinegar. Artificial (synthetic, non-brewed) vinegars are prepared by diluting synthetic acetic acid and are coloured with caramel.

Specific Gravity Determine

the specific gravity using a hydrometer

or pycnometer

(see

Chapter 15). This varies from 1.013-1.022 for good malt vinegar. Brewed vinegars containing 5% acetic acid should have a specific gravity of about 1.019.

Pickles, Chutneys and Vinegar

985

Acidity REAGENTS

1. 2.

0.5 N NaOH Phenolphthalein indicator

Total Acidity Dilute 10 ml of sample with water in a porcelain dish (about 5 in. in diameter), add phenolphthalein, and titrate carefully with 0.5 N NaOH. Total acidity as ace-

r.. Titre x Normality of alkali

tic acid % (w/v)

x 60 x 100

Volume of sample taken x 1000

Fixed Acidity Evaporate 10 ml of vinegar at least 5 times with water and titrate as in total acidity.

Volatile Acidity Express by difference between total and fixed acid. Alternatively, steam distil the volatile acids and determine the acidity in the distillate as given below: Transfer 30 ml of the vinegar to a distillation flask and connect with the condenser. Heat to incipient boiling and pass the steam. Receive the distillate in a 25-ml graduated cylinder. After 15 ml have collected, test the drops of distillate from time to time with litmus paper, as they fall into the receiving vessel, and, when free from acid, discontinue the distillation. Note the volume

of the distillate. Mix by shaking and determine the acidity in an aliquot. Express the volatile acids as acetic acid. Total

Pipette 25 ml of the sample into a evaporate to dryness on a boiling tends to remain in the total solids by 2.5 hr in an oven at 100° C. Cool in a termination in duplicate.

Solids

tared silica dish (5 cm in diameter) and water bath. Remove the acid which three evaporations with water. Dry for desiccator and weigh. Carry out the de-

Ash Analysis REAGENTS

1.0.1

N HCl

2. 0.1 N NaOH 3. Methyl orange indicator Total Ash After determination of total solids, ignite the residue in a muffle furnace at 450° C to get the ash. Cool in a desiccator and weigh.

986

Analysis of Fruit and Vegetable Products

Alkalinity of Ash* The alkalinity number of an ash is defined as the number of millilitres of normal acid required to neutralize 1 g of the ash. To the ash in one of the silica dishes, add an excess of 0.1 N HCl and warm

the mixture.

Cool and

back titrate with 0.1 N NaOH solution using methyl orange as the indicator.

Carry out a blank titration using a similar volume of acid as added to ash. Calculate alkalinity as indicated below:

ml of blank Alkalinity number _ of ash

: titre

Wt

—

ml of sample ) Normality

at titre of ash x 1 N

x

of alkali

ie

1g

Water-Soluble and Insoluble Ash To the ash in the second silica dish, add 25ml of water. Cover witha watch

glass to avoid loss by spattering, and heat nearly to boiling. Filter through an ashless filter paper and wash with an equal volume of water. Place the filter paper and the residue again in the dish, ignite, and weigh. This gives the insoluble ash. Water soluble ash = Total ash — Insoluble ash Make up the filtrate which contains water soluble ash to 100 ml with distilled water and use for determining alkalinity of water soluble ash and NaCl.

Alkalinity of Water Soluble Ash Pipette 25 ml of the filtrate from water soluble ash solution (described above) and titrate with 0.1 N HCl using methyl orange as indicator. Report the result as the number of ml of 0.1 N acid required to neutralize the ash from 1 g of the sample.

Nitrogen Concentrate 50 to 100 ml of vinegar to a syrupy consistency and determine nitrogen by the Kjeldahl method (see page 16). Sodium

Chloride

Pipette 50 ml of the filtrate from water soluble ash solution and titrate with 0.1 N silver nitrate using potassium chromate as indicator to a red brown end point or by Volhard’s method (see Chapter 30).

ml of AgNO, _ Normality Sodium chlo-_ __tequired

ride % (w/v)

‘ of AgNO,

Total volume of x 0-5. x

ash solution

mil of ash solution

ml of sample taken for

taken for titration

ashing

x- 100

xT, 000

" *Determination of alkalinity of ash is not useful if the sample contains added mineral acid.

