Modifying Lipids for Use in Foods [1 ed.] 0-8493-9148-2, 1-84569-168-7, 1-85573-971-2, 978-0-8493-9148-4

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Modifying lipids for use in food Edited by Frank D. Gunstone

CRC Press Boca Raton Boston New York Washington, DC

WOODHEAD

PUBLISHING LIMITED

Cambridge, England

Published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CB1 6AH, England www.woodheadpublishing.com Published in North America by CRC Press LLC, 6000 Broken Sound Parkway, NW, Suite 300, Boca Raton, FL 33487, USA First published 2006, Woodhead Publishing Limited and CRC Press LLC © 2006, Woodhead Publishing Limited The authors have asserted their moral rights. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publishers cannot assume responsibility for the validity of all materials. Neither the authors nor the publishers, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging in Publication Data A catalog record for this book is available from the Library of Congress. Woodhead Publishing ISBN-13: 978-1-85573-971-0 (book) Woodhead Publishing ISBN-10: 1-85573-971-2 (book) Woodhead Publishing ISBN-13: 978-1-84569-168-4 (e-book) Woodhead Publishing ISBN-10: 1-84569-168-7 (e-book) CRC Press ISBN-13: 978-0-8493-9148-4 CRC Press ISBN-10: 0-8493-9148-2 CRC Press order number: WP9148 The publishers’ policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp which is processed using acid-free and elementary chlorine-free practices. Furthermore, the publishers ensure that the text paper and cover board used have met acceptable environmental accreditation standards. Typeset by Replika Press Pvt Ltd, India Printed by T J International Limited, Padstow, Cornwall, England

Contributor contact details (* = main point of contact)

Chapters 1, 2 & 7 Professor Frank Gunstone 3 Dempster Court St Andrews Fife KY16 9EU Scotland UK

Chapters 4, 14 & 24 Professor Gudmundur G Haraldsson Science Institute University of Iceland Dunhaga 3 107 Reykjavik Iceland

Tel: +44 1334 479 929 Email: [email protected]

Tel: +354 525 4818 Fax: +354 552 8911 Email: [email protected]

Chapters 3 & 13 Dr Martin RL Scheeder ETH Zurich Institute of Animal Sciences ETH Zentrum LFW 56.1 CH-8092 Zurich Switzerland

Chapters 5 & 23 Professor Colin Ratledge Department of Biological Sciences University of Hull Hull HU6 7RX UK

Tel: +41-44-632 32 78 Fax: +41-44-632 11 28 Email: [email protected]

Tel: +44 1482 465 243 Fax: +44 1482 465 822 Email: [email protected]

xiv

Contributor contact details

Chapter 6 K Warner US Department of Agriculture National Center for Agricultural Utilization Research Peoria, IL USA

Chapter 11 Xeubing Xu BioCentrum-DTU Building 221 Technical University of Denmark DK – 2800 Lyngby Denmark

Email: [email protected]

Tel: 45-45252773 Fax: 45-45884922 Email: [email protected]

Chapter 8 Dr Alejandro G Marangoni Department of Food Science Guelph Ontario, N1G 2W1 Canada Email: [email protected]

Chapter 9 Gary R List 1815 N. University Street Peoria, IL 61604 USA Tel: 309-681-6388 Fax 309-681-6340 Email: [email protected]

Chapter 10 Dr Véronique Gibon Project Manager R&D De Smet Techologies and Services Da Vincilaan, 2, Bus G1 1935 Zaventeus Belgium Tel: + 322 716 1390 Email: [email protected]

Chapter 12 Professor Denis J Murphy Biotechnology Unit School of Applied Sciences University of Glamorgan Treforest CF37 1DL Wales UK Tel: +44 1443 483 747 Email: [email protected]

Chapter 15 Professor Jan Pokorný Department of Food Chemistry and Analysis Institute of Chemical Technology Prague Czech Republic Email: [email protected]

Contributor contact details Chapter 16 Dr HM Premlal Ranjith* and Miss Upuli Wijewardene Diotte Consulting and Technology The Conifers 36 Bishops Wood Nantwich Cheshire CW5 7QD UK

xv

Chapter 19 Dr Earl Hammond Department of Food Science and Human Nutrition Food Sciences Building Iowa State University Ames, Iowa 50011 USA Email: [email protected]

Email: [email protected]

Chapter 17 Dr Gerrit van Duijn* and Eckhard Flöter Unilever Research PO Box 114 3130 AC Vlaardingen Holland Email: [email protected] [email protected]

Chapter 18 Professor William E Artz Dept Food Science and Human Nutrition 382 Agricultural Engineering Sciences Bldg University of Illinois 1304 West Pennsylvania Avenue Urbana, Illinois 61801-4726 USA Tel: (217) 333-9337 Fax: (217) 333-9329 Email: [email protected]

Chapter 20 Staffan Norberg AarhusKarlshamn (AAK) SE-374 82 Karlshamn Sweden Tel: +46 454 829 77 Email: [email protected]

Chapter 21 Dr Christian Gertz Chemiedirektor Chemisches Untersuchungsamt Hagen Pappelstrasse 1 D-58099 Hagen Germany Tel.: +49-2331-2074726 Fax.: +49-2331-2072454 Email: [email protected]

Chapter 22 Kaustuv Bhattacharya International Food Science Centre A/S Herredsvej 60C, Apt 12 8210, Aarhus V Denmark Email: [email protected]

1 Introduction: Modifying lipids – why and how? F. Gunstone, Scottish Crop Research Institute, UK

1.1

Introduction

Annual production of oils and fats was 136 million tonnes in 2004/05 and is forecast to be 141 million tonnes in 2005/06. In the quarter-century 1976– 2000 consumption (virtually the same as production) rose at an average rate of 3.7 %, equivalent to doubling every 20 years or so (Anon, 2005). This marked increase has come mainly from five vegetable oils (soybean, the two products of the oil palm – palm oil and palmkernel oil – rapeseed/canola and sunflower) and to a lesser extent from eight other vegetable oils (cottonseed, groundnut/peanut, sesame, corn, olive, coconut, linseed and castor) and four animal fats (butter, lard, tallow and fish oil). These increases result partly from larger areas being devoted to their cultivation and partly from rising yields. The composition of these oils is described in selected chapters in Part I of this book. It is estimated that ~ 80 % of total oil and fat production is used for food and the balance for animal feed and by the oleochemical industry. The major food uses include frying oils, baking fats, cooking fats, shortenings, spreads, salad oils, mayonnaise, confectionery fats and ice cream. However, there are additional dietary fats not counted by market analysts in their assessment of commodity oils and fats. For example, figures for groundnut (peanut) oil do not include the commodity eaten as nuts, figures for butter do not include milk consumed as such or as cheese, and figures for lard, tallow and fish oil do not include fat consumed when eating meat (beef, lamb, pork, chicken) or fish. The consumption of oils and fats varies considerably between developed, developing and under-developed countries and demand is expected to grow. This is illustrated in the figures for selected affluent and non-affluent countries/

2

Modifying lipids for use in food

regions (Table 1.1). Increasing consumption in highly populated countries such as China and India will fuel the demand for oils and fats for many years to come. Apart from low levels of minor oils described in Chapter 2, consumption is confined to fewer than 20 commodity oils. These are not always ideal for food purposes, and have to be modified for their end use. It is useful in this preliminary chapter to consider the important properties required of an oil or fat for its use in foods, and to review briefly the major modifying procedures. The latter will be detailed in chapters in Part II and the application of modified lipids in foods is covered in Part III.

1.2

What properties are desired?

An oil or fat should have the optimum physical, chemical and nutritional properties dictated by its end use. The more important of these properties are indicated below, and some of the topics will be developed in later chapters. However, these factors are not always mutually compatible and compromises have to be made. The best physical and chemical properties may be achievable only at the sacrifice of some nutritional excellence. This provides an important challenge for food technologists, and this book will show how these challenges have been met in the past, how they are being met today and how they may be met in the future. Because nutrition is a developing science new problems appear, and what is practised and accepted today may be less so in the future. This is illustrated in the present concern about fats containing unsaturated acids with trans configuration. These are produced (among other ways) by partial hydrogenation, a process developed and exploited throughout the 20th century. Only in recent years have there been nutritional concerns about such acids that are now being addressed with greater or lesser urgency.

Table 1.1 Consumption of oils and fats for food and non-food purposes in selected countries in 1990/2000 and 2004/05. World

USA

EU-25

China

India

Personal (kg/person/year) 1999/00 2004/05

18.4 21.0

49.9 49.0

43.2 50.2

13.6 19.6

11.4 11.7

Total (million tonnes/year) 1999/00 2004/05

113.4 136.4

15.7 15.5

17.1 17.4

14.7 18.0

6.6 8.0

Source: Anon (2005).

Introduction: Modifying lipids – why and how?

3

1.2.1 Physical properties The most important physical properties of oils and fats for the food industry are thermal properties associated with crystallization and melting, with the formation of solids and liquids and with the behaviour of plastic fats that are mixtures of solid and liquid components. It is mandatory, for example, that salad oils do not contain lipids that will crystallize during storage in a refrigerator. Most frying oils and oils used as food coatings (and lubricants) should also be free of solid components. Few natural oils meet this requirement and appropriate modification has to be carried out. The successful production of spreads, on the other hand, depends on having appropriate levels of solid fat at refrigerator temperature, at ambient temperature and at mouth temperature. The solid fat content at 4 °C should not exceed 30–40 % if the fat is to be spreadable from the refrigerator; at 10 °C it should be 10–20 % so that the fat will ‘stand up’ (not collapse to a puddle of oil); and at mouth temperature the spread should melt completely to avoid a waxy mouth feel. Crystalline triacylglycerols, such as those present in spreads, show polymorphism. They exist in different crystalline forms with differing thermal properties and may change – quickly or slowly – from a physically less stable to a more stable form. It is important that solid triacylglycerols in a spread are in the most appropriate form (β′) and that this will not change during storage to the more stable β-form. β′-Crystals are relatively small, can incorporate large volumes of oil (liquid) and give the product a glossy surface and a smooth lustre. β-Crystals, on the other hand, though initially small, grow into needle-like agglomerates less able to incorporate oil and producing a grainy texture. Oils comprising acids of mixed chain length (generally C16 and C18) are more likely to remain in the β′ form while those containing almost entirely C18 acids are β-tending. 1.2.2 Chemical properties Food lipids are not usually considered to require defined chemical properties, but without oxidative stability of their lipid components foods would quickly become rancid and have a short shelf life. For this reason they should be oxidatively stable. An account of the deteriorative oxidation processes and of conditions under which these changes may be inhibited is an important part of Chapter 7.

1.2.3 Nutritional properties The selection and use of oils and fats in foods is strongly influenced by a range of nutritional properties. •

The total level of fat in a food with its marked effect on calorific value is important to consumers and has led to a demand for foods with lower fat content or for the use of fat or fat substitutes with reduced calorific value.

4

Modifying lipids for use in food

•

An appropriate balance between saturated, monounsaturated and polyunsaturated acids is desirable. There are many dietary recommendations given in terms of numbers (energy %) of total fat and of these three types of acids. This makes for simple messages that can be understood by the consumer and for relatively simple labelling. It has been argued, however, that this message is too simple and can be misleading (Gurr, 1999; Taubes, 2001). Saturated acids represent a group of acids that are of concern because they raise serum cholesterol levels. They can be described as cholesterolraising. The human diet contains saturated acids with 4–24 carbon atoms of which palmitic acid (16:0) dominates. Starting with Ancel Keys in 1957 (he died in November 2004, just two months short of his 101st birthday) several equations have been produced to correlate changes in serum cholesterol levels with changes in fatty acid dietary intake. Early equations took account only of changes for total saturated acids and total polyunsaturated acids, but the most recent include information for changes in (only) three individual saturated acids (lauric, myristic and palmitic), oleic acid, trans acids from partially hydrogenated vegetable oils and from partially hydrogenated fish oils and linoleic and linolenic acids taken together (Pedersen et al., 2001). This suggests that saturated acids outwith the 12:0–16:0 range are not cholesterol-raising. Among monounsaturated acids a clear distinction must be made between cis and trans isomers with the former being cholesterol-lowering and the latter cholesterol-raising. As is apparent in later chapters, much effort is now being put into producing high quality spreads with levels of trans acids approaching zero. There is also a growing concern about the quota for polyunsaturated fatty acids. In particular, the ratio of omega-6 to omega-3 acids should be between 5 and 10 to 1 (or less) but exceeds 25 in many countries. Since the presence of α-linolenic acid leads to oxidative instability and reduced shelf life, there is a need for development of more efficient antioxidant systems. Related to this is the efficiency or otherwise of the elongation–desaturation systems required to metabolize linoleic acid and linolenic acid to their very important C20 and C22 metabolites – arachidonic, eicosapentaenoic, and docosahexaenoic acids. Are materials containing these C20 and C22 polyunsaturated fatty acids a necessary part of the human diet or will the C18 members suffice? This question is discussed further in Chapter 7.

•

•

•

1.3

Methods of modifying oils and fats

It is convenient to divide the techniques for modifying oils and fats into technological and biological groups. In the former we accept what nature provides and seek to change fatty acid and/or triacylglycerol composition,

Introduction: Modifying lipids – why and how?

5

thereby modifying nutritional, chemical (mainly oxidative stability) and physical (mainly melting behaviour) properties to make them more suitable for their end-use. In the latter, we interfere at an earlier stage and either seek new and better sources or take plants which already produce large quantities of oil efficiently and try to modify the composition of the oils by conventional methods of seed breeding or by exploiting newer methods based on increasing genetic understanding.

1.4

Technological methods

Lipid technologists have developed several methods of modifying oils and fats. None of these is very new, but they are subject to incremental improvement either based on a better scientific understanding of the process or through the development of improved equipment. The oldest method is blending. This is a simple mixing of existing oils to provide a mixture with improved qualities. Examples include: • • • •

adding a small proportion of an oil with high oxidative stability (such as sesame oil or rice bran oil) to a less stable commodity oil to enhance its stability; mixing oils to get a fatty acid composition believed to have optimum nutritional value – an increasing number of such blends are appearing in the market place (see Chapter 22 on speciality oils); blending oils prior to interesterifacation to get a product with different triacylglycerol composition; blending oils to give a product with desired properties at minimum cost (Block et al., 1997).

Fractionation is a procedure by which a commodity oil or one that has already been modified is divided into two or more fractions differing in fatty acid and triacylglycerol composition. This can be achieved without loss of material and without need for further refining. The two fractions extend the range of usage of the original oil. This technique is used mainly for palm oil but has other applications also (Chapter 10). It is not always easy to predict what happens to minor components during fractionation, and this may have unforeseen consequences for oxidative stability. Hydrogenation of an unsaturated oil gives a product of higher melting point (more suitable for spreads and cooking fats) and of enhanced oxidative stability through having less polyunsaturated fatty acid. These benefits are achieved only at some nutritional cost. The level of essential fatty acid is lowered and acids with trans configuration are produced. These modifications follow the molecular changes resulting from partial hydrogenation which include saturation of some unsaturated centres, stereomutation of unsaturated centres (conversion of cis to trans isomers), double bond migration and conversion of linoleate mainly to trans 18:1 isomers (Chapters 7 and 9).

6

Modifying lipids for use in food

Interesterification is a procedure for rearranging the fatty acids of an oil or a blend of oils so that triacylglycerol composition is changed. When an alkaline catalyst is used, fatty acids are randomly distributed in the product. This is in contrast to the natural vegetable oils produced by (enzymaticallycatalysed) biological processes where the fatty acids are not randomly distributed. These changes affect thermal behaviour and may also have nutritional consequences (Chapters 7 and 11). Interesterification can also be carried out with lipases. The enzymatic processes have several advantages in that they occur under milder conditions, may require less costly equipment and produce less by-product so that there is less waste and less effort is required to purify the product. However, the major benefit of using a lipase is the added control over the nature of the product as a consequence of the specificity shown by many lipases. Fatty acidspecific lipases relating to chain-length or double bond position can be used to confine changes to a particular group of acids, while other lipases are specific for glycerol esters (mono-, di- or triacylglycerols) or distinguish between the different glycerol ester groups. Thus many lipases are described as being 1,3specific implying that changes can be made at positions 1 and/or 3 but not at position 2 where the ester group remains unchanged (Chapters 7 and 11). Interesterified products are generally less stable than the original oils, probably through changes – not yet fully understood – in the balance of pro- and antioxidants. This holds with both chemical and enzymatic catalysts.

1.5

Biological methods

1.5.1 Domestication of wild crops The oil and fat business is based almost entirely on a limited number of commodity oils which vary in their fatty acid composition, but there are many other plant species with fatty acid composition not very different from the commodity oils. These could be used as food lipids, but there would have to be a special reason for developing them through the long chain of events from agronomical improvements to retail marketing. A few of these will be discussed in Chapter 2 under the section on minor oils. There are also plants producing uncommon acids such as epoxy acids, acids with conjugated unsaturation, or oils with a very high level (> 80 %) of a single acid. Attempts to domesticate and commercialize such plants and their seed oils have taken longer than originally thought, with some niche products of this kind taking 20 and more years to develop. Most, but not all, of these oils are of interest to the oleochemical rather than the food industry.

1.5.2 Oilseeds modified by conventional seed breeding or by genetic engineering As a consequence of the difficulties in domesticating wild plants, greater

Introduction: Modifying lipids – why and how?

7

effort has been directed to the modifying of plants that are already sown and harvested on a commercial scale and where the best agronomic procedures are well known. This has the disadvantage of minimizing the range of important plant species (limiting biodiversity). The changes to be sought are partly agronomic but, of greater interest for this book, they include changes in fatty acid composition, triacylglycerol composition and minor components. These changes should be achieved without sacrifice of yield and must be biologically stable from season to season. They have to be accompanied by procedures of identity preservation. The modified seed must be kept separate at all times from its more conventional form. This has consequences for harvesting, transporting and extracting the seed and for the subsequent handling of the oil. Such changes may be effected by conventional seed breeding or by newer procedures of genetic engineering. It is important to know which method has been used because of the concerns expressed by some about procedures involving transgenic modification. Modifications to fatty acid composition which have been sought include: reduced levels of saturated acid for nutritional reasons; reduced levels of linolenic acid and/or higher levels of saturated acids to avoid hydrogenation (with consequent formation of undesirable trans acids); and higher levels of oleic acid. These are detailed in Chapter 12. One exciting possibility is to develop plant systems that will produce long-chain polyunsaturated fatty acids such as arachidonic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). There have been interesting developments in a number of research laboratories but such plants, which probably have to be genetically modified, are 10–20 years from commercial development (Chapter 12) (Drexler et al., 2003; Green, 2004; Qi et al., 2004).

1.5.3 Animal fats modified through nutritional changes From a nutritional viewpoint land animal depot fats are perceived as having several disadvantages. They are generally rich in serum cholesterol-raising saturated acids, frequently contain acids with trans unsaturation, and have high levels of cholesterol. Their level of essential fatty acids is low, and they contain little if any antioxidant. Further, animal fats are not acceptable to vegetarians and to some ethnic groups. Nevertheless animal fats contain low levels of long-chain PUFA (polyunsaturated fatty acid) and are a valuable source of such acids in the human diet. Some of the perceived disadvantages in ruminant animals are the consequence of biohydrogenation processes taking place within the rumen, and dietary regimes have been proposed to circumvent these changes. There is also an interest in modifying the fatty acid composition of chicken eggs and meat by appropriate changes to the diet of the chicken. This has been seen as a way of enhancing the (human) dietary intake of CLA (conjugated linoleic acid) and of EPA and DHA (long-chain omega-3 acids) (see Chapter 13).

8

Modifying lipids for use in food

1.5.4 Single-cell oils As indicated at the beginning of this chapter, oils and fats produced by the agricultural supply industry come mainly from plant sources and also to a minor extent from animals. An alternative approach is to seek new lipid sources from micro-organisms. Some of these can be made to produce high levels of lipids with an interesting fatty acid composition. While it is unlikely that these will replace the more conventional commodity oils for traditional use, nevertheless, they are already providing supplies of high quality longchain PUFA for infant formula and other special purposes (Chapters 5 and 23).

1.6

References

(2005), Oil World Annual 2005, Hamburg, ISTA Mielke GmbH. and GOMIDE F A C (1997), Blending process optimisation into special fat formulation by neural networks, J Am Oil Chem Soc, 74, 1537–1541. DREXLER H, SPIEKERMANN P, DOMERGUE F, ZANK T, SPERLING P, ABBADI A and HEINZ E (2003), Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results, J Plant Physiol., 160, 779–802. GREEN A G (2004), Producing essential fatty acids in plants, Nat Biotechnol, 22, 680–682. GURR M I (1999), Lipids in Nutrition and Health – a Reappraisal, Bridgewater, Oily Press. PEDERSEN J I, MULLER H and KIRKHUS B (2001), Serum cholesterol predictive equations with special emphasis on trans and saturated fatty acids. An analysis from designed controlled studies, Lipids, 36, 783–791. QI B, FRASER T, MUGFORD S, DOBSON G, SAYANOVA O, BUTLER J, NAPIER J A, STOBART A K and LAZARUS C M (2004), Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants, Nat Biotechnol, 22, 739–745. TAUBES G (2001), The soft science of dietary fat, Science, 291, 2536–2545. ANON

BLOCK J M, BARRERA-ARELLANO D, FIGUEIREDO M F

Part I Understanding food lipid structure and composition

2 Vegetable sources of lipids F. Gunstone, Scottish Crop Research Institute, UK

2.1

Introduction

This chapter is devoted to the most important source of lipids, namely those derived from vegetable sources. The major commodity oils are described first, covering their fatty acid and triacylglycerol composition and their minor components. This is accompanied by some indication of the ways in which each oil is modified and the major food uses. There follows a shorter account of some significant minor vegetable oils after which the procedures by which all these oils may be extracted and refined are detailed. Finally there is a brief discussion of future trends in the supply of vegetable-based lipids. Dietary lipids may be ingested as part of a food as when nuts are eaten or when green vegetables are consumed. Although these last contain only low levels of lipids, they are frequently of high nutritive quality and are accompanied by valuable minor components. All living material contains membrane lipids that are mainly phospholipids. However, larger in quantity are those oils and fats of vegetable or animal origin consumed as frying oils, salad oils, spreads, baked goods or chocolate. Over the last half-century the availability of vegetable oils has increased much more than that of animal fats. One important commodity analyst reports weekly on 17 oils and fats of which 13 are of vegetable origin and make up 80 % of total supply while four of animal origin (butter, lard, tallow and fish oil) provide the balance. It is estimated that ~ 80 % of the total supply is used for food and the remainder as animal feed (~ 6 %) or by the oleochemical industry (~ 14 %). Details of the nature of the vegetable oils are given in Section 2.2 but it is appropriate to provide some production data in order to give balance to the ensuing discussion (Table 2.1). The major oils (soybean, palm and palmkernel,

12

Modifying lipids for use in food

Table 2.1 Commodity oils and fats: production levels (million tonnes) and major producing areas in 2004/05. Oil/fat

Production

Food use (%)a

Major producing countries/regionsb

Soybean

32.57

90

Palm Rape/canola

32.50 16.11

70 75

Sunflower

9.08

95

Cottonseed

5.00

95

Groundnutc Palmkernel Coconut Olive Corn Sesame Linseed Castor Cocoa butterd Four animal fatse World total

4.50 3.80 3.01 2.73 2.05 0.77 0.60 0.52 1.20

95 70 70 95 95 95 0 0 95

USA 8.60, Br 5.74, Ch 5.11, Arg 5.03, EU-25 2.63 Mal 15.16, Indon 12.90 EU-25 5.47, Ch 4.56, Ind 2.08, Can 1.36 CIS 3.20, EU-25 1.76, Arg 1.41 Cent Eur 0.72 Ch 1.55, Ind 0.69, Pak 0.51, CIS 0.44, USA 0.42 Ch 2.05, Ind 1.13, Nig 0.32 Mal 1.83, Indon 1.31 Pp 1.27, Indon 0.74, Ind 0.40 EU-25 2.06, Syria 0.20, Tun 0.13 USA 1.10, EU-25 0.23 Ch 0.20, Ind 0.14, My 0.11 EU-25 0.15, Ch 0.13, USA 0.13 Ind 0.32, Ch 0.11 Ivory Coast, Ghana, Indon, Br, Mal

23.16 136.4

(80)

a

These values are estimates made by the author based, in part, on information provided by USDA for 2001/02 [www.fas.usda.gov/oilseeds/circular/2000/oilstats.html]. b These are not necessarily the countries in which the plants are grown. Oils may be produced from imported seeds as well as from domestic supplies. c Also called peanut oil. d Not included in Oil World publications – information added by the author. e Total of tallow (8.19), lard (7.43), butter (6.47) and fish oil (1.07). Abbreviations: Arg = Argentina, Br = Brazil, Can = Canada, Cent Eur = Central Europe, Ch = China, CIS = Commonwealth of Independent States (Former Soviet Union), Indon = Indonesia, Mal = Malaysia, My = Myanmar (Burma), Nig = Nigeria, Pak = Pakistan, Pp = Philippines, Syr = Syria, Tun = Tunisia Source: Anon (2005).

rapeseed and sunflower seed) total 94.1 million tonnes (69 % of total production) (Anon, 2005). They represent an even larger proportion of trade (imports/exports) as many of the oils produced at lower levels are consumed predominantly in the country/region where they grow. Table 2.1 shows the total production of each oil in 2004/05, an estimate of the share of each oil used for food purposes and the major countries of production of the oil from domestic and/or imported oilseeds. Gunstone (2005) has reworked these production figures and calculated the annual production (from 13 vegetable oils, cocoa butter and four animal fats) of the major fatty acids. These total figures have been adjusted for the 80 % consumed as human food and for the changes that occur during partial

Vegetable sources of lipids 13 hydrogenation. Adjustment has not been made for wasted and discarded oil nor for the lipid consumed in forms not counted by market analysts, such as fat in nuts or cheese, or consumed as meat or fish. The results suggest that production of linoleic is in excess of our dietary needs and that production of linolenic is much below the desirable level leading to an omega-6/-3 ratio of ~ 30 that is far too high (Table 2.2).

2.2

Major vegetable sources of food lipids

The major vegetable oils are pressed and/or extracted from seeds or pressed from fruits such as olive or oil palm (Section 2.4). Crude oils are mainly triacylacylglycerols (usually > 95 %) accompanied by lower levels of free acids, monoacylgycerols, diacylglycerols, phospholipids (1–3 %), free and/ or acylated sterols (1000–5000 ppm in total), tocols (300–2000 ppm) and hydrocarbons such as alkanes, squalene and carotenes. Oils from different sources differ in fatty acid and triacylglycerol composition and in the detailed composition of the various minor components. Most oils used in the food industry have been subject to refining processes to raise the triacylglycerol level and to reduce the levels of other components. Ideally, refining (Section 2.4) should remove undesirable components as efficiently as possible but leave suitable levels of the desirable minor components. In some refining procedures materials such as free acids, phospholipids, sterols and tocols are recovered as valuable by-products and find further use in the food, feed, cosmetics, pharmaceutical and oleochemical industries.

Table 2.2 Annual production (million tonnes) for seven major fatty acids in the total oils and fats produced by the agricultural industry. Column 1 relates to total production in 2004/05, column 2 to that used for human food and column 3 after adjustment for changes accompanying partial hydrogenation of soybean oil and rapeseed oil. Fatty acid

1

2

Lauric Myristic Palmitic Stearic Oleic Linoleic Linolenic Other Total n-6/n-3

3.4 2.6 27.4 7.2 47.8 37.5 4.5 6.0 136.4 8.3

2.0 1.8 21.7 5.5 38.4 31.9 3.5 3.9 108.7 9.1

*After hydrogenation this figure includes significant quantities of trans isomers. Source: Gunstone (2005).

3 2.0 1.8 21.7 5.9 48.8* 23.8 0.8 3.9 108.7 29.8

14

Modifying lipids for use in food

2.2.1 Soybean oil Soybeans provide two important materials: oil extracted from the beans and a residual meal containing high quality protein. Whole beans contain protein (40 %), carbohydrate (34 %), oil (21 %), and ash (5 %) (Wang, 2002) with the levels of oil and meal produced commercially being 18 and 79 % respectively. Soybean meal is such an important commodity for animal feed and, to a lesser extent, for human food that the oil has been described as a byproduct. However, as already indicated, soybean oil is produced at a higher level than any other oil and is better described as a co-product. The major fatty acids in soybean oil are linoleic (53 %), oleic (23 %), palmitic (11 %), linolenic (8 %) and stearic (4 %) (Wang, 2002). The widelyaccepted view is that this is a healthy oil, low in saturated acids and rich in polyunsaturated fatty acids (PUFA), especially linoleic acid. However, attempts to modify this fatty acid composition by partial hydrogenation or by plant breeding suggest that it would be better to have less saturated acids and less linolenic acid. Opinion on linolenic acid is contentious. This triene acid is easily oxidized, and food products with linolenic acid have a short shelf life, which is a matter of concern for the food industry, the retailer, and the consumer. However, there is also a matter of nutritional concern. There is a growing awareness that omega-6/omega-3 ratios (Section 7.5) at levels exceeding 20:1 in some countries are far too high and that values of 5:1 or lower (Crawford, 2004) are desirable. It has been argued (Gunstone, 2005) that, compared with nutritional recommendations, the agricultural supply industry as a whole (plant and animal) provides too much linoleic acid and too little linolenic acid. According to Gunstone’s calculations the only significant commodity sources of linolenic acid for food purposes are soybean oil and rapeseed oil, and this is largely destroyed by partial hydrogenation (Table 2.2). He argues that more effort should be devoted to developing antioxidant packages that will allow the safe use of linolenic acid. Because of its high level of linoleic acid (> 50 %), over half the triacylglycerols in soybean oil contain two or three linoleic chains, and most of the remainder have one linoleic chain. In a typical analysis, triacylglycerols exceeding 4 % were LLL 17.6 %, LLO 15.3 %, LLP 10.2 %, LLLn 7.9 %, LLSt 4.2 %, PLO 6.9 %, OLO 6.3 %, LnLO 4.8 %, other 26.8 %. (These three-letter symbols stand for all the isomeric triacylglycerols containing the three acyl groups indicated, where Ln = linolenic acid, L = linoleic acid, O = oleic acid, St = stearic acid and P = palmitic acid.) Soybeans producing oil with a different fatty acid composition have been developed both through traditional seed breeding and by introduction of genes from other plant species. These important developments are discussed in Chapter 12. Crude soybean oil contains several valuable minor components that are recovered in some measure during refining. Degumming produces lecithin – a fraction rich in phospholipids which serves as the main industrial source of these materials. Deodorizer distillate contains tocopherols (vitamin E) (0.15– 0.21 % in the oil raised to ~ 11 % in the distillate) and phytosterols (~ 0.33 %

Vegetable sources of lipids 15 in the oil raised to ~ 18 % in the distillate). Both of these are valuable byproducts. For more details see Wang (2002). Soybean phospholipids (1.5–2.5 %) are removed during the degumming stage of refining. The crude lecithin obtained by this process is a mixture of triacyglycerols (35–40 %), phosphatidylcholines (10–15 %), phosphatidylethanolamines (9–12 %), phosphatidylinositols (8–10 %) and other minor components. The phospholipid content can be increased by ‘deoiling’ with acetone to remove triacylglycerols, and individual phospholipid classes can subsequently be concentrated by dissolution in ethanol. The desired amphiphilic properties of natural phospholipids can be further improved by chemical manipulation, such as partial lipolysis/hydrolysis to produce some lysolecithin or partial hydroxylation of double bonds to increase the polarity of the molecule (Chapter 15). Wang (2002) further reports that the difference between crude and refinedbleached-deodorized (RBD) oil is such that tocopherols are reduced by 36 %, sterols by 32 % and squalene by 38 %. However, the ‘lost’ materials can be trapped in the deodorizer distillate and be recovered for further use. The major sterols are β-sitosterol (125–236), campesterol (62–131) and stigmasterol (47–77) out of a total of 235–405 mg/100 g. Tocopherols in solvent-extracted soybean oil are given as 1370 ppm divided between the α (11 %), β (1 %), γ (63 %), and δ (25 %) compounds. Soybean oil in its native but refined form or in some partially hydrogenated form is widely used for food purposes such as frying and salad oils, margarine and shortening, and mayonnaise and salad dressing. It is used universally for these purposes, especially in those countries where soybeans are grown and extracted. For example, it is reported that in the USA 86 % of all food lipids are derived from soybean oil and that no other oil attains a level above 3 %.

2.2.2 Palm oil The oil palm produces two different oils – palmkernel oil (Section 2.2.5) and palm oil. The latter is extensively fractionated into palm olein and palm stearin. These differ in composition and so extend the range of uses for palm oil. The plant grows in wet tropical regions. Malaysia and Indonesia are the major producing and exporting countries (Table 2.2). Supplies of palm oil have increased rapidly in the last 20–30 years, mainly through increasing the area under cultivation. World average annual production of palm and palmkernel oils together for selected five-year periods is: 6.6 million tonnes in 1981–85 and 15.1 million tonnes in 1991–95 (Anon, 2002), and 31.0 million tonnes in 2001–05 (Anon, 2005). Though second to soybean oil in total production, palm oil exceeds it in volumes traded. Current figures for palm oil production and exports are given in Table 2.3. The average production of palm oil (3.5 tonnes/hectare) and of palmkernel oil (0.4 tonnes/hectare) makes the oil palm the most productive source of oil/

16

Modifying lipids for use in food

fat. These are average figures, and significantly higher yields are obtained from the best plantations. The fatty acid composition, triacylglycerol composition and iodine value for Malaysian palm oil and its fractions are listed in Table 2.4. Palm oil is unusual in containing about 5 % of diacylglycerols. In terms of molecular species, the major triacylglycerols in palm oil are given as POP 29 %, POO 23 %, PLO 10 %, PLP 9 % (Siew Wai Lin, 2002). Palm oil is considered as a saturated fat in comparison to the more highly unsaturated vegetable oils. With almost the same levels of saturated and unsaturated acids, the description saturated can be misleading. The oil is rich in nutritionally important minor components. Because of its fatty acid composition and its minor components which act as antioxidants, palm oil has high oxidative stability. Palm oil is not much used in North America, but it makes a significant contribution to dietary fat intake in the rest of the world. Table 2.3 Production and exports (million tonnes and percentage of world total) of palm oil and palmkernel taken together in 2004–05.

World Malaysia Indonesia Other

Production

Exports

36.30 16.99 13.91 5.40

27.44 14.57 9.93 2.94

(46.8 %) (38.3 %) (14.9 %)

(53.1 %) (36.2 %) (10.7 %)

Source: Anon (2005).

Table 2.4

Compositional data for palm oil and selected fractions.

Major fatty acids 16:0 18:0 18:1 18:2 othera

Oilc

Oleind

Stearind

Mid-fractiond

44.1 4.4 39.0 10.6 1.9

40.9 4.2 41.5 11.6 1.8

47–74 4–6 16–37 3–10

41–55 5–7 32–41 4–11

12–56 34–50 5–37 0–8 22–40

1–11 45–74 19–42 2–8 34–55

Major triacylglycerols by carbon numberb C48 8.1 3.3 39.9 39.5 C50 C52 38.8 42.7 C54 11.4 12.8 Iodine value 52.1 56.8 a

Other acids present at low levels include 12:0, 14:0, 20:0, 16:1 and 18:3. These tricacylglycerols consist mainly of 3, 2, 1, and 0 palmitic acid chains with 0, 1, 2, and 3 C18 chains respectively. c Mean of 244 Malaysian samples. d Mean of many Malaysian samples. Source: Siew Wai Lin (2002). b

Vegetable sources of lipids 17 Palm oil, palm olein and palm stearin contain 500–700, 600–760 and 380–540 ppm, respectively, of mixed carotenes (almost entirely a mixture of the β and α forms) when crude, but this is lost in RBD oils. Red palm oil, obtained by an alternative refining process, and still containing ~ 80 % of the carotenes originally present, is marketed as an oil with added nutritional value because of the carotenes which serve as pro-vitamin A. Crude palm oil is rich in tocols (600–1000 ppm), with the level of tocotrienols (~ 70 %) exceeding that of tocopherols. Refined oil still has ~ 70 % of the original tocols. Some loss occurs during deodorization, and palm oil fatty acid distillate (PFAD, equivalent to deodorizer distillate) is enriched in tocols (750–8200 ppm) and can be used as a source of tocotrienol-rich tocols. Many health claims have been put forward for these compounds. Palm oil contains sterols (200–600 ppm in crude oil) that are mainly β-sitosterol, campestrol and stigmasterol. These are present at lower levels in refined oil (70–300 ppm) and concentrate in PFAD at an average level of 6500 ppm. The acyclic C30 hydrocarbon squalene is present in crude palm oil (200–500 ppm) and concentrates in PFAD during refining (5000–8000 ppm). Palm oil is used for a wide range of food purposes including frying, in spreads and vanaspati and in shortenings. Palm olein is a major frying oil, and palm stearin finds increasing use as hard stock in fat blends which are interesterified to produce spreads with no hydrogenated oil and therefore containing little or no trans acids. The presence of palm oil or palm stearin in a spread helps to stabilize the β′-crystal form because of the mixed C16 and C18 chains. 2.2.3 Rapeseed/canola oil Rapeseed production is second only to soybeans, and rapeseed/canola oil is third after soybean and palm. Production levels have grown steadily in recent years and peaked in 1999/2000 at 14.5 million tonnes. Thereafter production declined somewhat through reduced planting and/or poor weather that reduced yield. After falling to 12.3 million tonnes in 2002/03 production is now rising steadily with figures of 14.4 and 16.1 million tonnes for 2003/04 and 2004/05, respectively (Anon, 2005). Rapeseed and canola are terms describing the seed and extracted oil from Brassica species including B. napus (formerly B. campestris), B. rapa and B. juncea. The seed oil from these species was typically rich in erucic acid (22:1), and the seed meal had an undesirably high level of glucosinolates. These components reduced the value of both the oil and the protein meal, but they have been bred out of the modern rapeseed, now known as double zero or canola. It is grown mainly in Western Europe, China, India and Canada (where the canola varieties were developed). Typically it contains palmitic (4 %), stearic (2 %), oleic (62 %), linoleic (22 %) and linolenic (10 %) acids and has less saturated acids than any other commodity oil. In one example its major triacylglycerols were LnLO (8 %), LLO (9 %), LnOO (10 %), LOO

18

Modifying lipids for use in food

(22 %), LOP (6 %), OOO (22 %) and POO (5 %) (Przybylski and Mag, 2002). With its low level of saturated acids, its high level of oleic acid and the presence of linoleic and linolenic acids at a favourable ratio (~ 2:1) rapeseed oil rates highly in the classification of healthy oils. Also, the plant system lends itself to genetic modification, and rapeseed varieties with modified fatty acid composition have been developed, although it is still not clear how many of these will be economically viable. Rapeseed oils with less linolenic acid, or enhanced levels of lauric acid, stearic acid, oleic acid, or with unusual acids such as γ-linolenic acid, ricinoleic acid or vernolic acid have all been developed for commercial exploitation. Oleic-rich varieties have about 84 % oleic acid. For more details see Przyblski and Mag (2002), Gunstone (2004b) and Chapter 12. Rapeseed oil contains ~ 3 % of phospholipid which is removed during degumming. Rapeseed oils contain sterols and sterol ester together at levels between 0.45 and 1.13 %. These are mainly β-sitosterol (47–52 % of total sterols) and campesterol (28–34 %). All brassica seeds are characterized by the presence of brassicasterol (12–16 %), a sterol virtually absent from other seed oils. Tocopherols occur in rapeseed oils at levels of 430–2680 mg/kg oil with the γ- (60–74 % of total tocols) and α-compounds (26–35 %) predominating. Brassica oils, particularly those grown during short seasons in Canada and Scandinavia, frequently contain chlorophyll (and related compounds) at levels up to 50 ppm, although RBD oil usually has a chlorophyll (and derivatives) specification of less than 25 parts per billion. High-erucic rapeseed is still grown with the high-erucic oil being used for a range of industrial purposes and particularly for the preparation of erucamide used in ‘clingfilm’ (Temple-Heald, 2004). Rapeseed oil is generally accepted as a healthy oil by virtue of its fatty acid composition, and is widely used for food purposes, sometimes after brush hydrogenation to reduce the level of linolenic acid and sometimes after partial hydrogenation in spreads. The oil is used as a salad oil and salad dressing and mayonnaise, in margarine and other spreads, as a frying oil, and in many minor food applications. The oil is widely used, particularly in China, EU-15 and India – all countries where the plants are grown extensively. Canada and Australia are major exporting countries of seed and/or oil. Both these countries have modest populations and therefore limited local demand.

2.2.4 Sunflower seed oil Oil obtained from sunflower seeds (Helianthus annuus) occupies the fourth position in the ranking of vegetable oils by production levels (Table 2.1). It became popular as the major lipid constituent of many margarines when animal fats were first replaced by linoleic-rich vegetable oils, but in recent years it has had to compete with increasing use of soybean oil.

Vegetable sources of lipids 19 Sunflower oil is available in three ranges of fatty acid composition. The traditional and still major sunflower oil is linoleic-rich, but two other forms have been produced by conventional seed breeding; these are a high oleic oil and a mid-oleic oil. The latter (NuSun™ oil – National Sunflower Association, USA) is much favoured in the USA where attempts are being made to replace the traditional oil. The fatty acid composition of these sunflower types is given in Table 2.5. Their oxidative stability increases as the ratio of oleic to linoleic acid increases. The major triacylglycerols of the traditional linoleicrich oil are typically LLL (14 %), LLO (39 %), LLS (14 %), LOO (19 %), LOS (11 %) and other (3 %) (S = saturated) (Gunstone, 2004a). Crude sunflower oil contains phospholipids (0.7–0.9 %), tocopherols (630– 700 ppm), sterols (~ 0.3 %) and carotenoids (1.1–1.6 ppm). The phospholipids removed during refining are mainly phosphatidylcholines (55–64 % of total phospholipids), with lower levels of phosphatidylinositols (15–24 %) and phosphatidylethanolamines (17–20 %). The tocols are almost entirely αtocopherol which makes sunflower seeds and the extracted oil good sources of vitamin E. The seeds are also a rich source of selenium compared to most other seeds and nuts. Sunflower seed oil contains some wax (esters of longchain alcohols and long-chain acids) coming from an outer protective seed coat. This makes the oil appear cloudy on standing and is usually removed by winterization if the oil is to be used as a salad oil. This is achieved by filtering oil that has been held at 7–8 °C for 12–24 hours.

2.2.5 Lauric oils (coconut, palmkernel) Coconut oil and palmkernel oil are similar to one another in their fatty acid composition and differ so much from other commodity oils that they can be considered together. Both have high levels of medium-chain saturated acids, especially lauric acid (12:0), hence the term lauric oils. As a consequence they have only low levels of unsaturated C18 acids and low iodine values. They are used extensively both as food and as non-food oils and serve as the major source of C8 (caprylic or octanoic) acid and C10 (capric or decanoic) acid. These short-chain acids are concentrated by distillation of the hydrolyzed oils. The production of coconut oil has generally exceeded that of palmkernel Table 2.5

Typical fatty acid composition of sunflower oils.

Saturated Oleic Linoleic Linolenic Iodine value (approx) Source: Gupta (2002).