Pickles, Chutneys and Vinegar

987

Phosphoric Acid

Evaporate 25 ml of the vinegar in a silica dish and ash the residue at 650° C. To the ash in the dish, add 25 ml of 10 % HCl, cover with a watch glass, boil

gently over a low flame for 5 min, filter through an ashless filter paper, wash with hot water, and make up the volume to 100 ml. Determine the phosphcrus in an aliquot by the method given under phosphorus (see page 125) and express the results as phosphoric acid (H,;PO,). Thirty-one parts of phosphorus are equal to 98 parts of phosphoric acid. Malic Acid REAGENTS

1. 10% Calcium chloride solution 2. 0.1 N HCl 3. 0.1 N NaOH 4, Phenolphthalein indicator 5. Alcohol PROCEDURE Cider vinegar can be detected by its malic acid content. For a qualitative test, add 1 ml of lead acetate solution to a few ml of the sample. If no precipitate settles in a few minutes (leaving a clear supernatant liquid), the sample is not cider viriegar. Add 1 ml of calcium chloride solution (10%) to 5 ml of the vinegar and make alkaline with ammonia. Then filter off the precipitate. Add 3 volumes of alcohol to the filtrate and heat just to boiling. In the presence of malic acid, a flocculent precipitate settles. For the determination of malic acid, add 10 ml of the ela chloride solution to 100 ml of sample, make alkaline with ammonia, and filter after let-

ting the solution to stand for 1 hr. Evaporate the filtrate to 25 ml and add 75 ml of alcohol. Heat to boiling and filter. Wash the calcium malate precipitate with alcohol (75%), dry and ignite. Boil the ash with excess of 0.1 N HCl and back titrate with 0.1 N NaOH. Carry out a blank titration using the same volume of HCl as that used for the sample.

Malicalic,

acid %% =

acid)

: : Normality (Blank titre — Sample titre) x of NaOH * 67 x 100

ml of vinegar solution taken x 1.000 Caramel

Caramel is detected by Fiehe’s reaction, but it is not a conclusive test as

the reaction is given by any liquor which contains furfural. Extract 100 ml of the vinegar with 50 ml of ether. Transfer the separated

ether and allow it to evaporate off spontaneously. To the residue, add 3 drops

988

Analysis of Fruit and Vegetable Products

of 1% solution of resorcinol in HCl. In the presence of caramel a rose colour is produced. When the presence of furfural is suspected, follow the procedure given below which is based on AOAC method. Neutralize 10 to 20 ml of the vinegar with 10% NaOH solution and filter if necessary. Transfer to a centrifuge tube, add 2 ml of 5% zinc chloride solution and 2 ml of 2% potassium hydroxide solution, stir well and centrifuge. Pour off the liquid and add 25 ml of boiling water to the residue. Mix, centrifuge and pour off the liquid. Repeat this operation until the wash water is colourless. Dissolve the precipitate in 15 ml of 10% acetic acid solution, concentrate, neutralize carefully and filter. Divide into two portions. To one, in a 50-ml glass-stoppered cylinder, add 3-5 volumes of formaldehyde and just sufficient alcohol to form a homogeneous solution (avoid excess). Caramel, if present, is indicated by the formation of a brownish precipitate on standing. To the other, add equal volume of freshly prepared reagent consisting of phenylhydrazine hydrochloride—2 parts; sodium acetate (CH, COONa.3H,O) —3 parts; and water—20 parts. Dark brown precipitate is formed in the presence 4 of caramel. Mineral Acids

To 5 ml of vinegar, add 5 to10 ml of water and 4 to 5 drops of methyl violet solution (mix 1 part of methyl violet 2B in 10,000 parts of water). A blue or green colour indicates the presence of free mineral acid. Polarimetric Studies

Light-coloured vinegars, free from turbidity, may be measured directly in a 100-mm tube. Slight turbidity may be removed by filtering twice through a filter paper. It is advisable to add 10% basic lead acetate solution to precipitate malic or tartaric acid’ and to filterbefore polarization. If the vinegar is turbid or dark-coloured, add 5 ml of about equal quantities of lead subacetate and alumina cream to 50 ml of the sample, shake, filter, and measure in a 200-

mm tube. Add 10% to the reading to account for dilution. The polarization value of the vinegar is conveniently expressed in terms of actual direct reading obtained by the undiluted sample in a 200-mm or 400-mm tube..

Formol Value Brewed vinegars give a definite formol value but artificial products do not. It is useful as a preliminary quick test but is not a specific test to differentiate between brewed and synthetic vinegars. Distilled vinegars do not give any formol value. REAGENTS

1. 40% Formaldehyde solution 2. 0.01 N NaOH 3. Phenolphthalein indicator

Pickles, Chutneys and Vinegar

989

PROCEDURE

Add 2 or 3 drops of phenolphthalein to 10 or 20 ml of sample in a stoppered conical flask and titrate with standard alkali to rose pink. Use normal alkali to start with if the product is very acidic but finish with 0.1 or 0.01. N alkali. Neutralize 5 ml of formaldehyde to the same tint. Mix the two solu tions in the stoppered conical flask and allow to stand for 5—10 min. If pink

colour ‘has disappeared, titrate back with 0.1 or 0.01 N NaOH to the same tint. Imitation vinegar gives no appreciable formol value. Albuminoid Albuminoid ammonia

Ammonia

value, commonly

Value

determined in water analysis, can

be used for differentiating between malt vinegars and artificial products.1 REAGENTS

1. Alkaline potassium permanganate solution : Mix together one litre of potassium permanganate (KMnO,) solution (0.8%) and 500 ml of NaOH solu‘tion (40%). Boil the mixture until the volume is reduced to one litre. Unless it is freshly prepared, the reagent should be diluted with an equal volume of water and boiled down to the original volume before use. 2. Ammonia-free water: Take a large volume of tap water and a little dilute H,SO, in a distillation flask and start distilling. When the distillate gives no yellow colour with Nessler reagent, start collecting the water. 3. Nessler reagent :Dissolve 100g of mercuric iodide (HgI,) and 70 g potassium iodide (KI) in a small quantity of ammonia-free water,and add this mixture slowly with stirring to a cool solution of 160 g NaOH in 500 ml ammonia-free water. The reagent is stable for a year if stored in Pyrex or Corning glassware. 4. Standard ammonium chloride solution: Dissolve 3.819 g of anhydrous ammonium chloride in ammonia-free water and dilute to 1,000ml. Dilute 10: ml of this stock solution to 1,000 ml with ammonia-free distilled water (1.0

ml = 0.01 mg of N = 0.0122 mg NH;). PROCEDURE Pipette 5 ml of sample into a 100-ml graduated flask, neutralize with 0.2 N NaOH and dilute to the mark with water. Use ammonia-free distilled water throughout. Pipette 20 ml into a distillation flask, add 230 ml of water, a pinch of ignited sodium carbonate and ignited pumice. Distil 100 ml and reject the distillate. Then to the liquid remaining in the distillation flask, add 50 ml of alkaline potassium permanganate solution and 100 ml of water, and distil again. Collect exactly 100 ml of distillate and determine the ammonia by Nessler’s colorimetric method (as given in “Water Analysis”, Chapter 13)

or by matching against standard ammonium chloride solutions. For purposes of matching, dilute an aliquot of the distillate (10 ml is usually suitable for malt vinegar) to 50 ml with water in a Nessler glass, add 2 ml of Nessler

990

Analysis of Fruit and Vegetable Products

reagent, and match the colour ammonium chloride solution.

Forge ey ppm as N

po cepa

after 5 min

against dilutions

of standard

ml of standard ammonium chlo- _ Total volume x 1,000 x 0.01 *" of distillate ride required for matching p ml of distillate taken x m

Oxidation

x, sample ml.of, distillate. taken..