Traditional

Mid-oleic

High oleic

11–13 20–30 60–70 PI > PE > PS (PC is phosphatidylcholine, PI phosphatidylinositol, PE phosphatidylethanolamine, PS is phosphatidyserine) (Mutua and Akoh, 1993). The nature of PL also affects the incorporation rates of caprylic acid by TLL in hexane in the following order PC > PE > PA > PI (Peng et al., 2002). PL can be obtained commercially at different purities. PC is the most abundant phospholipid in nature and has usually been the substrate selected for enzymatic modification. Incorporation of novel fatty acids has been made with both pure phospholipid compounds and de-oiled lecithin (Peng et al., 2002; Vikbjerg et al., 2005a). Purified compounds have considerably higher price compared to the de-oiled lecithin. Selection will depend on the purity requirements. In most cases, lipase-catalyzed acidolysis reactions have been conducted with the assistance of organic solvents such as hexane or toluene (Mutua and Akoh, 1993; Adlercreutz et al., 2002; Hossen and Hernandez, 2005). The use of solvents increases the capital investment when the process is scaled up. Furthermore, it has been reported that increasing the amount of solvent reduces the recovery of PL more strongly than it increases the incorporation during TLL-catalyzed acidolysis (Vikbjerg et al., 2005a). If possible, it is recommended that the reaction should be conducted under solvent-free conditions. A clear elucidation of side reactions is important for practical operation in order to minimize by-products during reactions. Recently we produced caprylic acid-containing PC in a batch reactor by RML-catalyzed acidolysis between PC and caprylic acid in a solvent-free system (Vikbjerg et al., 2005b). A typical time course of the acidolysis reaction can be seen in Fig. 11.10. By-products were formed due to parallel hydrolysis reactions and acyl migration in the reaction system. Usually there was a tendency for a decrease in yields along with an increase in acyl incorporation. Response surface design was used to evaluate the influence of

50

100

40

80

30

60

20

40

10

20

0

0

20 40 60 80 Reaction time (h)

PC recovery (mol%)

Incorporation of caprylic acid (mol%)

Chemical and enzymatic interesterification of lipids for use in food 255

0 100

Fig. 11.10 Time course for immobilized Rhizomucor miehei lipase-catalyzed acidolysis reaction between phosphatidylcholine (PC) and caprylic acid. Reaction conditions: substrate ratio, 6 mol/mol caprylic acid/PC; enzyme dosage, 30 % (wt. % based on substrate); reaction temperature, 50 °C; water addition, 2 % (wt. % based on substrate).

major factors (enzyme dosage, reaction time, reaction temperature, substrate ratio and water addition) and their relationships on a number of responses reflecting the turnover of main reactions as well as side reactions. Several parameters important for the main reaction also affect by-product formation resulting in lower recoveries. All parameters besides water addition had an effect on the incorporation of caprylic acid into PC and LPC. Increased reaction time and enzyme dosage showed increased effect on incorporation into PC, while increased substrate ratio and reaction temperature showed opposite effects. The PC content decreased with increase of all parameters except for substrate ratio. In the solvent system using immobilized TLL, the incorporation of the desired acid was seen to increase with increase in temperature and substrate ratio (Vikbjerg et al., 2005a). Clearly optimization must be individually performed in each case. The exchange of fatty acids in PL has mainly been performed batch-wise in small screw cap vessels or glass bottles with either orbital or magnetic stirring. For larger scale production, it would be more convenient to operate in PBR as this allows continuous operation. Furthermore, due to the high enzyme dosages usually applied for lipase-catalyzed PL modification (> 40 % based on substrate), the final product is not easily removed from the reaction mixture after batch operation. To obtain the same conversion degree with the same enzyme load in the PBR, a very long residence time is required. Trials have been made for the continuous operation of the acidolysis reaction (Vikbjerg et al., 2005c). If no water was added to the substrate during reaction in the solvent-free system, very low incorporation of novel fatty acids was observed. Operative stability was tested for several days. Incorporation was highest at the beginning and decreased in the first 30 hours, where it stabilized afterwards. Incorporation of novel fatty acids was slightly higher when the water content was increased. In the presence of hexane, incorporations into PL were considerably higher and increased continuously in the first two

256 Modifying lipids for use in food days, and thereafter were stable with only a slight decline in incorporation over several days. Using PBR with solvent-free systems, it seems that small amounts of water are beneficial for the incorporation of desired fatty acids. Due to the high PL concentration, the substrates probably remove bound water from the enzymes, thus reducing the catalytic activity. Increasing water content will at the same time decrease the yield. Therefore, some kind of compromise needs to be made.

11.3.4 Glycerolysis for partial glyceride production Partial glycerides, more commonly known as mono- and diglycerides, have been produced commercially for many years. Today, they are widely used in the food, cosmetic and pharmaceutical industries as well as in the textile, fibre and plastic industries (Bornscheuer, 1995; Coteron et al., 1998; ElfmanBorjesson and Harrod, 1999; Bellot et al., 2001; Ferreira-Dias et al., 2001; Kaewthong and H-Kittikun, 2004). Mono- and diacylglycerols of edible fatty acids are approved by the EU as food grade additives. They have been given ‘Generally Recognized As Safe’ (GRAS) status by the US Food and Drug Administration (FDA), and can be used quantis satis (no permitted maximum level is specified) according to the European Parliament and Council Directive (European Parliament Council, 2004). According to World Health Organization (WHO) and the EU directive, mono- and diglycerides of fatty acids are required to contain at least 70 wt % MAG + DAG (mono- + diacylglycerols), at least 30 wt % MAG and maximum 7 wt % glycerol (European Food Emulsifier Manufacturers’ Association, 2004; European Parliament Council, 2004). Pure monoacylglycerols have excellent emulsifying properties, superior to diacylglycerols and mixtures of partial glycerol esters (Bornscheuer, 1995; Peng et al., 2000; Kaewthong and H-Kittikun, 2004). This is due to the MAG molecular structure with a favourable distribution between one hydrophobic fatty acid moiety esterified to a hydrophilic glycerol moiety. This combination, as well as the dietary safety profile, makes monoacylglycerols and mono/diacylglycerol mixtures very popular additives for facilitating uniform quality of food products among other applications (Krog, 1997). Accordingly, mixtures of mono- and diacylglycerols and distilled monoacylglycerols contribute to a market estimated at around 75 % of the worldwide emulsifier production. This corresponds to approximately 200 000– 250 000 tonnes produced per year (Moonen and Bas, 2004). The main applications of mono-diacylglycerols in foods are typically in fat-based products such as margarine, spreads, bakery products, cake mixtures, confectionery and the like (Krog, 1997; Kaewthong and H-Kittikun, 2004). Mono/diacylglycerols are often added to industrial food formulations in combination with other more hydrophilic emulsifiers, for instance in combination with hydrocolloids in dairy emulsions such as ice cream (Krog, 1997).

Chemical and enzymatic interesterification of lipids for use in food 257 Recent studies on DAG nutritional properties and dietary effects suggest that oils with high DAG content play an important role in reducing levels of serum TAG (Flickinger and Matsou, 2003; Watanabe et al., 2005). Thus, to fight obesity and other lifestyle-related diseases, substitution of TAG oils with DAG oils has attracted much attention recently. The first DAG cooking oil entered the Japanese market after its introduction in February 1999. With more than 70 million bottles sold (year 2003) and launched in the USA in 2005, the interest in a global DAG-market has indeed begun (Flickinger and Matsou, 2003; Kristensen et al., 2005). Chemical glycerolysis Today, commercial mono-diacylglycerol mixtures are widely manufactured using a glycerolysis reaction between glycerol and fats or oils, as illustrated in Fig. 11.11. The currently used glycerolysis reaction is performed at high temperatures (220–260 °C) with inorganic alkaline catalysts, such as NaOH or Ca(OH)2. Approximately 30 to 60 minutes of processing leads to an equilibrium mixture with partial glycerol esters and excess glycerol. To remove impurities and achieve high-purity products, subsequent removal of excess glycerol by distillation followed by molecular/short path distillation (SPD) processing is often performed (Bornscheuer, 1995; Krog, 1997; Rosu et al., 1997; Elfman-Borjesson and Harrod, 1999; Xu et al., 2000, 2002; Xu, 2000b; Bellot et al., 2001; Kaewthong and H-Kittikun, 2004; Lee et al., 2004). O O C O O C OH O

O O C

OH

O C

OH Catalyst

OH

OH

O C

O

2

+2

O C O

O

O C

Trillinolein (TAG)

Glycerol OH O O C OH OH OH O O C

Mixture of partial acylglycerols (MAG + DAG)

Fig. 11.11

Reaction scheme for production of mono-diacylglycerols by glycerolysis.

258 Modifying lipids for use in food The reaction can, to a certain extent, favour the formation of certain glycerol esters depending on the reaction conditions such as glycerol to oil ratio, reaction time and pressure (Peng et al., 2000). The effect on the product distribution of MAG, DAG, TAG and glycerol at equilibrium conditions after glycerolysis of different blended glycerol to oil ratios is illustrated in Fig. 11.12. Current chemical glycerolysis processing is usually conducted with a relatively low molar ratio of glycerol to oil of about two (Rendon et al., 2001). This provides an amount of non-reacted glycerol of about 8 wt% (Fig. 11.12b) and a distribution between MAG, DAG and TAG of 45–55 %, 38–45 % and 8–12 %, respectively (Krog, 1997). Enzymatic glycerolysis Today, it is well known that the Western diet is characterized by a general low intake of essential n-3 polyunsaturated fatty acids (PUFAs) and an 100 %

(wt %)

80 % 60 %

MAG DAG TAG

40 % 20 % 0% 0

10

20 30 Molar ratio gly: oil (a)

40

50

100 %

(wt %)

80 % Glycerol MAG DAG TAG

60 % 40 % 20 % 0%

0

10

20 30 Molar ratio gly: oil (b)

40

50

Fig. 11.12 Calculated product distribution at equilibrium conditions after glycerolysis reaction with different molar ratios of glycerol to oil. (a): Distribution between MAG, DAG and TAG (fat phase). (b): Distribution in the complete reaction mixture (fat phase and glycerol). Calculations are based on binomial random distribution with probability calculations of ester formation between mole fatty acids (FA) and mole hydroxyl groups (OH) and expressed as weight percentages (adapted from Damstrup et al., 2006).

Chemical and enzymatic interesterification of lipids for use in food 259 overabundance of n-6 fatty acids compared to international recommendations (Volker and Garg, 2001). In Australia and the USA, the current intakes of n-3 PUFAs are for instance less than 100–200 mg/day (Peters and Nurmikko, 2002). The intake is far from the recommendations of 1–2 g/day from organizations such as WHO, FDA and American Health Association (AHA) (Anselmino, 2004). This has led to a great deal of commercial interest in producing health-promoting and functional lipids with specific fatty acid profiles to overcome the gap between actual and recommended n-3 PUFA intake. Vegetable oils are considered as easily accessible, relatively cheap and neutral flavoured triacylglycerol raw materials for carrying nutritionally important PUFAs. However, the current chemical glycerolysis process makes the use of raw materials based on PUFA-vegetable oils difficult due to their sensitivity to oxidation at high temperatures (Peng et al., 2000). In contrast, enzymatic glycerolysis requires only a low temperature, below approximately 80 °C, which makes the use of heat-sensitive MAG and DAG with PUFAprofile feasible. Furthermore, the gentle enzyme technology reduces some of the heat-accelerated problems with development of bad flavour, dark colour and unwanted side reactions (Bornscheuer, 1995; Elfman-Borjesson and Harrod, 1999; Bellot et al., 2001; Kaewthong and H-Kittikun, 2004). Accordingly, lipase-catalyzed glycerolysis of vegetable oils is believed to be a potential alternative/supplementary industrial method for the production of nutritional high-valued monoacylglycerols with important n-3 PUFAs. This opportunity for product and process development has led to a great interest in the enzymatic glycerolysis process, not only from academia but also from industry since the mid-1980s. Glycerolysis under enzymatic catalysis has similar reaction routes to chemical glycerolysis, as shown in Fig. 11.11. Reaction equilibrium is still a central determinant for the evaluation of the product yield as shown in Fig. 11.12. However, because of its use of low temperature and its possible use of solvents, reaction equilibrium is not a target, which is imperative to achieve through the enzymatic glycerolysis process. This is because the operating conditions make it possible for a product ‘fishing’ strategy. All possible effort is being made to speed up the procedure of reaching reaction equilibrium on the other hand. This is illustrated in Fig. 11.13. Possible methods for such ‘fishing’ have been tested using crystallization, chromatographic separation, extraction, etc. as a step to break the reaction equilibrium and force the reaction to form more product (Peng et al., 2000). Recent progress has been made with the idea of using solvent partitioning to push the reactions beyond reaction equilibrium (Guo and Xu, 2005; Yang et al., 2005b, Damstrup et al., 2005, 2006). In such systems with the selected media, the reaction efficiency and effectiveness have been dramatically improved, and higher yields of monoacylglycerols and diacylglycerols have been obtained. Figure 11.14 compares the reaction behaviour of glycerolysis of sunflower oil using ionic liquid and tert-butanol as system media with a solvent-free system. The first

260 Modifying lipids for use in food Lipases Fats and oils + glycerol

Monoglycerides + Diglycerides

• Membrane separation • Extraction • Fractionation • Adsorption • etc.

Illustration of product ‘fishing’ for the enzymatic production of partial glycerol ester glycerides.

Yield of monoglyceride (mole %)

Fig. 11.13

100 80

60 40 20 0 0

2

4

6 8 10 12 Reaction time (h)

22 24

Fig. 11.14 Different reaction behaviours of glycerolysis of sunflower oil in ionic liquid, tert-butanol and the solvent-free system (0.5 mmol sunflower oil and 5 × 0.5 mmol glycerol for reaction at
, 40 °C; ◊, 50 °C; 䉭, 60 °C; , 70 °C in 2.2 g ionic liquid; 䉱, 50 °C in 2.5 g tert-butanol; •, 2 mmol sunflower oil and 5 × 2 mmol glycerol for reaction at 70 °C in solvent-free system) (adapted from Guo and Xu, 2005).

two systems have been shown to have good potential for industrial implementation. Recent progress in enzymatic glycerolysis In general, the major challenge in using enzymes in low-temperature glycerolysis reactions is that the system comprises of three heterogeneous phases: a hydrophobic oil phase, a hydrophilic glycerol phase and a solid enzyme phase, as illustrated in Fig. 11.15. Since enzymes in their native forms have hydrophilic characteristics, glycerol often binds to the enzyme particles and makes the access of the hydrophobic oil molecules to the enzyme difficult (Kristensen, et al., 2005; Yang et al., 2005b). Poor miscibility of the glycerol and oil at low temperatures in combination with high reactant viscosity results in a reaction system with high mass transfer limitations. As a result, long reaction times and/or low conversion of reactants generally make the reaction inefficient.

Chemical and enzymatic interesterification of lipids for use in food 261 Enzyme (Solid)

Glycerol (Hydrophilic) Inhomogeneous system

Oil (Lipophilic)

Fig. 11.15

Illustration of the heterogeneous reactant mixture used for enzymatic glycerolysis.

However the sustainability of an effective system has, to a certain extent, relationships with the content of glycerol in the system. In the case of DAG production, the theoretical stoichiometric amount of glycerol in the reaction system is low. Although the reaction time remains long, it is possible to have a sustainable system for DAG production without the usage of any medium assistance (Kristensen et al., 2005). With a sufficiently low glycerol amount, a conversion of TAG up to 60–70 % can be achieved (Fig. 11.16). However, for MAG production, assistance of media or involvement of alternative techniques is normally required to improve the formation of MAG in reasonable time and amounts. Solid phase crystallization (McNeill et al., 1990; Bornscheuer et al., 1994; Rosu et al., 1997), glycerol adsorbed to silica gel (Aha et al., 1998; Elfman-Borjesson and Harrod, 1999; Rendon et al., 2001), usage of protected glycerol (Akoh, 1993) and reactions performed in different media such as supercritical CO2 (Jackson and King, 1997) or organic solvents (Bornscheuer et al., 1994; Kwon et al., 1995; Rendon et al., 2001) are among the many interesting approaches to improve reaction efficiency and/ or product quality. Many of the approaches work successfully on a laboratory scale, and a great deal of knowledge, revealed from 20 years of progressing research, has been accessible. However, due to practical difficulties, the big industrial breakthrough for MAG production has still not taken place. For instance, solid phase crystallization and glycerol adsorption are difficult to handle continuously, and the reaction time required is still lengthy from an industrial point of view. At present, the high cost of the enzyme also makes the use of lipases in industrial applications uneconomic (Yang et al., 2003a; Kaewthong et al., 2005). To overcome some of the problems with high enzyme costs, a widely used strategy is to employ the lipase in an immobilized form. Immobilization of the enzyme onto a solid support material allows easy separation and reutilization of the enzyme. Furthermore, the use of immobilized enzymes on porous supports eliminates some of the problems arising with the use of suspended enzyme powders, such as the tendency to aggregation and attachment to the wall of the reactor, as the enzyme spreads on a large surface area (Barros et al., 1998).

262 Modifying lipids for use in food

Composition (wt %)

Composition (wt %)

100

80 60 40 20 0

5

10 15 20 Time (h)

25

60 40 20

30

Composition (wt %)

40 20 5

10 15 20 Time (h)

5

10 15 20 Time (h)

25

30

25

30

100

60

0

0

Novozym 435

Lipase F-AP15

80

0

80

0 0

100

Composition (wt %)

Lipase AK

Lipase PS-D

100

25

30

80 60 40 20 0 0

5

10 15 20 Time (h)

Fig. 11.16 Time courses for enzymatic glycerolysis with different lipases in batch reactors (䉭, TAG; 䊊, 1,3-DAG; 䊉, 1,2-DAG; 䊐, MAG; ■, total DAG (adapted from Kristensen et al., 2005).

Other benefits from immobilized enzymes compared to non-immobilized enzymes are improved activity and long-time stability in various organic media (Goto et al., 2005; Nakaoki et al., 2005). For instance, it has been shown that when lipases are immobilized onto a hydrophobic matrix, their activity increases 51-fold and exhibits superior heat resistance compared to the native lipase in esterification reactions of lauric acid in isooctane system (Goto et al., 2005). Since Zaks and Klibanov discovered that lipase enzymes act well in organic solvents (Zaks and Klibanov, 1984, 1985), many investigations of lipase-catalyzed interesterification reactions in solvents confirm the benefits of using non-conventional media. Among the useful solvents in interesterification reactions are dioxan, n-hexane, n-heptane, acetonitrile, acetone, isooctane and tert-butanol and tert-pentanol (Goto et al., 1995; Hess et al., 1995; Elfman-Borjesson and Harrod, 1999; Bellot et al., 2001; Rendon et al., 2001; Kaewthong and H-Kittikun, 2004; Yang et al., 2005b). In our experience, the usage of selected media is believed to provide potential approaches for glycerolysis with industrial applications (Guo and Xu, 2005; Damstrup et al., 2005). In such systems, commercially available immobilized CALB is shown to be one of the most stable enzymes. In spite

Chemical and enzymatic interesterification of lipids for use in food 263 of lowered productivity due to the presence of solvent, glycerolysis reaction in the tertiary alcohols, tert-pentanol (2-methyl-2-butanol) and tert-butanol (2-methyl-2-propanol), is found to have great potential for efficient glycerolysis (Damstrup, et al., 2005, 2006). These solvents offer benefits beyond the drawbacks related to lowered productivity. Amongst these advantages are high MAG yield/purity in the product mixture and very short reaction time together with a well-preserved PUFA profile of the MAGs (Damstrup et al., 2005, 2006; Yang et al., 2005b). In addition, fluid solvent systems make continuous operation in PBR feasible. The obvious benefits of using continuous PBR are: ease of separation between reactant mixture and catalyst; reuse of the enzyme without the need for a prior separation; a cost-effective reactor design; and long-term production due to high density loading of the immobilized enzyme into the reactor (Kaewthong et al., 2005; González Moreno et al., 2005). These advantages indeed promote the arguments for using solvents in enzymatic glycerolysis. Finally, a process design including reusability of the solvent minimizes the drawbacks from lowered space-time yield. A good review of lipase-catalyzed production of high-quantity monoacylglycerols by Peng et al. (2000) is recommended for further reading. In addition, a recently published and excellent patent review on lipid technology including patent literature involving monoglycerides by Lai and Lo (2005) is also recommended.

11.4

Remarks and future trends

The concept of interesterification, as defined in this chapter, with catalysis either by enzymes or chemicals, can be widely explored for unlimited product or process development. The subject is becoming more interesting and attracting more attention. Chemical interesterification, to a large extent, is a mature technology that offers the prospect of many new applications in industry. Chemical randomization of oils and fats for plastic fat production, although in practice for about a century, is gaining new momentum with the coming ‘trans-free’ ruling for the future market. The method has been widely used but the ‘share’ of industrial capacity using this method for plastic fat production is expanding. Chemical glycerolysis is a common method for monoacylglycerol production in the present food industry, and it is expected to dominate monoacylglycerol production in the foreseeable future. Another case of chemical interesterification, which is receiving more and more importance, is chemical methanolysis for the production of biodiesel. Energy crises in the past year have led to the expansion of biodiesel production capacity from oils and fats. Enzymes as catalysts for the interesterification of oils and fats, although far from mature and a long way from achieving widespread implementation

264 Modifying lipids for use in food in industry, have been a focus for extensive studies for decades. Even though nobody expects a fast increase in the applications of enzymatic interesterification in industry, interest in using enzyme processes and realization of their benefits have increased in the industrial sector. More and more industrial think-tanks are including this area in their development strategy. As an alternative to chemical randomization for plastic fat production, enzyme processes, even with such a low-priced product, have made an entry in the oils and fats industry in very recent years. Fats produced from the enzyme process in general can be used for margarine production without significant change of properties, though there are some unusual aspects compared to either blended or chemically randomized fats (Zhang, 2005). From the point of view of oxidation during storage, the enzymatically-produced fats have advantages over the chemically randomized product; the latter develops high peroxide values during storage. With regard to physical properties, the former can form the required crystal type and textures along with suitable solid fat profiles which are comparable to those of the chemically randomized fats. However, margarines produced from the enzymatically-interesterified fats become more physically stable with a higher conversion degree of the interesterification. The control of the conversion degree, therefore, becomes highly critical to achieving good quality products. Furthermore, the optimization of margarine formulation, such as the use of diacylglycerols to postpone crystal transformation and the optimization of process conditions for margarine production, needs more studies to cater for the new requirement of using the partially interesterified fats from the enzymatic processes. For the enzymatic production of structured lipids with structural specificity concerning the locations of fatty acids, enzyme processes are the only potential methods for large-scale production. Products of high value intended for functional foods and pharmaceutical applications provide only a small market. So far, a few products, such as fats for infant formula, fats for confectionery products, etc. have been available in the market. The initial criteria are the functional values of the products and their market demands. The economical balance of the process is the crucial point for industrial consideration. So far, technical issues for enzymatic interesterification concerning the production of structured lipids are not, in general crucial. The benefits of such products in human nutrition remain to be explored. Process optimization for a defined product needs a lot more work to reduce the cost of the process at engineering and management levels. Concept development for process engineering is a high priority for future research. A package of knowledge for building up a new plant is still far from available. For enzymatic modification of PL, an understanding of the reaction systems is just the beginning. Before these types of reactions can be implemented industrially, a lot more work will need to be done to increase efficiency. For these reactions to be applicable, their operative stability needs to be better understood and a longer lifetime of the immobilized enzyme is desired.