Value, Alkaline Oxidation Value, Iodine Value and Ester Value

Oxidation, alkaline oxidation, iodine and ester values are useful in diffe-

rentiating various types of vinegars.8 In the case of malt vinegar, alcohol and acetyl methyl carbinol have been found to be mainly responsible for the

distinctive oxidation and iodine values obtained.4® The oxidation value is the number of ml of 0.01 N KMnQ, required by 100 ml of the vinegar in 30 min for oxidation. The alkaline oxidation value is

the number of parts by weight of oxygen required to oxidise 1,00,000 parts of sample under standard conditions.’ The iodine value is the number of ml of 0.01 N iodine absorbed by 100 ml of the vinegar. The ester value is the number of ml of 0.01 N’ KOH required to saponify the esters from 100 ml of the vinegar. All determinations must be made under standard conditions. REAGENTS

. 0.1 N Standard potassium permanganate - 10% Potassium iodide solution 0.02 N Standard thiosulphate . Dilute H,SO, . Phenolphthalein indicator 0.1 N and 1 N Standard potassium hydroxide 0.1 N Standard iodine . 0.1 N Standard HCl . Starch indicator . 10% NaOH —sSEONIAKNAYWDHD —

PROCEDURE

In a 150-ml distillation flask fitted with a stoppered funnel, distil 60 ml of vinegar. After collecting 45 ml of the distillate, add 15 ml of waterand con-

tinue distillation until the total volume of the distillate is 60 ml. Oxidation value : Pipette 5 ml of distillate from malt or grape vinegar or 10 ml of spirit vinegar or artificial vinegar into a 250-ml glass-stoppered conical flask. Add 10 ml of dilute H,SO, and pipette 15 ml of 0.1 N potassium permanganate solution. Stopper the flask, allow to stand for 30 min, and add 5 ml of 10% potassium iodide solution. Titrate the liberated iodine with 0.02 N sodium thiosulphate. When most of the iodine colour has been discharged, add starch near the end point and complete the titration. Carry out a blank using distilled water in place of the sample.

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Pickles, Chutneys and Vinegar 991

992

Analysis of Fruit and Vegetable Products

Oxidation value

(Blank titre — Sample titre) x 2 x 100 ml of sample taken for estimation

Alkaline oxidation value: Pipette 2 ml of the distillate into a 250-ml glassstoppered conical flask. Add 100 ml of water and 10 ml of 10% NaOH solution. Pipette 10 ml of 0.1 N standard potassium permanganate solution. Stopper the flask and allow to stand for 30 min. Then acidify with 10 ml of dilute H,SO, (1+3). Add 0.5 g of potassium iodide and titrate the liberated iodine with 0.02 N sodium ‘thiosulphate using starch as indicator mear the end point. Carry out a blank titration similarly using 2 ml of distilled water in place of the distillate. Alkaline oxidation value = 8 x (Blank titre — Sample titre) Todine value : Pipette 5 ml of the distillate from malt vinegar or 10 ml from spirit or artificial vinegar and make it just neutral to litmus using 1 N potassium hydroxide. Add 10 ml of 1 N potassium hydroxide and pipette 10 ml

of 0.1 N iodine standard solution. Stopper the flask and allow to stand in the dark for 15 min. Then add 10 ml of dilute H,SO, and titrate the excess iodine with 0.02 N standard sodium thiosulphate solution using starch as indicator. Carry out a blank similarly using distilled water in place of the sample. (Blank titre — Sample titre) x 2 x 100 Iodine value = —— ml of sample taken for estimation Ester value : Pipette 25 ml of the distillate intoa 100-ml ground glass roundbottomed flask (suitable for heating with a reflux condenser). Make the distillate neutral with 1.0 N potassium hydroxide using phenolphthalein indicator. Discharge the pink colour by dropwise addition of 0.05 N HCl. Then pipette 10 ml of 0.1 N potassium hydroxide and heat under reflux for2 hr on a boiling water bath to saponify the esters. Cool, add phenolphthalein (water, if necessary) and titrate with 0.05 N HCl. Carry out a blank test similarly using 25 ml of water instead of the distillate.

Ester value = (Blank titre — Sample titre) x 2 x 4. Interpretation of Results Determination of total solids, nitrogen, nature of acids present, formol titration, volatile substances, ash content and its nature, and certain specific tests are of value in recognizing a genuine vinegar from an artificial one. The physico-chemical characteristics of different vinegars are given in Tables

31-2 and 31-3.”" Malt Vinegar

Volatile reducing substances ate very high in malt vinegar and very low in artificial vinegar. In spirit vinegars, the values, though higher than artificial vinegars, are much lower than malt vinegars. The following approximate values have been\recorded by Pearson.®

Pickles, Chutneys and: Vinegar

993

ee

Alkaline. Oxidation

_ oxidation

Todine

Ester

value

value

value

value

70-180

380—1500

30—140

Malt vinegar

500-1800

Cider vinegar

Upto 3500

Grape vinegar

600-2000

60—180

380—1000

50—220

Spirit vinegar

g0—650

3-20

$-30

0+20°

0-20

0-10

O—25

O—T5

Artificial vinegar

A send

ee

ee

TABLE 32-3: Overall Rangeof Variation in the Chemical Composition of Brewed and Synthetic Vinegars®

Physico-chemical characteristics ,

1. 2.

°Brix Total acidity%

Synthetic vinegars (20) gaguriad

Min.

Max.

1.0 3.75

3.0 6.46

Brewed vinegars (100)

Av.

Min.

Max.

Av.

ees 5-47

iz

10.4

5-32 4.28

1.96

8.65

3.

Volatile acidity%

3.75

6.46

5-47

1.58

8.57

4.01

4.

Fixed acidity%

0.00

0.00

0.00

0.02

1.10

0.29 0.64

5.

Sugars%

0.00

0.00

0.00

0.00

2.03

6.

Nitrogen%

0.00

0.002

0.0005

0.001

0.072

7.

VRS (milli-eq/100 ml)

0.24

1.18

0.97

2.04

19.§2

10.03

8.

Oxidation value

22

168

288

812

567

20

112

9.

Iodine value

74-3

109

552

356

fo)

6

17

19

146

7755

0.19

1.29

0.69

1.02

8.60

3.81

Total ash%

ro)

0.12

0.034

0.09

1.05

0.53

P,Os

°

°

0.01

0.15

0.03

10.

Ester value

11.

Total solids%

12.

13.

‘49 ' i

0.21

°

Determination of ‘“‘albuminoid ammonia value” and total nitrogen are useful in differentiating malt vinegars from artificial products,! as can be seen from the following figures. ;

Malt vinegars Artificial

condiments

Albuminoid

Total

ammonia (as N, w/v) ppm

nitrogen (w/v) ppm

208-400 O-4

404-840 Y

11-34

994

Analysis of Fruit and Vegetable Products

In addition to these, the genuine malt vinegar contains significant proportions of thiamine, riboflavin, nicotinic acid, pantothenic acid and pyridoxin. There would,

of course, be no

traces of these in artificial vinegars.

Malt

vinegars are dextro-rotatory and usually contain not less than 0.2 g of ash in 100. ml. The water soluble ash from 100 ml of malt vinegar requires not less than 4 ml of 0.1 N acid to neutralize its alkalinity. Cider Vinegar

Specific gravity of cider vinegar varies from 1.013 to 1.015. It is distinguished from other vinegars by the presence of considerable quantity of malic

acid. It yields a strongly alkaline ash rich in potassium. The laevo-rotatory characteristics of the cider vinegar is so fixed and unalterable that a righthanded polarization of 0.5° may safely be assumed as evidence of adulteration. The polarization of cider vinegar expressed in terms of 200 mm of the undiluted sample should be between 0.1 and 4.0 Ventzke. Grape Vinegar

The specific gravity varies from 1.0129 to 1.0213 and the vinegar is slightly laevo-rotatory with polarized light. It usually contains a small proportion of free alcohol, traces of reducing sugars, and 0.1 to 0.4% acid potassium tartarate, and has a characteristic aroma by which it can easily be recognized. Molasses

Vinegar

It is a product of acetic fermentation of the sugar house wastes. With polarized light, -molasses vinegar is dextro-rotatory before and laevo-rotatory after inversion. Wood Vinegar or Pyroligneous Acid

It has a characteristic tarry taste and invariably contains traces of furfural.

Spirit Vinegar

Its specific gravity ranges from 1.008 to 1.013. Ash, nitrogen, phosphoric acid and total solids contents are insignificant and may be present only in traces. This vinegar invariably contains alcohol (non-acidified), aldehydes, traces of nitrogen and phosphoric acid, and has no optical activity in polarized light. References

t.

Mitra, S.N., Analyst, 78, 499 (1953).

2.