Chemical and enzymatic interesterification of lipids for use in food 265 Although solvent-free systems may seem to work well during batch operation, there is no information as to whether the lipase can actually be re-used. In terms of stability during continuous operation, it may be beneficial to use non-polar solvents as higher incorporation can be obtained for a prolonged time compared to solvent-free systems. Even though major by-product formation occurs during lipase-catalyzed acidolysis, it should be kept in mind that these by-products (LPC and GPC) are themselves valuable products having wide applications in the same area as the original material. These compounds can be purchased in purified form from different companies, and are usually sold at considerably higher price compared to natural PL. Concerning the production of partial glycerol esters, it is well documented that enzymatic glycerolysis has become a powerful method in terms of MAG/ DAG quality and functionality, which makes it possible to expand their fields of applications into functional foods, pharmaceuticals, etc. Of particular interest is the possibility of producing ‘new’ MAGs with PUFA profiles that differ significantly from MAGs produced by traditional chemical glycerolysis methods. With the introduction of efficient long-lasting commercially available immobilized lipases and the benefits of large-scale PBR, the industrial breakthrough for enzyme-catalyzed glycerolysis has moved closer. Accordingly, in the future, it is most likely that the fat and oil industry will, to a certain extent, adapt the enzyme-catalyzed glycerolysis into industrial plants, even although the chemical process will still occupy the main position for many years to come. As discussed in the above section, a medium, usually an organic solvent, has been an advantage in achieving a high degree of efficiency and effectiveness in the process and good product yields. The use of solvents, however, is contrary to the aim of an environmentally friendly and energy-reduced enzymatic process due to the extra processing required for solvent removal. From this point of view a solvent-free medium or the application of nonevaporative solvents such as ionic liquids could be a better solution. This certainly opens a new window for future visions.

11.5

Sources of further information and advice

Enzymatic interesterification has been attracting significant interest since the 1980s and chemical interesterification for even longer. A large amount of information has been collected and accumulated. A large number of recent review papers and chapters concerning each topic have been cited in the relevant sections. A number of company homepages are worth visiting, including technology providers (e.g. Desmet Ballestra, Alfa Laval), enzyme providers (e.g. Novozymes, Amano) and product providers (e.g. ADM, Unilever, Danisco, Degussa, Karlshmns, Aarhus United, Kao, Nisshin Olillio, Loders-Croklaan). These companies provide a real picture of interesterification in industry.

266 Modifying lipids for use in food

11.6

Acknowledgements

Financial support for our group research activity is acknowledged from the Research Council for Technology and Production, the Danish Dairy Foundation, Danisco, and the Centre for Advanced Food Studies as well as other assistance from industry and collaboration partners.

11.7

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270 Modifying lipids for use in food ROSU R, UOZAKI Y, IWASAKI Y

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272 Modifying lipids for use in food and KLIBANOV A M (1985), Enzyme-catalyzed processes in organic solvents, Proc Natl Acad Sci, 82(10), 3192–3196. ZHANG H (2005), Lipase-Catalysed Interesterification for Margarine Fat Production, PhD Thesis, Technical University of Denmark, Lyngby. ZHANG H, XU X, MU H, NILSSON J, ADLER-NISSEN J and HØY C-E (2000), Lipozyme IM-catalyzed interesterfication for the production of margarine fats in a 1 kg scale stirred tank reactor, Eur J Lipid Sci Technol, 102(6) 411–418. ZHANG H, JACOBSEN C, PEDERSEN L S, CHRISTENSEN M W and ADLER-NISSEN J (2006), Storage stability study for margarines produced by enzymatically interesterified fats compared to the margarines by the conventional methods – chemical properties, Eur J Lipid Sci Technol, 108(4) 227–238. ZHANG H, XU X, NILSSON J, MU H, ADLER-NISSEN J and HØY C-E (2001), Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosus lipase in a large-scale study, J Am Oil Chem Soc, 78(1), 57–64. ZHANG H, PEDERSEN L S, KRISTENSEN D, ADLER-NISSEN J and HOLM H C (2004a), Modification of margarine fats by enzymatic interesterification: evaluation of a solid-fat-contentbased exponential model with two groups of oil blends, J Am Oil Chem Soc, 81(7), 653–658. ZHANG H, SMITH P and ADLER-NISSEN J (2004b), Effects of degree of enzymatic interesterification on the physical properties of margarine fats – solid fat content, crystallization behavior, crystal morphology and crystal network, J Agr Food Chem, 52(14), 4423–4431. ZHANG H, JACOBSEN C and ADLER-NISSEN J (2005), Storage stability study for margarines produced by enzymatically interesterified fats compared to the margarines by the conventional methods I. Physical properties, Eur J Lipid Sci Technol, 107(7–8), 530– 539. ZAKS A

12 Plant breeding to change lipid composition for use in food D. J. Murphy, University of Glamorgan, UK

12.1

Introduction

In global terms, plant lipids are the second most important source of edible calories in the human diet (after carbohydrates). Plant lipids are also sources of several essential vitamins and nutrients. For example, plant lipids are the ultimate source of the so-called ‘essential fatty acids’ that are an obligatory component of the diet of all mammals – ever since that time many millions of years ago when our distant animal ancestors lost the ability to introduce double bonds beyond the ∆9 position in long-chain fatty acids. Since the dawn of agriculture, certain plant species have been cultivated specifically for their lipid compositions. For example, the earliest olive plantations have been dated to more than nine millennia before the present day, and maize may have been domesticated in Mesoamerica as early as ten millennia ago. In addition to their acyl lipid ingredients, plants are important dietary sources of a host of other lipophilic compounds, including vitamins A and E and a range of phytosterols. Most of our dietary plant lipid is derived from oil crops and is in the form of either ‘visible’ (e.g. oils, margarines, chocolate) or ‘invisible’ (cakes, confectionary, processed foods) fats. In the past, the lipid compositional requirements for these products have been provided by a variety of commodity plant oils that may be blended together and/or chemically modified (e.g. by hydrogenation) for a particular edible application. More recently, there has been a move towards a greater segmentation of the commodity plant oils market, with far more stress placed upon the initial composition of the plant oil itself. Hence, the increasing demand for plant oils that are enriched in monounsaturates, very long-chain ω-3 fatty acids, carotenoids, phytosterols

274 Modifying lipids for use in food and tocotrienols. With an increased willingness by buyers to pay a premium for such nutritionally enhanced oils, it is becoming more economic for growers and processors to segregate such value-added products. This in turn is driving plant breeders to select new varieties of oil crop designed for consumers who are becoming ever more aware of lipid-related nutritional issues, such as the presence of trans-fatty acids and saturates in foodstuffs of all kinds. In this chapter, I will describe how plants are being manipulated through various forms of breeding in order to supply this wide range of dietary lipids, with a special focus on fatty acid composition.

12.2 General perspective 12.2.1 Margarine – the beginnings of an industry for plant lipids Prior to the late 19th century, there was no plant lipids industry and the few dedicated oil crops that were grown tended to be consumed locally. In Northern Europe, rapeseed had been grown as an oil crop since Roman times, but the oil was mainly employed for lighting, as a supplement to tallow and beeswax. The few edible oils that were transported and traded, such as olive oil, were treated very much as luxury products away from their region of origin around the Mediterranean. In much of Europe and the New World, virtually all of the visible dietary fat was derived from dairy and meat products, and edible vegetable oils were a distinct rarity. Indeed, a few thousand years ago, Northern Europeans became so dependent on milk products that most of them still carry a mutation in the lactase gene that enables it to be expressed throughout life, rather then ceasing its activity after weaning as it does in most members of our species. This is the reason that 98 % of adult Thais and 100 % of Amerindians are lactose-intolerant, while in milk-drinking Sweden the figure is only 2 % (Enattah et al., 2002). Plant lipids were first propelled into mainstream food production by a technological innovation in the late 1860s. This happened when a French chemist called Hippolyte Mège Mouriès produced what we now know as margarine. Even in those days, plant lipid researchers had to respond to the needs of industry and government and the work of Mège Mouriès followed a call by French emperor, Napoleon III, for research into a possible alternative to butter as a high-calorie foodstuff for the French army. After a considerable amount of experimentation, the raw material that Mège Mouriès selected for the new product was a solid fatty acid fraction called margaric acid, because of the lustrous pearly drops of the crystalline form that are reminiscent of pearls, which are called margarites in Greek (this is also the derivation of the name, Margaret). The earliest forms of margarine were mixtures of animal and plant fats but, initially, this mixed lipid product was not a great commercial success. Two technical advances tipped the balance towards using only plant fats in margarine and allowed the new fatty spread to compete more effectively

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with butter. Firstly, improved refining methods allowed the purification of a greater variety of liquid oils and solid vegetable fats that could be blended to make a good spreadable margarine. Secondly, the process of hydrogenation, which was invented in 1901 by German chemist William Normann, enabled the large-scale conversion of relatively cheap plant oils into solid fats. Not only did the hydrogenation process produce a good, inexpensive butter substitute, it also significantly reduced the amount of oxidation-prone polyunsaturates in the solid margarine, which greatly extended its shelf life and therefore its utility for consumers. Margarine soon spread around the world and, a few years later, the American author, Mark Twain, overheard the following conversation between two businessmen aboard a Cincinnati riverboat: Why, we are turning out oleomargarine now, by the thousands of tons. And we can sell it so dirt-cheap that the whole country has got to take it – can’t get around it, you see. Butter don’t stand any shows – there ain’t any chance for competition. However, US dairy farmers soon mounted an effective counter-attack against a product that they believed would ruin their livelihoods. Thanks to their political clout, especially in strong dairy States like Wisconsin, the farmers managed to get margarine classified as a ‘harmful drug’ that was subject to restricted sales. Margarine was also heavily taxed; stores had to be licensed to sell it and, like alcohol and tobacco, it was promptly bootlegged. Ironically, in view of its origins as a food for the French army, the US government refused to purchase margarine for the use of its own armed forces. Finally, as a way of making it even less attractive, some states did not allow yellow margarine to be sold, so the shopper was faced with purchasing an unsightly, off-white slab of fat (van Stuyvenberg, 1969). Surprisingly, the punitive federal taxes on margarine were not abolished until 1950; yellow margarine could not be sold in Wisconsin until 1967; and, to this day, the sale of yellow margarine is prohibited in the Canadian Province of Quebec. Some of this hysteria about margarine might appear somewhat comical, but there are still echoes of similar attitudes to food innovation in some of the current debates about aspects of the use of genetic engineering for crop improvement, including the modification of fatty acids in oil crops.

12.2.2 Diversity of plant lipids Before looking at plant lipid modification in more detail, it will be useful to consider the historical role of these products as part of our diet and to examine the crops that are grown specifically as sources of dietary lipid. Globally, there are now just 15 major crops that supply most of the human diet and five of these are crops with relatively high oil contents, namely soybeans, oil palm, maize, peanuts and coconut (Harlan, 1992). About two-

276 Modifying lipids for use in food thirds of the 110 million tonnes of commercially produced plant oil is from soybean, palm and canola (genetically improved rapeseed). The major fatty acids from the world oil supply are palmitic, linoleic and oleic acids. In addition to these major edible fatty acids, many unusual fatty acids can accumulate in seed oils of other plant species, as is shown in Table 12.1. Sometimes these unusual fatty acids might comprise in excess of 90 % of the seed oil (Hildebrand et al., 2005). As we can see from Table 12.1, unusual fatty acid modifications include variations in carbon chain length and degree of unsaturation. Most naturally-occurring fatty acids have double bonds in the cis configuration, although occasionally trans double bonds are also found, e.g. in photosynthetic membrane lipids. There are also different positional isomers, conjugated unsaturated, acetylenic, hydroxy, epoxy and keto fatty acids. Cyclopropenoids, cyclopentenoids and even fluoro fatty acids add more diversity to the list of fatty acid species. These more exotic fatty acids are rarely used in edible products; rather they are part of the 20 % of plant oils that are used for non-food purposes, i.e. as oleochemicals in the manufacture of detergents, resins, paints, polymers and pharmaceuticals or as animal feedstuffs (Murphy, 1994).

Table 12.1

Accumulation of novel fatty acids by some oil-producing plants.

Fatty acida

Amountb

Plant species

Actual and potential uses

8:0 10:0 12:0 14:0 16:0 18:0 20:0 22:0 24:0 18:1∆6 cis 18:1∆9 cis 22:1∆13 cis 18:2∆9,12 cis α18:3∆9,12,15 cis γ18:3∆6,9,12 cis 18:1–hydroxy 18:1–epoxy 18:2 9c12a 18:3–oxo 18:3–conj 20:1/22;1wax

94 95 94 92 92 65 33 48 19 76 78 58 75 60 25 90 60 70 78 70 95

Cuphea avigera Cuphea koehneana Litsea stocksii Knema globularia Myrica cerifera Garcinia cornea Nephelium lappaceum Brassica tournefortii Adenanthera pavonina Coriandrum sativum Olea europaea Crambe abyssinica Helianthus annuus Linum usitatissimum Borago officinalis Ricinus communis Crepis palestina Crepis alpina Oiticica Tung Simmondsia chinensis

Fuel, food Detergents, food Detergents, food Soaps, cosmetics Food, soaps Food, confectionery Lubricants Lubricants Lubricants Nylons, detergents Food, lubricants Plasticisers, nylons Food, coatings Paints, varnishes Therapeutic products Plasticisers, cosmetics Resins, coatings Coatings, lubricants Paints, inks Enamels, varnishes Cosmetics, lubricants

% % % % % % % % % % % % % % % % % % % % %

a Fatty acids are denoted by their chain length followed by the number of double bonds or nature of other functionalities. b Percentage of total seed or mesocarp fatty acids; data are taken from Murphy (2001).

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12.2.3 Markets for plant lipids Despite recent increases in the global consumption of animal products, for most people, plants are still the major source of dietary fats, although they are often unaware of exactly how much and what type of lipid they are consuming (this is especially true for ‘invisible’ fats). Plant lipids are normally obtained in the form of liquid vegetable oils either from oilseed crops like soy or rape, or from oil-rich fruits like oil palm or olive. In 2005, the ten most important commercial oil crops produced a total of 107 million tonnes of oil with a value of about $70 billion (Oil World, 2005). Therefore, plant sources supply about 80 % of the total global demand for traded fats and oils – the non-plant fats and oils are mainly obtained from animal, fish and dairy sources. Plant-derived oils tend to have relatively narrow fatty acid profiles, being mainly dominated by C16 and C18 saturates, and by the C18 mono-, di-, and tri- unsaturates. Such a profile has suited the treatment of plant lipids as generic commodity oils, to be produced and transported in bulk and to be blended and/or hydrogenated as necessary to fit a particular end use. This is especially apparent in the processed food sector where most plant lipids are used. Hence, different plant oils may be blended in different proportions to produce the various types of solid fat that are used in products such as spreads and shortenings. Since the 1980s, there has been an increasing segmentation of the plant oils market as food producers seek to highlight oils from particular crops, which may have special attributes that can add value to an end product. For example, high linoleate sunflower oil is favoured for certain ‘high polyunsaturate’ margarines, while cold-pressed, unprocessed ‘virgin’ olive oil is favoured for its organoleptic qualities. In contrast, other plant oils, such as soy and rape, have tended to remain as generic commodity products. In the case of rape oil, this is rather odd because the oil has a very high oleic acid content, which makes it suitable to be branded as ‘high in monounsaturates’. There are also varieties of oilseed rape that have less than 4 % linolenic acid, which avoids the need for hydrogenation and potentially allows the oil to be marketed as ‘low in trans-fatty acids’. Despite these favourable attributes, however, rape oil still tends to be treated as a low-cost commodity, rather than as a higher value, segmented-market product like olive oil. This brings us to an important point about the reasons for the manipulation of plant lipid composition. Since the early 1990s, many new types of plant oil crop have been developed, and many more are in the pipeline. However, many of these new plant oils have not been taken up by the market, or have not been exploited to their full potential. Part of the reason for this is that many of the modifications of plant lipid composition, especially by genetic engineering, have been technology-driven, rather than being market-led. This means that markets may be unprepared, unaware or simply unwilling to take on the new products.

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12.3 Plant breeding Plant breeding, and indeed any other form of breeding, is based on two mutually dependent processes: namely genetic variation and selection. A given population must exhibit some heritable variation in a character of interest to a breeder before selection is possible. Hence one cannot usefully breed from excessively inbred or clonal populations. Equally, it is vital to have a system that enables recognition of variability in a character, especially if such a character is cryptic. Therefore, while the early farmers could readily select for seed size or taste, a character such as fatty acid composition could not be selected for directly until the advent of modern techniques of lipid analysis in the 20th century.

12.3.1 Discovering variation There are at least 500 000 species of higher plants and many of these accumulate storage oils in either their seeds and/or fruits. Unlike membrane lipids, which are extremely constrained in their acyl chain lengths and functionalities, storage lipids appear to be able to contain virtually any type of acyl moiety with chain lengths extending from as little as C8 all the way up to C24 (see Table 12.1). From the 1960s, the regional research division of the USDA at Peoria, Illinois has been undertaking a survey of some of the enormous diversity in acyl lipid composition of oils from plants collected from around the world. It has been found that there are many hundreds of plants, which are currently not grown as crops, but which have oil-rich seeds that accumulate novel and potentially useful fatty acids. Since the 1990s, much of the focus on such plants has entailed the isolation of genes that regulate the formation of the exotic fatty acids and their transfer to mainstream oil crops like soy or rape. However, as we shall see below, this genetic engineering approach has not been without its problems. Quite apart from the technical problems of persuading an existing crop to accumulate novel fatty acids in the right place and in the appropriate quantity, there is the problem of managing their cultivation and processing. After all, a soy seed that contains regular soy oil looks exactly the same as one that has been engineered to contain a non-edible, and possibly toxic, industrial oil. An alternative strategy that is now beginning to receive more deserved attention is to domesticate the original plants that made the exotic fatty acids in the first place, so that they can be grown as commercial crops in their own right.

12.3.2 Domesticating new oil crops The great advantage of domesticating existing plants rather than developing transgenic crops is that these existing plants are already adapted to accumulate their exotic fatty acids in the appropriate cellular compartment, namely in the triacylglycerols of their storage oil bodies. In the native plants, these

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exotic fatty acids are hardly ever found in cell membranes or in any other acyl lipids where their presence could be damaging. A further advantage of novel oilseed crops is that the seed oils already contain accessory stabilizing agents, such as antioxidants, which prevent the breakdown of some of the more highly reactive fatty acids such as conjugated polyunsaturates and those containing acetylenic bonds. Although many of the potential new crops may already be excellent sources of useful products, such as novel fatty acids, they are often not suitable for large-scale agriculture. The reason for this is simple: these plants have not been optimized for agronomic performance over centuries or even millennia, as have some of our more familiar crops. They suffer from the usual characteristics of wild plants; for example, they tend to flower asynchronously throughout the summer and therefore do not produce their seeds at a single time, which makes harvesting very difficult. They often produce seed pods that are prone to shatter before or during harvest, resulting in a loss of many of the seeds. Often, the canopy architecture of the plant is not suitable for existing harvesting machinery. They may be susceptible to a variety of diseases or pests, including fungi and insects. Finally, in the case of oilseeds, although they may contain as much as 90 % of a novel fatty acid in their seed oil, the overall oil yield in tonnes per hectare (T.ha–1) may be relatively low. The improvement of these important agronomic characters requires the manipulation of numerous complex traits. Companies are often dismayed by the prospect of domesticating new species, citing the example of major crops such as wheat, which is still being improved after more than 10 000 years of domestication. Nevertheless, we can now be more optimistic about the prospects for crop domestication. Many of our newer commercial crops have been improved at a much more rapid rate than wheat since the mid-20th century, thanks to the use of modern breeding techniques. Examples of such crops include hybrid maize, rapeseed, sunflower and soybean, which have only been grown as mainstream commercial crops for a century or less. There is also now the prospect of using biotech methods, such as marker-assisted selection (see below) to accelerate the development of new oil crops.

12.3.3 Creating new variation The domestication of new crops is a relatively recent option for plant breeders. In the past, breeders had to rely on the existing portfolio of crops in order to select for useful variation. Remarkably, almost all of our major crops were domesticated many thousands of years ago and no new mainstream crops have been domesticated since Roman times. The major revolution in plant breeding over the past hundred years has been the development of techniques that enable breeders to create additional variation in populations of the existing domesticated crops, even ones that are relatively inbred. To this we can add the development of a huge range of technological tools that facilitate selection for genetic characters that were invisible to our forebears. Before the early

280 Modifying lipids for use in food 20th century, breeders had to rely on existing variation resulting from ‘spontaneous mutations’ caused by errors in DNA replication. Such spontaneous mutations may be due to environmental insults or to high-energy electromagnetic radiation (X- or γ-rays), normally from cosmic rays. However, the rate of such spontaneous mutations is very slow indeed. Even the discovery of hybridization in the 18th century only modestly improved the capacity of breeders to improve variation. By the early 20th century, two developments revolutionized the ability of breeders to effect such genetic manipulations and to greatly extend their ability to create more novel and useful variations in crop genomes. Firstly, it became possible for the first time to cause mutations to occur deliberately and thereby to increase the rate of mutagenesis in a population by many thousand-fold. This is the technique of induced mutagenesis. Secondly, the efficiency of hybridization was vastly increased in the mid-20th century by the invention of tissue culture and the use of plant growth regulators. These developments greatly extended the potential gene pool that could be accessed by the breeder of a particular crop. Novel genes could be acquired from very distantly related species, and transferred into an elite crop cultivar by interspecific hybridization, followed by repeated backcrossing, in order to create new genetic combinations that have been of great utility to farmers. This is the technique of wide crossing. Induced mutagenesis was first used for the genetic manipulation of crops in the 1920s when agents such as X-rays and chemical mutagens were successfully used in maize and wheat. The most commonly used chemical mutagens in plant breeding are alkylating agents that directly react with DNA bases and modify their structure. One of the best known of these alkylating agents is ethyl methane sulphonate while another useful mutagen is sodium azide. Since the 1950s, γ-radiation from cobalt-60 or caesium-137 sources has been used with considerable success in crop breeding, most particularly in developing countries. An example of induced mutagenesis being applied to oil crops is the conversion by a group at CSIRO, Australia, of the non-edible, high α-linolenate oilseed, linseed, to a new edible, high linoleate variety that has been called ‘Linola™’. In this case, many tens of thousands of seeds from a conventional high α-linolenate variety of linseed were subjected to treatment by the mutagen, ethyl methane sulphonate (Green and Marshall, 1984). The aim was to produce a few mutagenized seeds in which the gene(s) encoding or regulating the fatty acid desaturase responsible for the conversion of linoleate to α-linolenate had been disrupted. It was found that there were several mutants that produced about half of the normal amount of α-linolenate, implying that the character was controlled by two genes. By crossing some of these single mutants, a population of double mutants was created in which the α-linolenate level was as low as 2 % of seed fatty acids. This was a great improvement on the situation in normal linseed oil, where linolenate is typically about 50 % of total fatty acid.

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As a result of this process of mutagenesis-assisted breeding, linseed oil, which is non-edible and oxidation-prone and is used in the manufacture of paints and putties, was converted into a premium quality edible oil with a similar acyl composition to that of sunflower. Unfortunately for the breeders who developed Linola in the 1980s, the market for high-polyunsaturate oils was already well supplied by the likes of sunflower and safflower, and demand was already moving away from polyunsaturates towards highmonounsaturate, low-trans oils. This has meant that the new variety was not as much of a commercial success as originally hoped (global Linola crop areas are only about 100 000 ha, whereas sunflower and rapeseed areas are in the tens of millions of hectares). However, this example does illustrate how an existing non-edible crop can be genetically modified by induced mutagenesis to create a completely different oil profile for food use (note that such mutagenized genetic manipulations are not officially classified as ‘GM’ because the resulting plants are not transgenic). Another method for the introduction of novel variation into a plant is by so-called ‘wide crossing’. In this case, a crop is hybridized with another plant that carries the desired genetic characters in order to create a hybrid that resembles the original crop parent, but also has the novel traits from the other parent. Wide hybridization can be done between plants of different species and even different genera. Normally, the progeny of such crosses are sterile and often the embryonic plants do not develop into seeds. However, since the 1930s, breeders have used a variety of tissue culture methods, such as chromosome doubling and embryo rescue, to obtain fertile progeny from wide crosses. Wide crossing has been used successfully to transfer genes into crops from other species, most notably for characters like disease resistance and salt tolerance. Several efforts have also been made to use wide crosses for the manipulation of plant lipid compositions, but so far no commercial products have been developed. As I hope it is apparent from this brief account of modern plant breeding, the intrusive manipulation of crop genomes by humans, including the transfer of genes from other species and the use of chemical- and radiation-induced mutagenesis, has been underway for over a century. Since the 1930s, all of the principal crop species have been modified using these methods, and it is estimated that radiation mutagenesis alone has produced over 3000 new varieties that are grown in some 60 countries around the world (Maluszynski et al., 2000). It is a moot point as to whether such long-standing intrusive breeding techniques carry intrinsically greater or lesser risks, or are more or less ‘natural’, than the more recently developed technologies of direct DNA manipulation via transgenesis.

12.3.4 Selecting for variation One of the major problems confronting breeders who are trying to produce crop varieties with modified lipid profiles is the identification of the desired

282 Modifying lipids for use in food lipids in seed populations that may number in the tens of thousands. Lipid analysis is a destructive technique, unless a small fragment of the seed tissue can be accurately assayed without damaging the rest of the seed. The development of routine gas–liquid chromatography (GLC) in the 1960s, and the later automation of this technology to allow for round-the-clock analyses, has greatly facilitated the task of the oilseed breeder. Another innovation, made possible by the sensitivity of GLC, is the use of half-seeds for analysis. This involves the dissection of a small fragment of seed tissue for analysis, while the rest of the seed is retained for germination to produce a new adult plant. This combination of methods was used in the breeding of the most successful new oil crop of the past half century, namely the canola varieties of oilseed rape as discussed below in Section 12.4.2. Some idea of the improvement in selection efficiency represented by the combination of the half-seed method and GLC analysis can be gleaned from the following figures. Prior to the development of GLC, it required about 200 000 whole seeds (1 kg) and two weeks to perform just one fatty acid analysis. Now, it is possible to analyze a fragment of a single seed, weighing just 2 mg, in 15 minutes. Thanks to this 650 million-fold improvement in analytical efficiency, it became feasible to accurately screen many thousands of seeds in the search for that most rare of events; a spontaneous mutation in just one or two genes in a genome that might contain from 25 000 to over 100 000 genes. Similar automated methods of analysis have now been developed for the mass screening of variants in other lipidic molecules of nutritional interest, ranging from sterols and carotenoids to tocopherols, lycopenes and xanthins. However, not all selection methods need to be high-tech in order to be effective. In the case of the new high linoleic linola variety of linseed described in Section 12.3.3, the tens of thousands of mutagenized seeds were initially screened using a method that was simple, rapid and very cheap, i.e. permanganate staining. Each seed was pressed lightly onto a strip of filter paper so that some of the oil was absorbed onto the paper. Next, the filter paper was dipped in a solution of potassium permanganate, which oxidized any α-linolenate in the absorbed seed oil to yield a purple colour. Nearly all of the seeds produced bright purple spots. However, the tiny number of seeds in which the α-linolenate genes had been mutagenized, produced a much lighter coloured spot that was instantly recognisable. These seeds were then grown up and the seed oils of their progeny were analyzed by the more accurate and quantitative method of GLC. The use of the preliminary permanganate screen saved the breeders from tens of thousands of relatively expensive and time-consuming GLC analyses. These screening tools allow breeders to select variants from much larger populations than was previously possible, thereby increasing the chances of identifying rare mutations that result in sometimes rather subtle, but nevertheless very useful, changes in lipid composition in a crop. As we have seen, such methods of selection have resulted in the development of at least two new edible oil crops, linola and canola, but similar approaches are now used

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across the seed industry for the selection of improved lipid profiles in all the major oil crops.

12.4 Oil crop modification 12.4.1 Introduction Since the early 1960s, several established oilseed crops have been successfully modified in order to improve their edible qualities, while in other cases new crops have been more or less developed from scratch. Such developments have been facilitated by the sorts of advances in plant breeding that were outlined in the previous section. Another important set of tools has been provided by improved analytical techniques that have allowed for the rapid and accurate mass screening of thousands of plant samples for possible changes in lipid composition. All of the major analytical techniques are chromatography- or spectroscopy-based, beginning with relatively crude and insensitive (but often useful) methods like column and thin-layer chromatography and progressing to GLC and mass spectroscopy.

12.4.2 Canola – a new oilseed crop Probably the most impressive example of the manipulation of an oilseed to enhance its edible performance was the modification of the existing high erucic form of oilseed rape in the 1960s to produce the current high oleic canola oil. Prior to this time, oilseed rape, and indeed all the other brassica species, produced a seed oil that consisted mainly of the very long-chain C22 erucic acid. In Europe and North America, this oil was normally used for non-edible purposes and oilseed rape was very much a minor crop with a limited and not very profitable market. In the 1960s, the Canadians were looking for new crops to grow on their huge prairie farms and one possibility was to breed an edible form of oilseed rape. Plant breeder Keith Downey led a small team in Saskatoon that was looking for a way to reduce the amount of erucic acid and instead to increase the amount of a much more useful fatty acid such as oleic acid (18:1). Oleic acid is the main ingredient of olive oil and is the premium monounsaturated fatty acid recommended by nutritionists. Downey’s genetic and biochemical analysis of rapeseed plants had already indicated that the unwanted erucic acid was formed by elongation of much more desirable oleic acid. If the elongation pathway could be disrupted, the seeds should accumulate large amounts of oleic acid and would be readily marketable for their new edible oil. The genetic analysis showed that this elongation pathway was controlled by only a few genes and was thus potentially amenable to manipulation by a classical breeding approach (Downey and Craig, 1964). All Downey’s group needed was a plant that had one or two mutations that prevented it from elongating oleic acid so that it accumulated oleic acid instead.

284 Modifying lipids for use in food By using the mass-screening method outlined in Section 12.3.3, it was eventually possible to find spontaneous mutants of conventional high erucic rapeseed that had lower levels of erucic in their seed oil. Because several genes were involved, it was necessary to cross some of the mutants with intermediate amounts of erucic to produce varieties with very low levels of the fatty acid. Although this took a few years of hard work, by 1964 the project was eventually rewarded with success as the team developed the first zero-erucic acid variety of oilseed rape, which they christened ‘canola’. The remarkable achievement of these breeders has been highlighted by recent advances in molecular genetics, which have allowed us to discover the exact nature of the mutations that the Canadian group selected in order to create the low erucic acid phenotype of modern rapeseed/canola. Their canola plants contain single point-mutations in two genes encoding isoforms of the enzyme β-ketoacyl CoA synthase: this protein is part of the fatty acid elongase complex now known to mediate the formation of erucic acid from oleate (Fourmann et al., 1998). This means that they succeeded in the alteration of just two nucleotides in a genome that contains over 1.2 billion nucleotides. It also demonstrates the power of genetics as applied to plant breeding. Such a result would be the envy of any latter-day biotechnologist and is a useful reminder that genetic engineering is not the only way to achieve the precise genetic manipulation of a crop. Since the late 1970s, canola has been a mainstay of Canadian prairie agriculture and a major export earner for the country. Canola-standard oilseed rape has also been adopted enthusiastically as an edible oil crop around the world with an annual value in excess of $6 billion (Oil World, 2005). Thanks to this small team of Canadian breeders, oilseed rape is now a globally important crop that is used to make salad oil, cooking oil, margarine, as well as being a key ingredient in all manner of food products from biscuits and cakes to curries and pies. It is also interesting to reflect that this single rather modest new crop, developed over about a decade by breeders, has already earned far in excess of all the profits of the agbiotech industry in the two decades after 1985 (James, 2005). Since the development of high oleic canola varieties in the 1960s, rapeseed has been improved further as an edible oil crop by reductions in its content of oxidation-prone linolenic acid so as to avoid the need for hydrogenation and the accumulation of trans fatty acids, as we will now discuss.

12.4.3 Other modified oilseed crops The need to reduce levels of trans fats in foods has driven breeders to develop several new high oleic varieties of the major oil crops since the 1990s. For example there are now commercial high oleic varieties of the ‘big-three’ oilseed crops, soy, rape and sunflower, all of which have been produced by conventional breeding. Efforts are also underway to produce a high oleic version of oil palm, both by screening for existing variation and

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by transgenic methods but, with such a slow growing tree crop, this process may take several decades. A particular attraction of high oleic, plant oils is that they have applications both as premium edible oils and as high-grade feedstocks for the manufacture of many oleochemicals. An example of the latter market is the use of high oleic soybean oils as biodegradable lubricating fluids that have relatively long working lives and low susceptibility to oxidation at high temperatures (Cahoon, 2003). High oleic soybean varieties with as much as 83 % oleate and less than 3 % α-linolenate have been developed (Rahman et al., 2001) and are now being marketed by major seed companies. Breeders have also developed other lines of soybean that have high levels of stearic acid (Rahman et al., 2003) and other nutritionally relevant fatty acids. Several high oleic canola/ rape varieties have been developed that typically contain about 70–80 % oleate, 15 % linoleate and only 3 % α-linolenate. Major seed companies such as Cargill, Dow Agrosciences and Bayer are now developing such varieties for various end-use markets in the edible and non-edible sectors. By 2004, high oleic rape/canola was already being planted on about 250 000 ha in Canada, which is 5 % of the total area of canola cultivation (AgCanada, 2004). Sunflower oil, once the high polyunsaturate edible oil par excellence, is also steadily being rebranded as a high oleate oil. Traditional sunflower oil consists of about 68 % linoleic acid, 20 % oleic acid and 10 % saturates, which means that hydrogenation is still necessary for many food uses. During the 1990s in the USA, there was great interest in very high oleic sunflower varieties (with 80 % oleic acid, 10 % polyunsaturates and 10 % saturates) that had already been developed by breeders. However, these varieties were only available to farmers in very limited quantities because of a patent on hybrid planting seed with oleic levels at or above 80 %. The patent holders chose not to license their breeding material to other companies; thus high oleic sunflower production was very limited and the price of the oil was quite high compared to commercial oils. By 1999, these problems were resolved with the development of a new intermediate oleate hybrid variety called NuSun™ (National Sunflower Association, USA), which was commercially launched in that year. NuSun oil contains about 65 % oleic, 25 % polyunsaturates and 10 % saturates, which does not require hydrogenation and works especially well in commercial frying applications. By 2001, over 200 000 tonnes of NuSun™ oil were being produced and the hoped-for commercial breakthrough came in the same year with the announcement that Proctor & Gamble would be using NuSun™ oil exclusively in its popular Pringles® line of potato chips (Kleingartner, 2002).

12.4.4 Advanced breeding for oil crop modification During the mid–late 20th century oil crop breeding was driven by advances in analytical technologies and by a vastly improved knowledge of lipid

286 Modifying lipids for use in food metabolism. Since the 1980s, the task of the oilseed breeder has been facilitated further by new techniques of advanced breeding, such as the use of DNAbased molecular markers, greatly improved tissue culture methods and more recently by the application of genomics and proteomics (Murphy, 2005). DNA marker-assisted selection Plant breeding has always relied on the selection of agronomically favourable characters from the diverse gene pool that is present in any crop species, even if many elite commercial cultivars tend to be highly inbred. Often these agronomic characters are visible and easily identified, e.g. height or flower colour or resistance to fungal attack. In other cases, the characters can be much more subtle and sometimes can only be measured by sophisticated analytical techniques, e.g. the amounts of certain secondary products or the fatty acid composition of the seed oil. In all of these cases, it was formerly necessary for the breeder to grow up and analyze each new generation before it was possible to measure the character, or phenotype, and select the appropriate plants. The advent of marker-assisted selection has changed this as breeders can now select a few plants that are likely to express the required characters from amongst tens of thousands of progeny even before the plants have developed to maturity. The basis of the method is DNA-fingerprinting and it is in principle no different from the methods used with such great effect in modern medical diagnostics or in forensic science (Gill et al., 1985). Molecular markers such as microsatellites, RFLPs (restriction fragment length polymorphisms) and RAPDs (random amplified polymorphic DNA) have now been developed for many oil crops, including trees like oil palm. These markers can be assembled into genetic maps that have considerable utility both in basic biological research and in commercial breeding programmes. The markers can be used to track the presence of valuable characters in large segregating populations as part of a crop-breeding programme. For example, if a useful trait like disease resistance, improved nutritional quality or higher yield can be linked with a specific marker, many hundreds or even thousands of young plantlets can be screened for the likely presence of the trait without the necessity of growing all the plants to maturity or doing costly and time-consuming physiological or biochemical assays. While the earlier molecular markers like RFLPs were relatively expensive, newer markers like microsatellites and SNPs (single nucleotide polymorphisms) are considerably cheaper and easier to use. The use of molecular markers can decrease the timescale of crop breeding programmes by several years and can substantially reduce costs. Although largely limited to the major temperate crops at present, the same technology can be applied to assist the breeding of any crop and even to domesticate entirely new crops. A good example of the potential for marker-assisted selection can be seen with edible tree crops, many of which are major export earners for developing countries. Examples of such crops include oil palm, coconut, coffee, tea, cocoa and the many commercial fruit tree species, such

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as bananas and mangoes. In the case of oil palm, the fruits that are the source of the palm oil are not produced for 5–7 years after planting. This means that a breeder must wait for at least 5–7 years before being able to determine the oil composition of a particular experimental cross. In contrast, breeders of annual crops have to wait only a few months before a plant like soy or oilseed rape sets seed. However, by using DNA markers, the oil palm breeder can now (in theory) identify whether new plantlets carry the required gene when they are only a few weeks old. This type of molecular marker technology is reasonably well developed for the major annual oilseed crops like soy or rape (Quiros and Paterson, 2004), but is still very much under development for more complex crops like oil palm (Basri et al., 2004). Tissue culture and mass-propagation The use of modern techniques of cell, tissue and organ culture is central to many crop improvement programmes in both industrialized and developing countries. Indeed the limiting step to the successful development of transgenic varieties of the major edible crops has not been transgene insertion itself, but rather the regeneration of viable plants from the transgenic explant material. Tissue culture has been widely used in crop breeding programmes since the 1950s (Phillips, 1993). For example, the use of embryo rescue techniques has enabled the incorporation of characters like disease resistance from wild relatives of crops into elite breeding lines. It is now possible to make wide crosses between hexaploid wheat and barley, rye or diploid wheat. The hybrids of such crosses are sometimes sterile due to embryo abortion but can be ‘rescued’ by culturing or transplanting the embryos. Another important technique that is increasingly used in crop breeding programmes is the production of doubled haploids. The repeated selection of heterozygous materials in a breeding programme can increase uniformity, but many generations are required to reach homozygosity in loci associated with agronomic traits. The artificial production of haploid plants followed by chromosome doubling offers the quickest method for developing homozygous breeding lines from heterozygous parental genotypes in a single generation. Haploid gamete cells from anthers or ovaries can be converted into diploids after colchicine treatment and then regenerated to yield doubled haploid plants. This technique is now used widely for the improvement of many of our most important oil crops, including maize, rapeseed and soybean (Forster et al., 2000). Yet another useful application of tissue culture methods is the mass clonal propagation of certain crops, in particular trees. Clonal propagation has not always been commercially successful, however. In the 1980s, a scheme to mass propagate millions of oil palm plantlets from a superior breeding line foundered when many of the maturing trees were discovered to have an abnormality in their floral development (Corley, 2000). This led to a failure of fruit formation and, since the major products of the crop are fruit oils, the trees were effectively useless. The abnormality is now known to be due to a

288 Modifying lipids for use in food tissue culture effect whereby the expression of homeotic genes regulating meristem identity is disrupted. Although the problem is now in the process of being rectified by further research, commercial confidence in clonal propagation has not recovered and relatively little commercial planting of clonal oil palm has been done since the late 1980s (Corley, 2000). The continuing scope for crop improvement, following the identification of higher yielding germplasm and its multiplication by mass-propagation, can be exemplified once again by considering the case of oil palm. Oleic acid rich oil from palm mesocarp is the most important edible oil crop produced in Asia. Moreover, palmkernel oil is also the most widely used oleochemical feedstock for the manufacture of detergents and other lauricbased products. Since the early 1980s, the average yield of Malaysian palm oil on plantations has more or less stagnated at 3.5–4.0 T.ha–1 (USDA, 2004). This is despite the availability of new clonal lines that can yield as much as 7.5 T.ha–1 (Ginting et al., 1995). Malaysia currently produces about 13.5 MT.yr–1 of palm oil worth an annual $4 billion: this is in a country with a total GNP (gross national product) of $60 billion. To put this figure of $4 billion into context, the estimated entire revenue generated by the US agbiotech sector in 1999 was just $2.3 billion – and this included all the companies supplying inputs to the sector or its employees (Ernst & Young, 2000). Therefore, the effective doubling of the palm oil yield that could be implemented following a successful mass-propagation programme could contribute a significant 6.6 % extra to the overall gross national product of this single Asian country. The application of a similar strategy with other tree crops, or even relatively undomesticated annual crops, could also yield equally striking results that would particularly benefit developing countries. Genomics Genomics is the term given to the massively parallel study of the DNA and protein sequences in an organism and the specification of when and where such sequences are expressed. However, genomics is much more than the mere assembly of DNA or protein sequence information or gene expression catalogues. It can also be used a tool in crop breeding programmes and even for the domestication of new plant species as future crops. Many characteristics of agricultural importance in crop plants, including some fatty acid traits, appear to be regulated by a large number of genes and therefore do not segregate into simple Mendelian ratios, as would be expected if only one or two genes were involved. Examples of such complex traits include height, branching, seed oil and protein yield, oil quality and flowering time. The use of more sophisticated genomic tools since the 1990s has shown us that, although dozens of genes may underlie such complex traits, sometimes much of the variation in their phenotypic expression can be caused by a small number of key regulatory genes. These genes can now be identified and mapped based on sequence similarities, expression profiles and molecular markers. Genes that play a major role in regulating agronomically relevant

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complex traits in model plants like Arabidopsis, and also some crops like maize, are now being isolated at an ever-increasing pace. Examples include height, flowering time, vernalization, shattering of seed pods and stem branching. Many of these plant genes encode transcription factor proteins that in turn regulate the expression of large sets of other genes. For example, transcription factors can switch on entire metabolic pathways or patterns of cell division, resulting in the formation of new tissues or organs and the accumulation of new storage products (Murphy, 1998a). The application of information from genomics is enabling crop scientists to identify key genes that regulate the accumulation of specific lipids that breeders may wish to either eliminate (e.g. α-linolenate for trans-free foods) or up-regulate (e.g. oleate for high-monounsaturate oils). This information can then be used to develop DNA-based markers for marker-assisted selection as described above.

12.5 Transgenic oil crop modification Transgenesis is the addition of exogenous (i.e. externally derived) DNA sequences and their incorporation into the genome of a recipient organism, such as a plant or animal. In the case of plants, the DNA can be added to cells directly by propelling small gold particles coated with DNA into plant tissues. This technique, called biolistics, can be used for any plant, crop or otherwise, but is a rather hit-and-miss affair that does not always result in the incorporation of the DNA into the plant genome. Alternatively, the DNA can be added in a more controlled fashion by means of a bacterial vector, e.g. Agrobacterium tumefaciens, that is able to insert a specific region of DNA into the genome of the plant. Even with their uncertainties, both of these methods of DNA transfer, or transgenesis, are much more efficient than alternative methods of crop genetic manipulation, such as induced mutation or wide crosses. For example, as discussed in Section 12.3.3, the creation of new crop varieties via radiation, chemical or somaclonal mutagenesis normally involves the repeated treatment of tens of thousands of tissue explants or seeds. These extremely drastic procedures result in hundreds of mutated genes, nearly all of which will be undesirable, and perhaps even lethal, to the crop. It then takes many years of backcrossing and selection to obtain a plant that carries a mutation in the desired gene(s), but not in other essential genes. Even then, it is still possible that there may be undetected hidden, or cryptic, mutations that only manifest themselves in later generations as the crop is tested or grown in commercial cultivation. Another significant drawback of mutagenesis is that the breeder can only manipulate genes that already exist in the crop genome. Furthermore, nearly all mutations result in a loss of gene function so mutagenesis is nearly always about reducing the effect of unwanted genes, rather than increasing the expression of desirable genes.

290 Modifying lipids for use in food These drawbacks in the existing technology of creating variation in plants made the prospect of a new and more direct method very appealing to plant researchers. The first experimental transgenic plant cells were produced by several European and US groups in 1983 (Barton et al., 1983; Caplan et al., 1983). By 1987, the commercial utility of the technology was demonstrated when it was shown that copies of a bacterial gene could be transferred to plants and thereby confer resistance to certain insect pests. In 1992, the first transgenic crop, the Flavr Savr® (Calgene Inc, USA, now part of Monsanto) tomato, was released in the USA. This tomato variety was not successful, however, mainly due to the use of poorly performing breeding lines and commercial mismanagement (Martineau, 2001). As detailed below, several transgenic oilseeds were developed at this time, but were also commercial failures. However, the next group of transgenic crops, that have been released on a steadily increasing scale after 1996, proved to be much more successful. Four major crops, soybean, maize, oilseed rape and cotton, have been bred to express two groups of simple traits, namely herbicide tolerance and insect resistance. For the first five years or so, commercial cultivation of these transgenically bred crops was largely limited to North America, but the technology has now been adopted more widely, especially in South America and China.