Edwards, F.W. & H.R. Nanji, Analyst, 63, 410 (1938).

Pickles, Chutneys and Vinegar

995

Illing, E.T. & E.G. Whittle, Analyst, 64, 329 (1939).

Whitmarsh, J.M., Analyst, 67, 188 (1942). Lyne, F.A. & T. McLachlan, Analyst, 71, 203 (1946). ae eS Pearson, D., The Chemical Analysis of Foods, J. & A. Churchill, London, 6th edn., p. 365

(1970). Nagarathnamma, M., J.S. Pruthi & G.S, Siddappa, Indian Food Packer, 18{2), 11 (1964). Nagarathnamma, M. & G.S. Siddappa, Indian Food Packer, 18 (2), 15 (1964).

CHAPTER 33 \

Statistical Methods

PARAMETERS AND STATISTICS Sample and Population Population is the totality of items under consideration and sample is the ‘material taken from the population for investigation. For example, a truck load of fruits used for processing represents the population, and fruits taken for analysis represent the sample. A sample is not a miniature replicate of the population. Hence, in making decisions about a population, necessary allowances are required to be nade for the role of chance. If a sample is to be representative of the population, each member of the population should have an equal chance of being included in the sample. A sample of size n is said to be random of each combination of » items in the population has an equal chance of being chosen.

Average Values Arithmetic Mean: It is a common measure of an average number of observations and is calculated by dividing the sum of the observations by the total number. It is calculated using the expression; eo

2x

Cae

where, x = arithmetic mean xx= sum of individual observations n = number of observations

As n becomes larger, the mean approaches more and more closely to the true mean of the population to which the observations belong. Population values are represented by the symbol u in the case of the mean value. In order to distinguish between quantities computed from observed data and quantities which characterize the population, the term statistic will be used to designate a quantity computed from the sample data, and the term parameter will be used to designate a quantity characteristic of the population.

Statistical Methods

, 997

Variance

Variance (v or s?) is the parameter used to measure the amount of scatter about

the mean values of a distribution. It is defined as the mean square deviation of all individual values from the arithmetic mean, i.e., in a sample of n observations. v= s2

= 2(x-u)? nN

Since the population mean, y, is not generally known, the sample mean, x, has to be used. Hence the variance of the population is given by the expression g2

_ wx = x)? er

|

where, x = score xx% = mean of the scores nm = number of observations

To solve 2(x-%)?, each score is required to be subtracted from the mean. This

could be simplified by using the expression

Zaz =(Xx)? (2) nN

The expression used for calculating the variation of the population is the following: Dx?

n TP

ey

Standard Deviation

Variince is usually calculated in the mathematical treatment of frequency distributions. It is an estimate as to how far the sample is likely to differ less from the true value of the population. The variance is not a linear function of the observations. Hence, standard deviation, s, which is the square root of variance,is usually employed. It is calculated using the following expression:

or s=

v/s?

Standard Error of the Mean

It is directly related to the standard deviation and variance by the formula S.E.. (x). =

S

Sa

998

Analysis of Fruit and Vegetable Products

where, S.E. x = standard error of the mean s = standard deviation m = number of observations /

Coefficient ofVariation and Percent Variability When two or more sets of data having widely differing arithmetic means are to be compared, it is not correct to compare the standard deviations. In such cases coefficient of variation (C.V.) is made use of. It is statistically found by dividing the standard deviation, s, of the sample by the mean, x . Per cent variability is the coefficient of variation expressed as percentage. The expressions used are:

CV. =

—3 % Variability a X 100

S

x

Standard Error of the Difference

It is a measure of the degree to which the difference between two mean would be expected to vary due to random error. It is estimated from the standard errors of the two distributions using the following expression which is applicable only when the distributions are independent. S.E.a-B)

3

a

a

Example 1 The computation of the statistics discussed above is illustrated in Table 33-2 using the data given in Table 33-1. TABLE 33-1:Computation of Certain Statistics Given sets of scores A and B as follows:

No. of observations

XA

xq

xB

xp

1

5

ps

3

9

2

7

49

2

4

3

6

36

3

9

4 5

5 6

25 36

6 5

36 25

6

4

16

4

16

7 8

3 7

9 49

5 6

25 36

9

5

25

4.

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10

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