12.5.1 The concept of ‘designer oil crops’ During the late 1980s and early 1990s, oilseeds like soy and rape were at the forefront of attempts to produce commercial transgenic (‘genetically-modified’) crops. These efforts were pioneered by researchers in small biotech companies, such as Calgene, as well as the large multinationals, such as DuPont, Monsanto and Zeneca (now Syngenta). Part of the rationale for these efforts was a belief that fatty acid biosynthesis was well understood at the biochemical level and that relatively few genes would be needed to effect substantial changes in oil profiles in a seed. This led to the concept of ‘designer oil crops’ as described in the book of the same name that appeared in 1994 (Murphy, 1994). As pointed out at that time, there are three major challenges that confront those who wish to use transgenesis to modify plant lipid composition. These are to ensure that the new transgene is only expressed in the appropriate place (normally the fruit or seed); to ensure that the novel fatty acid is segregated into storage lipids and away from membrane lipids; and to ensure that the transgenic crop varieties and their products are adequately segregated from other identical-looking varieties of the same crop that are producing a different oil. In those optimistic days of the early 1990s, oilseed rape was often talked about as an archetypical ‘designer oil crop’ that could be engineered to produce as many as a dozen different oils to supply products ranging from margarines and pharmaceuticals to bioplastics and lubricants (Murphy, 1994). Despite the initial optimism of many researchers (including the author),

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the use of agbiotech to manipulate the fatty acyl composition of oils has turned out to be more complex than was first thought. Indeed, recent findings suggest that our understanding of even the basic pathway of triacylglycerol oil biosynthesis is far from complete and that there are probably several alternative parallel biosynthetic pathways rather than just one (Murphy, 2004). The consequence of these complexities of plant lipid metabolism has been that, although there have been many impressive achievements in isolating oil-related genes and producing transgenic plants with modified oil compositions, it has not yet been possible to achieve the kind of high levels, i.e. 80–90 %, of novel fatty acids that will make possible their widespread commercial exploitation (Murphy, 1999). As of the 2006 season, there were no significant commercial plantings of transgenic oil crops with modified fatty acid profiles. Although there were over 90 Mha of transgenic oilseeds such as soybean, canola, maize and cotton planted in 2005, these varieties were all modified to carry input-related genes involved in herbicide tolerance or insect resistance traits, rather than alterations in seed oil content (James, 2005). The only commercially grown transgenic crop with modified seed oil is the ‘laurical’ variety of canola (rapeseed) originally marketed by Calgene in 1995. From an original level of 40 % lauric acid newer ‘laurical’ varieties have been produced with 40–60 % lauric acid by the insertion of several additional transgenes (Voelker et al., 1996). However, this crop remains far from being a commercial success and cannot compete with cheaper tropical lauric oils from coconut and oil palm. Many genes that regulate the formation and accumulation of other exotic, non-edible fatty acids were isolated during the 1990s, including hydroxylases, conjugases, desaturases and epoxidases, but so far it has not been possible to use any of these genes to effect the production of any commercially useful oils in transgenic plants. Two of the major challenges facing designer oil crops are to prevent the novel fatty acids from leaking into cellular membranes and to segregate the seeds and oils during cultivation and processing, as we will now consider.

12.5.2 Segregation of novel fatty acids from membrane lipids The cellular membranes of all organisms are crucial to their metabolism and survival. Biological membranes are made up of a lipid bilayer into which are embedded the many proteins that mediate such processes as transport, respiration, photosynthesis and signal transduction. The fatty acid composition of a given membrane is closely regulated and the presence of inappropriate acyl groups often leads to serious disruption of membrane, and hence cellular, function. In plants that accumulate fatty acids that differ significantly from those of membrane lipids, specific mechanisms have evolved that prevent the ‘leakage’ of unwanted acyl groups into membrane lipid pools. Biochemically speaking, this is not a trivial task, because storage lipids and many membrane lipids are assembled on the same organelle – the endoplasmic reticulum.

292 Modifying lipids for use in food As yet, we do not completely understand the mechanisms by which some plants are able to segregate unusual fatty acids away from membranes (Millar et al., 2000). We know that this mechanism is very important because the leakage of a novel fatty acid, namely the saturated species, stearic acid, resulted in very poor seed germination rates in a transgenic variety of oilseed rape that had been engineered to have an elevated stearate oil for use in the manufacture of edible spreads (Thompson and Li, 1997). In this study, it was found that a small amount of stearate had leaked into cell membranes, resulting in a reduction in membrane fluidity and impairment of function that affected the development of the entire plant. Although, in this case, the transgenic rape seeds only accumulated about 40 % stearate and just 3–5 % leaked into cell membranes, other oilseeds like mangosteen (Garcinia cornea) can accumulate over 65 % stearate in their seed oil without any detectable leakage into cell membranes. Two hypotheses have been proposed to explain the sort of lipid segregation that plants like mangosteen seem capable of, but which seems to be lacking in major oil crops like oilseed rape. The first hypothesis is that there is a compartmentation of membrane and storage lipid synthesis in specific membrane domains of the endoplasmic reticulum. The other hypothesis is that there is a selective accumulation of the novel fatty acids in triacylglycerols after synthesis (Millar et al., 2000). Research is currently underway to address these issues but, until we understand more about fatty acid segregation, the production of most exotic fatty acids in transgenic crops will remain more of an aspiration than a reality (Thelen and Ohlrogge, 2002).

12.5.3 Segregation of transgenic crops from other crops Unless a transgenic variety of an oil crop completely replaces all non-transgenic varieties of the same crop, it will require complete segregation at every stage of production from seed storage and planting to harvesting and downstream processing. This can add at least 10–20 % to costs and imposes considerable (and often overlooked or under-estimated) management problems. These challenges can be rather formidable, given the complexity of the supply chain from breeder to grower to crusher to processor and so on, all the way to the retailer and ultimately to the consumer. The difficulties in ensuring strict segregation of otherwise indistinguishable transgenic crops have been pointed out (Murphy, 1994, 1996) but have consistently been under-estimated by many in the industry. However, several well-publicized failures in the segregation of transgenic crops since 2000 have thrown this issue into much sharper focus. The latest episode in a growing list of such failures involved a variety of transgenic maize that was widely grown for several years in the USA (and exported for human consumption to Europe) although it had not been granted regulatory approval (Macilwain, 2005). Such incidents do not inspire confidence in the ability of sections of industry to regulate and manage themselves.

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They have also called into question the wisdom of growing crop varieties that contain therapeutically active compounds alongside major commodity varieties of the same crop. An example would be a maize variety engineered to produce high levels of omega-3 fatty acids growing in the same region as regular maize. However, these management problems can and should be resolved. After all, since the late 1970s, UK farmers have been growing two (non-transgenic) oilseed rape varieties that produce respectively a major edible commodity oil and an industrial oil that is prohibited for human consumption. Despite the potential for cross-pollination between the two crops and the fact that over 400 000 ha of rape is grown in the UK, careful management by farmers, seed crushers and oil processors has ensured that there have been no reported instances of cross-contamination. So, segregation can work, but everybody in the food chain must cooperate to ensure that strict standards are observed.

12.6 Plant lipid manipulation in the 21st century There are two apparently contradictory trends in the current development of plant lipids for edible production. First, there has been a huge consolidation and concentration of both the major oil crops and the suppliers of plant material for growing such crops. This trend leads to a more generic, commoditybased market. Second, however, there is a trend towards segmentation and identity preservation of individual oils that may carry a considerable price premium. These divergent tendencies are driven by different forces that favour the cheapest generic oils, especially for use in mass-produced processed food, on one hand, while also favouring the creation of niche products with specialized identity-preserved oil compositions that are often based on health claims on the other. The speed with which such dietary fads can come and go makes it difficult for breeders to produce appropriate varieties, especially given the decades-long timescale of most breeding programmes. For example, since the 1980s, we have gone from advice to reduce all dietary saturates, then to increase polyunsaturates, then monounsaturates were favoured, and now omega-3 acids are the ‘flavour of the month’ for fatty acid nutritionists. The most recent consumer interest seems to be in high-monounsaturate oils and in omega-3 acid oils, ideally with some lipophilic antioxidants like tocols or carotenoids thrown in for good measure. Plant oils with each of these profiles are currently being developed by breeders, as we will now see.

12.6.1 Low-trans oils The market for oils that contain reduced or zero levels of trans fatty acids is currently driven by health concerns that have led to the imposition of labelling requirements revealing whether a product contains over a given threshold of these fatty acids. Such labelling requirements were introduced into the USA

294 Modifying lipids for use in food on 1 January 2006, and are likely to be required in the European Union in the near future. Typical threshold levels of trans fatty acids that would trigger compulsory labelling are in the region of 0.5–1.0 %, whereas some existing foods can contain as much as 40 % trans fatty acids. The solution in most cases will be to develop high oleic oil crops and, as we have seen above, breeders have been gradually producing such varieties of the major oilseeds over the past decades. There are still challenges for breeders in attempting to reduce further, or to eliminate altogether, α-linolenate from seed oils and to ensure that the high oleic traits are crossed into their highest performing elite commercial lines. For most crops, it is possible to produce high oleic varieties by nontransgenic routes and, given the sentiment about GM crops in Europe, such a route is obviously preferable at the present time. Given the huge potential market for foodstuffs that are free of trans fatty acids, it is possible that high oleic oil crops will become the mainstream commodities over the next decade, rather than existing polyunsaturate-rich varieties. Such a development would certainly be welcome to processors as it would obviate the need to segregate the oil, but it would be less well received by farmers and seed suppliers who would lose their premium for a value-added variety. The types of product, e.g. a spread or a liquid oil, that would result from a high monounsaturate plant variety would also be potentially suitable for the delivery of omega-3 fatty acids, as long as they were kept down to about 10 % to avoid oxidation problems. However, if customers required higher levels of omega-fatty acids, e.g. in a neutraceutical format, it would be better to produce dedicated high omega-3 oil crops. We will now look at the rationale for, and recent progress towards, this objective.

12.6.2 Can plants substitute for ‘fish oils’? Oils rich in omega-3 fatty acids include the so-called ‘fish oils’ (or more correctly ‘marine oils’), which are characterized by relatively high levels of very long-chain polyunsaturated fatty acids (VLCPUFAs) such as eicosapentaenoic acid (20:5ω-3, EPA) and docosahexaenoic acid (22:6ω-3, DHA). These compounds are part of the group of omega-3 fatty acids that are essential components of mammalian cell membranes, as well as being precursors of the biologically active eicosanoids and docosanoids (Funk, 2001; Hong et al., 2003). There have been numerous reports concerning the importance of dietary supplementation with these fatty acids for human health and well being. For example, dietary VLCPUFAs have been shown to confer protection against common chronic diseases such as cardiovascular disease, metabolic syndrome and inflammatory disorders, as well as enhancing the performance of the eyes, brain and nervous system (Crawford et al., 1997; Spector, 1999; Benatti et al., 2004). It is worth mentioning here that none of these VLCPUFAs are strictly ‘essential’ in the diet in the same way that vitamins are. The only unequivocally essential fatty acid is cis 9, 12

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linoleic acid, an omega-6 fatty acid that mammals are unable to synthesize. All of the VLCPUFAs can be synthesized from linoleic acid in a wellnourished and healthy individual. Unfortunately, most Western diets do not provide an adequately balanced fatty acid intake and this often necessitates the inclusion of VLCPUFAs to maintain optimum health. Consumption of fish is therefore currently recommended in most Western countries to provide a balanced diet, and much of the nutritional benefit of the fish actually comes from the VLCPUFAs of the fish oils. These fatty acids are not only synthesized by the fish themselves, but are also derived from micro-organisms, especially photosynthetic micro-algae, that are ingested as part of their diet. As an alternative to fish consumption, therefore, it is possible to purchase VLCPUFA dietary supplements that are derived from cultured micro-algae or fungi. However, low oil yields and high costs of oil extraction have limited the scope for this production method, and everdwindling fish stocks are also threatening supplies of the main source of marine oils. This situation has led to renewed interest in the possibility of breeding oilseed crops that are capable of producing significant quantities of VLCPUFAs in their storage oils. Higher plants do not normally accumulate such fatty acids, but they can accumulate C22 and C24 monounsaturates and C18 polyunsaturates in their seed oils, so it seemed possible that C20 and C22 polyunsaturates might also be accumulated providing the plants were able to synthesize these fatty acids. The most serious of several technical challenges to the engineering of VLCPUFA production in plants is the number of enzymes that are needed for the conversion of a typical plant C18 PUFA, such as linoleate or linolenate, to the C20 and C22 VLCPUFAs with up to six double bonds that are characteristic of fish oils, as shown in Fig. 12.1. Other key challenges are similar to those that have confronted previous attempts to engineer transgenic oilseed, namely to ensure seed-specific expression of the transgenes and to channel the novel fatty acids towards oil accumulation and away from membrane lipids. During 2004 and 2005, there were several reports that encourage the view that the economic production of VLCPUFAs in transgenic plants might be possible (Abbadi et al., 2004; Qi et al., 2004; Wu et al., 2005). In one rather heroic experiment, no fewer than nine genes from various fungi, algae and higher plants were inserted into the oilseed, Brassica juncea, with the resultant accumulation of as much as 25 % arachidonic acid and 15 % eicosapentaenoic acid (Wu et al., 2005).

12.6.3 Transgenic oil palm – the emerging behemoth? Although the three major edible oilseed crops have been produced in steadily increasing amounts since the 1980s, their rate of growth is dwarfed by that of oil palm. From being a very minor crop in the 1960s, palm oil production has steadily increased until in the 2005–06 season it finally caught up with soy oil (the long-time industry leader) as the major global source of plant

296 Modifying lipids for use in food ω-6 (∆ ∆6) pathway Oleic acid ∆12 desaturase Linoleic acid

ω-3 (∆ ∆3) pathway

18:1 n-9 ∆15 desaturase 18:2 n-6

18:3 n-3

α-Linolenic acid

18:4 n-3

SDA

20:4 n-3

ETA

20:5 n-3

EPA

∆6 desaturase GLA

18:3 n-6

DGLA

20:3 n-6

AA

20:4 n-6

∆6 desaturase

∆6 desaturase

∆5 elongase 22:5 n-3

DPA

∆4 desaturase 22:6 n-3

DHA

Fig. 12.1 Biosynthetic pathways for very long chain polyunsaturated fatty acids. This figure shows the complexity of the various pathways involved in the synthesis of the kinds of omega-6-and omega-3-enriched ‘marine oils’ that various groups are attempting to engineer into oil crops. The starting substrates are linoleic acid and α-linolenic acid (ALA) in the omega-3 and omega-6 pathways, respectively. The ∆6 desaturation of LA and ALA gives rise to γ-linolenic acid (GLA) and stearidonic acid (SDA). Arachidonic acid (AA) and eicosapentaenoic acid (EPA) are synthesized by further ∆6 fatty acid elongation and ∆5 desaturation steps. An omega-3 desaturase interconnects the omega-6 and omega-3 pathways for more efficient EPA production. Finally EPA is elongated by a ∆5 fatty acid elongase and desaturated by a ∆4 desaturase to produce docosahexaenoic acid (DHA). Other abbreviations are: DGLA = dihomo-γ-linolenic acid, ETA = eicosatetraenoic acid and DPA = docosapentaenoic acid.

oils. Annual production of edible palm oil in 2005–6 was in excess of 34.6 MT (USDA, 2005), with China and Europe as the principal importers. An additional 3.7 MT of palmkernel oil was also produced in 2005. Although most of this high lauric oil is used in non-edible applications such as detergents, some of it is also used in high-energy sports drinks and in infant formulations. What is most impressive about oil palm, however, is not its current production figures but its future potential as the world’s main source of edible and nonedible oils. The average mature oil palm plantation currently yields about 3.5–4.0 T.ha–1, bears fruit throughout the year and lasts for 25–30 years of productive life. Moreover, there are higher yielding cultivars that already produce over 10 T.ha–1 and individual trees have been identified that could be clonally propagated, and which yield the equivalent of over 50 T.ha–1. In contrast, a typical oilseed rape or sunflower crop needs to be replanted each year, can only be harvested during a short period in summer and yields a

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paltry 0.5–1.0 T.ha–1. Yields of soy oil are even lower at about 0.3 T.ha–1. This means that if we satisfied the world’s annual plant oil requirement of 120 MT by only growing oil palm at 10 T.ha–1 instead of temperate oilseed crops, we could save as much as 70–80 million hectares for other uses. Oil palm is also a much more economic crop that has a lower environmental impact than the other oil crops. The main thing that is holding back oil palm from competing in the developing niche markets for added-value edible oils is the difficulty in manipulating its fatty acid profile. Palm oil has a relatively high content of the saturate, palmitic acid, as well as the more desirable oleic acid. This means that, to date, the food industry has treated palm oil as a relatively lowvalue generic commodity. For example, the April 2005 oil prices in Rotterdam (the main global spot market) showed palm oil trading at a discount of $100– $200 per tonne compared to its major rivals, soy, rape and sunflower (Oil World, 2005). However, unrefined palm oil is also characterized by high level of nutritionally desirable carotenoids and tocols that give this oil an attractive but, unusual (for a vegetable oil), red colour. The renewed interest in these antioxidant and otherwise desirable lipids has led to attempts to market red palm oil as a high-value niche product in the health food sector (see more on these non-acyl lipids in the next section). Despite these developments, palm oil is still something of a Cinderella product in some quarters. There are two factors that may enable breeders to turn this position around for the oil palm industry. First, worldwide sampling studies are beginning to show more genetic diversity in fatty acid profiles than had been suspected hitherto. Coupled with improving breeding and clonal propagation methods, this may mean that new varieties of oil palm, including high oleate genotypes, might be developed in the coming decades. The second development is the use of transgenesis to produce ‘designer’ oils from this crop. Although such studies are still at an early stage, efforts are proceeding to produce a new transgenic high oleate variety, as well as several varieties aimed at the market for renewable and biodegradable industrial feedstocks. These programmes may take several decades to come to fruition, but oil palm is certainly an interesting crop to watch for the future.

12.6.4

Modification of non-acyl lipids in plants

Golden rice Probably the best-known recent example of a nutritionally enhanced crop is the development of the transgenic ‘golden rice’ by a Swiss-based group (Ye et al., 2000). The grains of this GM rice variety are yellow because they have been engineered to accumulate the lipophilic pigment, β-carotene (pro-vitamin A), which is normally absent from rice grains. The transgenic rice contains three inserted genes encoding the enzymes responsible for conversion of

298 Modifying lipids for use in food geranylgeranyl diphosphate to β-carotene. It is claimed that consumption of this rice by at-risk populations may alleviate the vitamin A deficiency (leading to night blindness) that currently afflicts some 124 million children worldwide. Such claims are hotly disputed by anti-GM groups (e.g. Greenpeace http:// www.greenpeace.org/~geneng/) and the ‘golden rice’ has yet to prove itself in large-scale field and nutritional trials in the target developing countries. Interestingly, the rights for the commercial exploitation of ‘golden rice’ in developed countries, including the USA and Europe, have now been acquired by Syngenta. It is possible that this could lead to the marketing of ‘vitaminenhanced’ food products derived from golden rice, e.g. in breakfast cereals, which may be more acceptable to the public than the current generation of food from input trait modified GM crops. One of the reservations expressed about the original varieties of golden rice was the relatively low content of pro-vitamin A, which might require the daily consumption of several kilograms of rice to meet dietary requirements for vitamin A. More recently, this problem has been solved by replacing the daffodil phytoene synthase gene in the original varieties of transgenic golden rice with a similar gene from maize. Use of the maize transgene in rice led to a 23-fold increase in pro-vitamin A levels and grains of ‘golden’ rice that were bright orange, rather than an insipid yellow (Paine et al., 2005). This improved variety of golden rice still has many years of backcrossing into local varieties and field testing before it will be known whether it is a viable crop. Not the least of the challenges is to ensure that the pro-vitamin A is in a form that can withstand processing, storage and cooking, and is also completely bioavailable following consumption by people. There are many cases of vitamins and mineral nutrients that are either lost during postharvest treatments or pass through the digestive system, e.g. due to chelation or other forms of complexing. Probably the best-known example of this is spinach, where only 2 % of the iron is actually bioavailable due to the presence of oxalates – sadly, a real-life Popeye would not garner much strength from canned spinach! (Although spinach is not a good provider of dietary iron, it is an excellent source of omega-3 polyunsaturates, due to the α-linolenic acid in its abundant thylakoid membranes.) Notwithstanding the many challenges that face golden rice, the development of several new cultivars is well underway in Asia and this crop may yet have a modest impact on human nutrition in some parts of the world. The vitamin E group The vitamin E group of compounds includes four tocopherols and four tocotrienols, all of which have significant antioxidant properties. These lipophilic vitamins can be found in most non-processed seed oils but are often lacking in foods made from processed oils. There is interest in trying to increase the levels of this group of lipidic vitamins in plant oils using a variety of approaches. For example, transgenic plants that accumulate 10–15 fold higher levels of vitamin E compounds have been engineered by adding

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homogentisic acid geranylgeranyl transferase genes from several cereals to Arabidopsis plants (Cahoon et al., 2003). As described above, unrefined palm oil also contains significant amounts of vitamin E group compounds (Han et al., 2004). Since 2000, several varieties of oil palm have been identified that produce oil that is highly enriched in tocols to levels in excess of 1500 ppm, which would be of great interest as potential health food products. Phytosterols and stanols Another category of plant lipid of interest to the food industry is the phytosterols. Margarines enriched in phytosterols extracted from (non-transgenic) wood pulp or vegetable oils have recently been marketed and, despite an appreciable price premium compared to conventional margarines, they have enjoyed modest commercial success. The appeal of the phytosterol-enriched margarines is based on evidence that they may help to reduce blood cholesterol levels and hence combat heart disease. Such products could be made more cheaply if more of the phytosterols were synthesized in the same seeds as the oil from which the margarine is derived. Efforts are now underway to up-regulate phytosterol biosynthetic pathways in transgenic plants. The impact on human health of such products could be considerable. Indeed, it has been surmised that the widespread availability and consumption of low-cost, phytosterolenriched margarines could eventually lead to a quantifiable reduction in national rates of cardiovascular disease, which is still the most common cause of mortality, especially in low-income groups, in all industrial societies (Plat and Mensink, 2001).

12.7 Future trends We have seen that modern plant breeding technologies are capable of providing a wide spectrum of altered lipid profiles in plant oils that can potentially satisfy the demands of what has been a rather fickle and inconstant food industry. As the industry produces an ever wider range of foodstuffs that are targeted at different sorts of customer (e.g. health foods, organics, highquality ranges, cheaper ranges, etc.) it will become more economic for specific plant oils to be segregated and identity preserved for certain applications. This in turn will signal plant breeders to focus on satisfying such niche markets. It seems likely that we will gradually move away from the current low-value commodity oils that are bought in bulk and blended to order, and towards a more sophisticated segmented market that will include addedvalue oils from transgenic crops. Examples of the latter will include oils that are highly enriched in nutritionally desirable lipids, such as long-chain omega3 fatty acids and antioxidants such as carotenoids and tocols. There are two additional developments that may perturb the present market structure, namely biodiesel and oil palm. The potential impact of new high-

300 Modifying lipids for use in food yielding oil palm varieties has already been addressed in Section 12.6.4 and this may affect the industry very significantly in the long term. A shorter term trend that is already having an impact is the increasing diversion of oil crops away from food markets towards biodiesel production (Murphy, 1998b). This is beginning to affect the prices of plant oils and may have even more effect as Europe in particular seeks to use this renewable biofuel to help fulfil its obligations under the Kyoto agreement. In the longer term, however, it seems unlikely that society will countenance such a profligate waste by burning of a semi-refined product that can serve as either a nutritious food or a source of valuable oleochemicals. Besides, even if we used all of the available arable land on the earth for biodiesel crops, we would still only produce a fraction of the fuel needed to sustain current rates of usage for transportation. It is more likely that biodiesel will be a significant, but temporary, perturbation in the use of oil crops and that in the longer term, much of the production of such crops will shift to more efficient tropical systems such as oil palm.

12.8 Sources of further information and advice Links to databases and other resources for plant lipids (much of this information comes from the Lipid Analysis Unit site, as listed below) General lipids •

• • • • • •

The American Oil Chemists’ Society – the largest global society of oils and fats chemists, technologists and biologists with much useful information on nutrition and labelling (www.aocs.org). Of particular interest to the general reader is their monthly magazine, INFORM (http://www.aocs.org/press/inform/) and the journals JAOCS and Lipids. The European Federation for the Science and Technology of Lipids (http://www.eurofedlipid.org/index.htm) publishes the European Journal of Lipid Science and Technology (www.ejlst.de) and organizes conferences. Cyberlipid – a website containing much useful information on lipid chemistry, biochemistry and analysis (http://www.cyberlipid.org/). Lipid Nomenclature – this is the IUPAC guide http:// www.chem.qmw.ac.uk/iupac/lipid. Conjugated linoleic acid – Wisconsin Food Research Institute (http:// www.wisc.edu/fri/clarefs.htm). These pages give a comprehensive list of references to papers dealing with CLA. Compilation of trivial names of fatty acids (by RO Adlof and FD Gunstone) (http://www.aocs.org/member/division/analytic/fanames.htm). Lipidat – a relational database of thermodynamic and associated information on lipid mesophase and crystal polymorphic transitions, including lipid molecular structures (glycero- and sphingolipids) http:// www.lipidat.chemistry.ohio-state.edu/.

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Lipid Bank for Web – a database of information on lipid structures and properties with thousands of references (http://lipid.bio.m.u-tokyo.ac. jp/). Lipid Analysis Unit – this site is a general information resource on lipids supported by the Lipid Analysis Unit at the Scottish Crop Research Institute (http://www.lipid.co.uk).

Plant lipids •

•

• •

• •

On-line chemical data base for new seed crops produced by the New Crop Research Unit at NCAUR, Peoria, IL, USA (http:// www.ncaur.usda.gov/nc/ncdb/search.html-ssi/) (see Abbot et al., J Am Oil Chem Soc, 74, 723–726 (1997) and correction on p. 1181 for instructions) – chromatographic, physical chemical and spectroscopic information on oil seeds. Similar but more extensive database to the one previously provided by BAGKF (Institute for Chemistry and Physics of Lipids), Munster, Germany – SOFA (Seed Oil Fatty Acids) (www.bagkf.de/sofa) (see Aitzetmuller et al., Eur J Lipid Sci Technol, 105, 92–103 (2003)). A catalogue of genes for plant lipid biosynthesis at Michigan State University (http://www.canr.msu.edu/lgc/index.html). NPLC (National Plant Lipid Cooperative) (http://www.msu.edu/user/ ohlrogge/). A further source of links to web-based lipid information, includes: NPLC Directory of Plant Lipid Scientists, The NPLC Electronic Mailing List, The NPLC Database of Plant Lipid Literature. The Plant Lipids Home Page (http://blue.butler.edu/~kschmid/lipids.html). Maintained by Katherine Schmid this page contains many useful lipidrelated links. The Malaysian Palm Oil Board (MPOB) – website devoted to all aspects of oil palm biology, technology, food and non-food uses and commercial matters (http://www.mpob.gov.my).

Food and industry related •

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Loders Croklaan, once a subsidiary of Unilever Ltd, is now part of the IOI group in Malaysia. The site covers science, technology and nutrition related to lipids in general and to oil palm products in particular (www.croklaan.com). ITERG – French Research Institute dealing with oils and fats research and technology (www.iterg.com). Natural of Norway – manufacturers of conjugated linoleic acid for health food and other applications (www.natural.no). The website of the British Nutrition Foundation carries information on lipids in addition to other food components (www.nutrition.org.uk).

302 Modifying lipids for use in food • • • • • • •

• • • •

USDA – US government site with information on fat-related nutritional advice (http://www.nalusda.gov/fnic/dga/dga95/lowfat.html). CTVO – a European group devoted to the chemical and technological utilization of vegetable oils (www.danet.de/fnr/ctvo). IENICA – an Interactive European Network for Industrial Crops and their Applications (www.csl.gov.uk/ienica/). ACTIN – a UK group devoted to non-food uses of oils and fats (www.actin.co.uk). European website for the American Soybean Association (www.asaeurope.org). International Food Science and Technology – contains information on various food problems including those involving lipids (www.ifst.org). Oil World is a German company producing data on a weekly basis for oilseeds, oils and fats and oil meals and covers production, imports, exports and disappearance. Information is based on different commodity oils and fats and is presented on the basis of individual countries (www.oilworld.de). FFA Sciences is a company manufacturing probes to measure free fatty acid levels in oils and clinical samples (www.ffasciences.com). Britannia Foods has some articles ‘By Invitation Only’ of interest to lipid technologists mainly – (www.britanniafood.com). Peter Lapinskas – consultant to the oils & fats industry – some interesting data on unusual seed oils (www.lapinskas.com). Plant Lipids – an Indian company specializing in products derived from a range of plant lipids (http://www.plantlipids.com).

Lipidomics-related websites • • • • •

http://www.ksu.edu/lipid/lipidomics http://hcc.musc.edu/research/shared_resources/lipidomics.cfm http://medschool.mc.vanderbilt.edu/brownlab/comlip.html h t t p : / / w w w. w i s s e n s c h a f t - o n l i n e . d e / g b m / h o m e p a g e / abstract_detail.php?artikel_id=265 http://www1.elsevier.com/gej-ng/29/50/lipids/119/47/26/article.pdf

12.9

References

ABBADI A, DOMERGUE F, BAUER J, NAPIER JA, WELTI R, ZAHRINGER U, CIRPUS P

and HEINZ E (2004), Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation, Plant Cell, 16, 2734–2748. AGCANADA (2004), The United States Canola Industry: Situation and Outlook, Agriculture and Agri-Food Canada Bi-weekly Bulletin, February 27, 17(4), available at http:// www.agr.gc.ca/mad-dam/e/bulletine/v17e/v17n04_e.htm. BARTON KA, BINNS AN, MATZKE AJM and CHILTON MD (1983), Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission to R1 progeny, Cell, 32, 1033–1043.

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13 Modifying fats of animal origin for use in food M. R. L. Scheeder, ETH Zurich, Switzerland

13.1

Introduction

Fatty acids in animal lipids originate from endogenous de novo synthesis, from dietary sources or from microbial synthesis and modification in the digestive tract. The essential polyunsaturated C18 n-6 and n-3 fatty acids cannot be synthesized by vertebrate organisms and are exclusively supplied by the diet. Subsequent modification of fatty acids occurs in the digestive tract by microbes or after absorption by endogenously produced enzymes. The activity of endogenous enzymes can in turn be affected by specific fatty acids. Therefore, genetic disposition for de novo synthesis and enzyme activity, diet composition, microbial activity and the interactions between these factors contribute to the highly complex fatty acid composition of animal lipids. These factors also provide manifold opportunities for considerable biomodification of animal lipids. However, desired objectives may be very different. One can be to increase the content of potentially health-beneficial bioactive fatty acids and to decrease saturated fatty acids which are still considered to be potential health risk factors. On the other hand, typical and desired technological properties of animal fats, such as oxidative stability, firmness and plasticity, depend on the presence of a certain amount of saturated fatty acids and may be adversely affected by (poly)unsaturated fatty acids. The challenge, therefore, is to increase the dietetic value of animal lipids and at the same time to maintain physical and sensory characteristics at an acceptable level. Technological methods of adjusting physico-chemical properties can be applied to extracted meat or milkfats (i.e. rendered animal fats or purified butter fat) as for any other fat or oil. However, the bulk of animal lipids is consumed in the form of meat and milk or their products and is ingested

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without being chemically modified. The focus of this chapter will, therefore, be on bio-modification measures that aim to change the lipid composition in the animal organism.

13.2 Motivation for bio-modification of animal lipids The perception of food of animal origin is somewhat inconsistent. Animal products are highly esteemed as food, but they have a bad image as nutrient. The great demand for animal derived food despite its rather high price reflects eating pleasure, sensory value and status. With increasing income in developing and transition countries, demand for and production of animal derived food are predicted to grow appreciably (Delgado, 2003) and food disappearance data clearly illustrate the correlation between increasing demand for animal derived food and increasing wealth in the industrialized countries during the last decades (faostat.fao.org, 2005) – at least until a certain level of affluence is reached. Although no cause and effect relationship is implied, it may be mentioned that during the same period life expectancy increased considerably in the wealthy nations. In contrast, food of animal origin is often denounced as a health risk factor, blamed for its supposedly high content of fat, particularly saturated fatty acids, and cholesterol. There is still a widespread belief and public perception that ‘fat is bad’ and dietary cholesterol a major cause of coronary heart disease (CHD). These associations, however, have been severely challenged. It is clear now that the type of fat rather than the total amount in the diet is important for human health (Calder and Deckelbaum, 2003) and it seems clear that the importance of dietary cholesterol as CHD risk factor has, despite public perception, obviously been over-emphasized (Hu et al., 2001; Parodi, 2004). The proportion of saturated fatty acids in major animal products, e.g. lean meat and lard, is below 50 % and, moreover, not all saturated fatty acids (SFA) detrimentally affect plasma lipids to the same extent; short to medium-chain SFA (C4–C10) are not associated with risk of CHD, and stearic acid also seems to be related to a far lower risk than C12– C16 (Kris-Etherton and Yu, 1997; Hu et al., 2001). Not only may the health risk of SFA and cholesterol be over-stated but animal products contain several beneficial components, including bioactive fatty acids with specific physiological functions (Macrae et al., 2004). In this context, polyunsaturated fatty acids (PUFA) of the n-3 family, particularly the long-chain C20 and C22 fatty acids (LC-) and the so-called conjugated linoleic acids (CLA), deserve specific attention. Additionally, the multibranched-chain fatty acid phytanic acid recently gained some interest as a potential anti-diabetes agent (McCarty, 2001) and promoter of fatty acid βoxidation (Ellinghaus et al., 1999). The natural and common sources of these fatty acids in human nutrition are predominantly or even exclusively of animal origin. Because of the

308 Modifying lipids for use in food importance granted to these fatty acids, alternative sources have been developed. CLA for example are produced semi-synthetically and are commercially available. However, the isomeric composition of these products is very different from CLA occurring naturally in meat and milk of ruminants (AFSSA, 2005). LC-PUFA, particularly arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are commercially produced by the use of micro-organisms (Gunstone, 1999), and ARA and DHA single-cell oils are now used as dietary supplements in infant formulas (Gunstone, 1999; Ratledge, 2004). Another possibile way to produce specific fatty acids would be to modify oil-seed crops through genetic engineering techniques (Huang et al., 2004). However, food not only fulfills nutritional demands but is also strongly related to tradition, taste and joy. The basic idea of modifying animal lipids is, therefore, to provide food with optimized compositional characteristics with the aim of meeting nutritional recommendations without the need to change traditional eating habits. Producing health-promoting animal products has indeed become a recommended strategy to maintain economics of future livestock production (Macrae et al., 2004). This is, however, a rather challenging aim due to conflicts between the requirements and recommendations of dieticians (nutritive value), the technological properties requested by food processors (physicochemical properties) and the sensory characteristics desired by the consumers (palatability).

13.3 Specific bioactive fatty acids in animal products 13.3.1 Polyunsaturated n-3 fatty acids Omega-3 or n-3 unsaturated fatty acids contain the first double bond at the third carbon from the methyl end of the chain. Vertebrates normally do not possess the ability to synthesize fatty acids with double bonds further to the methyl end of the acyl chain than the n-7 position (e.g. ∆9-desaturation of palmitic acid). The availability of n-6 and n-3 fatty acids in the organism therefore depends on the dietary supply of these essential fatty acids. The basic n-3 fatty acid, α-linolenic acid (ALA, 18:3n-3), is found mainly in photosynthetic active tissue of plants, while the predominant PUFA in storage organs (oilseeds, cereals) is linoleic acid (LA, 18:2n-6). It is only in few seed oils, such as linseed or rapeseed oil, that considerable amounts of ALA are found. ALA has several specific functions in mammalian organisms, although its major metabolic fate is to be used as fuel in β-oxidation (Sinclair et al., 2002). A major metabolic function of ALA is carbon recycling, e.g. for the lipid synthesis in the brain. It also plays a role in the protection of skin and fur and it is probably involved in regulating water homeostasis (Sinclair et al., 2002). However, much more attention has been given to the cardioprotective effects of ALA, which have been confirmed by epidemiological

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studies and secondary prevention trials (cited in Hauswirth et al., 2004). Another highly relevant role of ALA is to serve as precursor of the very long-chain n-3 polyunsaturated fatty acids EPA, DPA (docosapentaenoic acid, 22:5n-3) and DHA. These n-3 LC-PUFA can be endogenously synthesized from ALA in avian and mammalian organisms via a series of elongation and desaturation steps (Leonard et al., 2004). It is important to mention that the same enzymes are also involved in the elongation and desaturation of n-6 PUFA with consequent competition of LC-PUFA synthesis between these two fatty acid families. The dietary n-6/n-3 ratio, therefore, is considered an important nutritional issue, although there are indications for duplicate enzyme sets in mitochondria (Infante, J.P., 1997 cited by Stordy, 1999) and recently it has been concluded from results of an epidemiological study that n-3 fatty acids decrease the risk of CHD irrespective of the n-6 intake (Mozaffarian et al., 2005). The effectiveness of conversion from ALA to n-3 LC-PUFA has been a matter of considerable discussion (Gerster, 1998; Brenna, 2002), because of the fundamental and highly specific physiological functions of EPA and DHA. EPA, like its n-6 counterpart ARA, is a precursor of so-called eicosanoids, a series of endogenous mediators (prostaglandins, prostacyclins, thromboxanes and leukotrienes) which are involved in inflammatory response, regulation of blood pressure, platelet aggregation and further physiological reactions. Recently, DHA was also recognized as a precursor of endogenous mediators, called docosanoids (Hong et al., 2003; Serhan, 2005). However, a major function of DHA is its role as a vital building block in membranes of brain, synapses, retina and spermatozoa (Blank et al., 2002; Broadhurst et al., 2002). Moreover, an impressive list of further beneficial effects of DHA can be cited (Horrocks and Yeo, 1999). Overall, it may be concluded that EPA and DHA have specific and physiologically important functions while their production from ALA, which is quite readily available from plant sources, is limited (Burdge and Calder, 2005). This makes animal derived food as a potential source of LC-PUFA interesting because farm animals, when supplied with dietary ALA, may convert it to the more valuable n-3 LC-PUFA to a certain degree.

13.3.2 Conjugated linoleic acids CLA came into the focus of nutritional research when it was identified as an anti-carcinogenic compound found in fried beef and effective in animal tumour models (Ha et al., 1987). The dominant CLA isomer in ruminant products is the 18:2 cis-9, trans-11 compound, aptly named ‘rumenic acid’. This is formed in the mammary gland mainly by endogenous ∆9-desaturation of trans-vaccenic acid (18:1 trans-11), which in turn is an intermediate product of microbial PUFA biohydrogenation in the rumen (Griinari et al., 2000). In contrast, the best synthetic CLA products, often used in feeding experiments, mainly contain the cis-9, trans-11 (c9, t11) and the trans-10,

310 Modifying lipids for use in food cis-12 (t10, c12) isomers in approximately equal proportions. Meanwhile, an increasing number of studies raised evidence for impressive beneficial effects like anti-atherosclerotic and anti-diabetic effects, inhibition of carcinogenesis, modulation of immune functions and reduction of adipose tissue (Belury, 2002). In consequence, CLA has become an interesting feed supplement in animal nutrition in order to fortify CLA in food of animal origin and to make use of its effects in terms of enhancing feed conversion efficiency and proportion of lean in the carcasses (Jahreis et al., 2000), although these effects are not always consistent (Bee, 2000; Dugan et al., 2001; Scheeder et al., 2002b; Corino et al., 2003). However, the value of strategies to enhance the CLA content of animal products by adding synthetic CLA to the feed may be questioned in the light of the rather disappointing results from human studies, which have been far less convincing than might have been expected from the effects exerted in animal models and in vitro studies (Calder, 2002). Meanwhile some detrimental effects have been attributed mainly to the t10, c12 isomer (Belury, 2002; Wahle et al., 2004) which is generally found as one of the two major compounds in synthetic CLA (Jahreis et al., 2000). In an exhaustive report about health risks and benefits of dietary trans fatty acids, including CLA, the authors recommend that the use of synthetic CLA mixtures in animal nutrition not be authorized (AFSSA, 2005). As outlined below, there might nevertheless be reasonable and safe ways to use synthetic CLA mixtures as a tool to modify the fatty acid composition of adipose tissue in pigs.

13.3.3 Phytanic acid More recently, another fatty acid, which is originally also derived from ruminant products, the so-called phytanic acid, has raised interest mainly because of a hypothesized anti-diabetic effect (McCarty, 2001). Phytanic acid is a branched-chain fatty acid (3,7,11,15-tetramethylhexadecanoic acid), derived from phytol, a side chain of chlorophyll which can be cleaved by microbes in the rumen. Because of the first methyl group at position 3 in the acylic chain (the β-carbon), phytanic acid has to undergo peroxisomal αoxidation before being further subjected to β-oxidation. It is, therefore, a key player in rare, inherited disorders of α-oxidation in humans (e.g. Refsum’s disease), which cause phytanic acid accumulation in tissues and serum and lead to degenerative changes in the retina and the nervous system (Mukherji et al., 2003). On the other hand, phytanic acid was shown to increase insulin sensitivity and it is hypothesized that it may mimic or complement various effects of CLA including inhibition of tumour growth (McCarty, 2001).

13.4 Genetic effects on the composition of animal lipids A major genetic effect on the fatty acid composition of animal lipids is based on the genetic disposition of the animal to synthesize fat. This involves the

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partition of nutritional energy beyond that required for maintenance and work, between protein (i.e. muscle) and adipose tissue synthesis. The animal organism can synthesize de novo only SFA, which then can be desaturated to monounsaturated fatty acids (MUFA). Greater fat accretion or secretion of lipids from de novo synthesis will ‘dilute’ the PUFA, which can only originate from the diet. Thus, the fatter an animal, the more saturated will the lipids in adipose tissue be (Malmfors et al., 1978; Nürnberg et al., 1997; De Smet et al., 2004). Due to successful breeding for a high lean meat content and a reduced amount of adipose tissue in the carcasses, the proportion of PUFA is generally rather high in modern pigs (Scheper, 1982). Even a century ago, butchers complained about the impaired meat and fat quality in the ‘modern’ lean pigs (Herter and Wilsdorf, 1914), and the basic conflict between nutritional and technological quality became evident: PUFA are desirable in human nutrition, but high amounts in lard are undesired because of an increased susceptibility to oxidation (Monahan et al., 1992; Flachowsky et al., 1997) and an impaired (soft) consistency of adipose tissue (Enser et al., 1984; Whittington et al., 1986; Gläser et al., 2004). Similar consequences as in adipose tissue can be observed in muscle tissue or lean meat. The intramuscular lipids mainly consist of phospholipids (PL) and triacylglycerols (TAG). PL normally contain more PUFA than TAG, while TAG contain appreciably more MUFA and somewhat more SFA than PL (Table 13.1). The amount (not the composition) of PL, which are the major structural lipids forming the membranes, is quite constant in the muscle. An increasing amount of intramuscular fat therefore is nearly exclusively due to an increase of TAG. Consequently, the fatty acid composition of meat Table 13.1 Fatty acid composition of neutral and phospholipids in M. longissimus dorsi of pigs fed either a control diet or a diet containing extruded linseed. Neutral lipids

Phospholipids

Treatment

Control

Linseed

Control

Linseed

SFA 16:0 18:0 MUFA 16:1 18:1 PUFA 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3

39.7 25.2 12.5 56.1 4.4 50.8 4.2 3.0 0.26 0.14 0.03 0.06 0.05

39.4 24.8 12.6 55.1 4.1 50.1 5.6 3.3 1.05 0.13 0.04 0.12 0.05

30.2 20.6 8.1 27.6 2.8 23.9 42.2 25.2 0.8 7.4 1.1 1.8 1.5

31.1 21.2 8.5 22.5 2.1 19.7 46.5 26.5 3.0 5.7 3.1 3.0 1.3

Source: Sottnikova and Scheeder (unpublished).

312 Modifying lipids for use in food changes to a higher proportion of MUFA and SFA at the expense of PUFA with increasing amount of intramuscular fat. Therefore, genetic differences between species and breeds can be related mainly to effects of the extent of fat accretion in muscle (De Smet et al., 2004). Further genetic effects may be attributed to differences in the activity of desaturases and elongases, synthesizing LC-PUFA from their essential C18 PUFA precursors or MUFA produced from palmitic or stearic acid. To our knowledge, not much work has been done in this area probably because measuring fatty acid composition in a huge number of individual animals as required for breeding is still laborious and expensive. This might also apply to dairy cows and milk fat, where some work has still to be done for a full understanding of lipogenesis, lipid accretion and modification in the mammary gland (Clegg et al., 2000). New technologies for efficient analyses, such as portable near-infrared devices to enable on-line measurement of fatty acid composition related traits, or modern breeding tools, such as marker-assisted selection, together with the increasing demand for optimization of the fat composition are likely to trigger future activity in this field. The fatty acids in milk fat are derived from (i) the diet and rumen microorganisms, (ii) adipose tissue stores, and (iii) de novo synthesis in the mammary gland. Because de novo synthesis in the mammary gland results predominantly in short- and medium-chain fatty acids but not in C18 fatty acids, genetic correlations of milk fat concentration and proportion of short-chain fatty acids are positive, while the correlations with long-chain fatty acids, derived either from body stores or from diet/micro-organisms, are negative (Palmquist et al., 1993a). Accordingly, milk from Jersey cows, a breed known for its high milk fat percentage, was higher in C6–C14 fatty acids than milk from Holstein cows, while it was the other way round for 18:1. Both 18:1 and short-chain fatty acids help to lower the melting temperature of milk fat, which is supposed to be necessary to successfully deliver the milk to the offspring (Gibson, 1991). Thus, it can be assumed that 18:1 compensates for a shortage in short-chain fatty acids (Palmquist et al., 1993a). The desaturation capacity might, therefore, be another genetic disposition important both for milk fat composition and for formation of cis-9, trans-11 CLA from transvaccenic acid (trans-11-18:1) (Griinari et al., 2000). Thus there are some starting points for the alteration of milk fat composition by means of breeding, but it has been questioned whether milk fat composition will become an important element of genetic improvement of dairy cattle, because of the gradual and slow changes which can be achieved. There may also be unfavourable antagonistic correlations between desired improvements in milk fat quality and other product traits leading to overall unclear economic incentives (Gibson, 1991). A highly interesting field of research opens at the interface of genetics and nutrition. Several fatty acids have been identified as signalling molecules and transcription factors which may affect gene expression (Crestani, 2004). A specific example is phytanic acid, which has been recognized as a natural

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ligand of nuclear receptors and was shown to act as transcriptional activator (Ellinghaus et al., 1999). It has also been hypothesized that phytanic acid could induce fatty acid catabolism (Ellinghaus et al., 1999) and may exert anti-diabetic effects (McCarty, 2001), making it a potentially interesting compound in animal feed and functional foods. Recently, it has been reported that phytanic acid is increasingly incorporated in liver, heart and muscle of finishing pigs with increased amounts of phytol up to 2 % in the diet (Raes et al., 2004a). Phytanic acid reached impressive levels of about 20 % of total fatty acids in liver and heart, predominantly replacing PUFA, while the incorporation in muscle tissue was much lower and no phytanic acid was found in lard. In a pilot study, we found a linear increase of phytanic acid in erythrocytes of finishing pigs from not detectable to 0.6, 1.2 and 1.86 g/100 g total fatty acid methylesters (FAME) with increased duration of exposure to 0.5 % phytol in the diet for 0, 20, 40 or 60 days prior to slaughter (Scheeder et al., 2005). In plasma TAG and PL, phytanic acid rose to remarkable 31.9 and 22.5 g/100 g FAME. The liver mass increased significantly with exposure time, whereas no consistent effect on liver glycogen content was detected. Despite the high phytanic acid levels in plasma TAG and PL, no effect on growth or adipose tissue accretion and no clear effects on medium(fructosamine) and long-term markers (glycosylated haemoglobin, HbA1c) of blood glucose levels were observed. The outcome could have been different in other, perhaps more appropriate, models than young, intensively growing pigs. However, phytanic acid can be increased in pork, depending on dose and duration of phytol intake, up to concentrations above those commonly found in beef. It must nevertheless be reported that phytanic acid is also suspected to promote prostate cancer (Mobley et al., 2003). Thus, further research in this field is certainly needed in order to elucidate the beneficial potential of fortification in animal products, and the drawbacks of phytanic acid. CLA is another, perhaps more promising, example of fatty acids potentially acting as transcriptional activator. The potential of CLA, administered as feed supplement, to modify lard composition, is outlined below. Genetic engineering would be another approach to modify the fatty acid composition in animal tissues. Spinach ∆12-desaturase has been successfully transferred and expressed in pigs, increasing LA from 9.9 to 11.6 % in adipose tissue (Saeki et al., 2004). The reported effect, although significant, seems quite limited compared to simple dietary interventions. Increasing n3 PUFA was achieved in mice engineered to carry a fat-1 gene from the roundworm Caenorhabditis elegans encoding for an enzyme that can add a double bond into an unsaturated fatty acid hydrocarbon chain and thus convert n-6 to n-3 fatty acids (Kang et al., 2004). Both groups argue that this approach could be a way to improve the dietetic value of animal products, however, there may be problems arising concerning the acceptance of genetically engineered farm animals.

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13.5 Housing and temperature effects on adipose tissue composition Ambient temperature can affect distribution and fatty acid composition of adipose tissue in the animal organisms. Pigs held at 12 °C developed more subcutaneous fat than pigs held at 28 °C. The subcutaneous fat was at the same time more unsaturated in the pigs held at lower temperatures (Lefaucheur et al., 1991). The endogenous desaturases are obviously activated at low temperatures to decrease the melting temperature and keep the subcutaneous fat sufficiently mobile. Even rather moderate temperature differences or seasonal influences when pigs have access to outdoor areas can lead to significant differences in 18:1 proportion in the backfat (Lebret et al., 2003). Housing and temperature can therefore influence pig fat quality under commercial production conditions.

13.6 Methods of modifying animal fats by changes in diet Dietary effects on the composition of adipose tissue or milk lipids are manifold and can be very powerful. In principle, all factors which increase or inhibit fatty acid de novo synthesis will change the share of fatty acids from endogenous versus dietary sources and therefore lipid composition. Dietary compounds, among which are certain fatty acids themselves, may also affect the activity of enzymes involved in the modification of fatty acids, as well as the microbial modification of fatty acids in the digestive tract. However, a major effect is the direct influence of dietary fatty acids by incorporation into triacylglycerols of adipose tissue, milk fat and/or phospholipids of the cell membranes, e.g. in muscle. Particularly in non-ruminants, in which most of the dietary fatty acids are absorbed unchanged, adipose tissue well reflects the composition of dietary fats. In ruminants, however, the feed is ‘processed’ in the rumen like in a fermentation vat. Feeding ruminants, therefore, actually means feeding the rumen microbes and this has fundamental consequences for dietary effects on the lipid composition.

13.6.1 Effects of dietary fatty acids on the composition of milk and adipose tissue lipids from ruminants The fermentation process in the rumen and its interactive response to feed compounds is highly complex and only a brief, simplified description of the main mechanisms can be given here. For more detailed information and accurate descriptions topical reviews are available (Harfoot and Hazelwood, 1988; Sutton, 1989; Palmquist et al., 1993a; Chilliard et al., 2000; Walker et al., 2004). Dietary carbohydrates and protein can be fermented by rumen microbes to volatile fatty acids, which are predominantly acetate, propionate and butyrate. Acetate is derived mainly from fermentation of cellulose while

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rapidly degradable carbohydrates, e.g. from cereals, preferentially yield propionate. Acetate-CoA and 3-hydroxybutyrate (formed from butyrate in the rumen wall) are the starting substrates for fatty acid de novo synthesis, propionate is a precursor of lactose. It is, therefore, likely that effects on ruminal acetate production affect fatty acid de novo synthesis and thus milk fat concentration and the proportion of short- and medium-chain fatty acids in milk fat. Propionyl-CoA instead of acetyl-CoA or methylmalonyl-CoA instead of malonyl-CoA can serve as starting substrate for the synthesis of odd-chain fatty acids (15:0 and 17:0) or methyl-branched isomers. However, the contribution of these processes to odd- or branched-chain fatty acids in milk is assumed to be very small. The major source of these fatty acids is rumen microbes, which contain a large proportion of odd- and branchedchain fatty acids in their membrane lipids (Vlaeminck et al., 2005). Oddchain fatty acids are formed through microbial elongation of propionate or valerate, while branched-chain amino acids (valine, leucine and isoleucine) and branched short-chain carboxylic acids (isobutyric, isovaleric and 2-methyl butyric acid) are assumed primers of microbial (iso and anteiso) branchedchain fatty acid synthesis (Kaneda, 1991). Dietary lipids may decrease microbial fermentation and, furthermore, provide long-chain fatty acids, which may lead to a shift from C6–C14 to C16/ C18 fatty acids in milk fat with the C16/C18 ratio depending on the C16/C18 ratio in the diet (Palmquist et al., 1993a). Dietary lipids are rapidly hydrolyzed in the rumen leading to free fatty acids. Unsaturated free fatty acids then undergo severe microbial biohydrogenation and commonly only a small portion of PUFA escape the rumen unchanged and are available for absorption at the duodenum. On average 80 % of LA and 92 % of ALA are hydrogenated, although with high concentrate diets these proportions may drop to 50 and 65 %, respectively (Chilliard et al., 2000). Biohydrogenation pathways for LA and ALA are different, resulting in stearic acid as end product but also various intermediates including fatty acids with trans and conjugated double bonds (Harfoot and Hazelwood, 1988). Due to the specific pathways of microbial biohydrogenation also a specific distribution of double bonds in the 18:1-trans isomers with predominantly ∆11-trans-18:1 (trans-vaccenic acid; vacca: Latin for cow) occurs in ruminant fats. In partially hydrogenated plant oils, in contrast, elaidic acid (∆9-trans-18:1) is the predominant isomer (Aro et al., 1998; Wolff et al., 2000). Trans fatty acids (TFA) are identified as a highly relevant risk factor for ischemic heart disease and further deleterious effects (Stender and Dyerberg, 2004). Because trans-vaccenic acid can be desaturated endogenously to c9, t11 CLA, it is often argued that TFA of animal origin might be less harmful than technologically produced TFA. However, this hypothesis has not been approved yet (Weggemans et al., 2004), although it may be assumed from the epidemiological studies cited in the review of Weggemans et al. (2004) that the amount of trans fatty acids of animal origin seldom reaches levels likely to exert a health risk. Furthermore, a shift from the less harmful vaccenic acid to the more harmful 18:1-trans10

316 Modifying lipids for use in food may occur when higher proportions of concentrate are fed (Dannenberger et al., 2004). Thus the health risk potential might be different for meat and milk fats derived from different production systems, depending on type and amount of concentrates used. The extensive biohydrogenation of unsaturated fatty acids is the key factor for the high degree of saturation of ruminant fats, and the idea of increasing the polyunsaturated/saturated fatty acid ratio (P/S) to improve the dietetic value of ruminant fats has, therefore, been of interest for quite some time (McDonald and Scott, 1977) and remains so (Scollan et al., 2005). Various attempts to protect dietary lipids from microbial lipolysis and unsaturated fatty acids from biohydrogenation have been undertaken: the use of whole or only coarsely crushed oilseeds, formaldehyde-protein-protected fat and selected fatty acids in fat prills or as calcium soaps (Sutton, 1989). The two latter sources mainly contain saturated fatty acids, and formaldehyde treatment is often not permitted or accepted. Crushed oilseeds in contrast provide a natural source of partially protected (poly)unsaturated fatty acids and significantly alter the fatty acid composition of adipose tissue in fattening bulls (Casutt et al., 2000). Fatty acids typical for the respective oilseeds (oleic, linoleic, linolenic acid in canola, sunflower and linseed, respectively) were increased significantly, although to a rather limited extent. Due to the still high biohydrogenation, the most obvious effect was an increase in stearic acid. This also led to the apparently contradictory effect that supplementation with highly unsaturated fatty acids produced tallow with a higher solid fat content at 20 °C compared with the control (Casutt et al., 1999). The supplementation of oilseeds thus affected the melting properties of the tallow, but the effect was too small to exert significant effects on other physical consistency traits (Casutt et al., 1999) and did not markedly affect properties of beef patties produced therewith (Scheeder et al., 2001). This is somewhat different for milk fat. Because of the high desaturation capacity of the mammary gland, a high availability of dietary 18:0 increases 18:1 in milk fat. Together with the decrease of 16:0 the melting temperatures are concomitantly decreased, leading to a lower solid fat content at 5 °C and therefore improved spreadability of butter at refrigeration temperatures (Banks cited by Palmquist et al., 1993b). The ruminal biohydrogenation of PUFA is a severe obstacle when trying to fortify n-3 fatty acids in the products. Wachira et al. (2000) reported that the extent of hydrogenation and therefore the transfer efficiency is higher when the n-3 source is forage where ALA is esterified to glycolipids, which are less prone to lipolysis. Feeding grass-based diets indeed increased the proportion of n-3 fatty acids in milk fat compared with a diet containing maize silage and concentrates (Leiber et al., 2005). In cheese from Switzerland, where mainly grass-based diets are still common, clearly higher proportions of n-3 fatty acids have been found than in e.g. cheddar (Hauswirth et al., 2004). There was also a specific n-3 enhancing effect of alpine grazing observed, which could not be explained with a higher ALA content in the

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alpine flora but was probably due to a reduced ruminal hydrogenation of ALA as a potential consequence of nutritional energy shortage and/or inhibiting secondary plant metabolites in the alpine flora (Leiber et al., 2005). DHA and EPA were also higher in milk from grass fed cows, but did not specifically respond to alpine grazing. Similarly, intramuscular lipids of grass fed bulls contained more n-3 PUFA including EPA and DHA compared with concentrate fed bulls (Dannenberger et al., 2004), whereas it was reported that including ALA-rich linseed in cattle diets increased ALA and EPA, but not DHA (Scollan et al., 2005). Attention must also be paid to the sensory properties of the products, as the susceptibility of the highly unsaturated fatty acids to oxidative degradation may result in deleterious flavour (Scollan et al., 2005). Because the main CLA isomer in milk is mainly built by desaturation of trans-vaccenic acid in the mammary gland or adipose tissues, increasing the amount of ALA in the diet will also increase CLA in milk and tissues of ruminants. Besides the cis-9, trans-11 isomer, a number of other isomers are also naturally occurring in ruminant products, but to a much lower extent (Jahreis et al., 2000; Nürnberg et al., 2002). The trans-11, cis-13 isomer might deserve special attention, because the amount of this isomer in the milk fat seems to respond to the altitude at which the cows are grazing and it could therefore be a typical ‘alpine’ CLA-isomer (Collomb et al., 2004). This has been supported by results of a controlled feeding experiment and alpine sojourn of dairy cows (Leiber et al., 2005). Increasing CLA is still claimed to be beneficial to human health and strategies to enhance its content in milk fat have been described recently (Chilliard et al., 2000; Lock and Bauman, 2004). Interestingly, dietary EPA and/or DHA specifically increase the ruminal production of trans fatty acids and CLA by a mechanism unknown so far (Chilliard et al., 2000). Supplementation of EPA and DHA to cattle diets could, therefore, increase these valuable n-3 LC-PUFA as well as CLA. However, the transfer efficiency of EPA and DHA from diet into milk is very low (2–4 %), and the supplementation of these fatty acids to the diet can massively reduce milk fat synthesis (Lock and Bauman, 2004). More detailed background information about strategies to increase EPA and DHA in milk and explanations for the low transfer efficiency are given by Rymer et al. (2003). A more efficient way might be to add EPA and DHA formulations directly to the milk. Such fortified products are already commercially available, marketed for example as ‘Einstein milk’, referring to the claims that DHA helps brain development and improves learning ability.

13.6.2 Modifying fats from non-ruminant (monogastric) animals In non-ruminant (monogastric) animals there is less microbial modification of dietary fatty acids in the digestive tract, although traces of 18:1 trans and CLA (18:2 9c, 11t) can be found in adipose tissue of pigs fattened on diets not containing these fatty acids (Gläser et al., 2000). This indicates microbial

318 Modifying lipids for use in food biohydrogenation already at sites in the gut where absorption of long-chain fatty acids takes place. Nevertheless, in monogastric animals the bulk of dietary fatty acids is absorbed as such (which also applies to humans) and the body lipids may well reflect the fatty acid composition of the feed. This is well established knowledge, and scientific evidence for the considerable influence of the fatty acid composition in the feed on the animal’s body fat composition dates back to the first quarter of the last century (Ellis and Ishikawa, 1926). Feeding diets with different fatty acid composition to pigs is surely one of the most certain ways to conduct an experiment resulting in significant differences. In fact, lipids are the only macronutrients for which Feuerbach’s philosophical proverb ‘You are what you eat’ (‘Der Mensch ist was er isst’) (Lemke, 2004) also applies in a physiological manner and this provides vast opportunities for manipulating the lipid composition in animals – but also bears the risk of undesired effects on fat quality. Medium-chain fatty acids (MCFA), naturally occurring in milk fat, coconut and palmkernel oil, can be used in animal nutrition for different purposes: MCFA have been shown to decrease methane emission from ruminants (Soliva et al., 2004); they can be used as growth promoting feed additives, because of their antimicrobial effect (Dierick et al., 2002); and MCFA are known to increase firmness and oxidative stability of lard when fed to pigs (Jaturasitha et al., 1996). MCFA could, therefore, be used to improve lard firmness, which is desired by meat processors, but the concomitantly increased amount of SFA is undesirable in terms of dietetic value. Furthermore MCFA bear another risk, which has made industrial processors of poultry fat for use in dehydrated soup concentrates or stock cubes establish a restrictive upper limit of maximum 0.25 % lauric acid. These convenience products are nowadays usually produced without heat treatment, leaving lipases from included cereals and spices sufficiently active to hydrolyze triacylglycerols. MCFA are particularly prone to hydrolysis, and lauric acid produces an undesired ‘soapy’ flavour making the soups unacceptable. If the aim is to avoid lauric acid in poultry fat, the close and linear relationship between lauric acid in the feed and in the abdominal fat should be considered (Fig. 13.1). A concentration of 2.1 g lauric acid per kilogram feed already resulted in 1.35 % lauric acid in abdominal fat. Accordingly, only traces below 0.3 g lauric acid per kilogram feed would be tolerable to cope with the threshold of 0.25 % in poultry fat (Scheeder et al., 2002a). It seems worth mentioning that similar effects can cause feed intake refusal, when MCFA-containing oils are used as ingredient in animal feed and mixed with ground cereals. One of the most striking conflicts in pig production arises from the contradictory demand of meat processors for firm lard and lean meat and of dieticians for fat low in saturated fatty acids and high in PUFA. Because pigs readily incorporate dietary PUFA in adipose and muscle tissue, it would be easy to produce high-PUFA lard and pork. Additional PUFA, however, markedly increase the oxidative potential of muscle and adipose tissue lipids. Autoxidation of PUFA increases specific volatile flavour compounds and may lead to

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12:0 in abdominal fat (g/100 g FAME)

5 4 3 2 1 0

Fig. 13.1

y = 0.142x + 0.070 R2 = 0.997

0

5

10

15 20 25 Total 12:0 intake (g)

30

35

Correlation between lauric acid (12:0) intake and proportion in abdominal fat of broiler chicken fattened to 2.3 kg live weight.

deterioration of the flavour of animal products (Elmore et al., 2000). While the oxidative stability can be controlled to a certain extent by the use of antioxidants in the feed (Wood et al., 2004) or added to products, an undesirably soft consistency of adipose tissue can hardly be corrected by means of food technology. Therefore, controlling consistency seems to be the most important issue when fortifying pork with PUFA. To control the PUFA content in lard in order to ensure a sufficiently high processing quality of pork and pig adipose tissue, various guidelines have been developed independently limiting PUFA concentrations in diets for growing-finishing pigs (Warnants et al., 1996). The Swiss guideline is one of the most restrictive with a recommended maximum of 0.8 g PUFA/MJ digestible energy or 12 g PUFA/kg feed (Perdrix and Stoll, 1995). To our knowledge, Switzerland is the only country where fat quality is routinely measured in pig carcasses and plays a part in the payment system. For this purpose, the so-called ‘fat score’, a semi-automated on-line method to measure the amount of double bonds in backfat, has been established in Swiss slaughter plants (Häuser et al., 1989). The analytical principle of the fat score is based on the iodine value according to Margosches (Margosches et al., 1924), and pooled samples of each batch of slaughter pigs are analyzed (Scheeder et al., 1999). When the current threshold for tolerable fat quality is exceeded (fat score 62) price deductions will be applied to the whole batch. Because of the mentioned correlation between lean meat content of the carcass, which generally is a main breeding goal, and the PUFA content in pig fat, these are challenging conditions for pig breeders. As shown in Table 13.2, pigs in the highest lean class often exceed the threshold for acceptable fat quality (Schwörer, 2004). Lard consistency, however, is more dependent on the saturated to unsaturated fatty acid ratio than on PUFA content alone (Gläser et al., 2002a, 2004). In a feeding experiment with finishing pigs a low fat control diet or the control diet supplemented with either lard, olive oil or soybean oil to achieve a similar amount of double bond in the diet were fed (Gläser et al., 2002a).

320 Modifying lipids for use in food Table 13.2 Relation between the proportion of lean meat in pig carcasses and the degree of saturation in the backfat. Lean classes [% Proportion of valuable cuts]1

Fat score2

46.95–49.99 50.00–52.99 53.00–55.99 56.00–62.31

58.6 60.4 61.6 63.4

± ± ± ±

1.2 2.0 2.6 2.5

1

Trimmed prime cuts as proportion of cold carcass weight, a measure of lean meat content in the carcass. Fat quality criterion based on the iodine value. Source: Schwörer (2004). 2

The fatty acid composition of backfat clearly reflected the dietary fatty acid composition in the supplemented groups (Table 13.3), but only the diet high in MUFA decreased SFA below 30 %. PUFA from soybean oil were mainly incorporated at the expense of MUFA, because LA as well as ALA inhibit the activity of ∆9-desaturase (Kouba et al., 2003). Therefore, although the fat score (like the iodine value) is highest in the soybean oil group, firmness of lard is not lower than in lard of pigs fed olive oil and the crystallization time was highest for the lard high in MUFA and low in SFA. The impact of the dietary-induced difference in fatty acid composition can also be seen in the melting curves and the development of solid fat content (SFC) between –6 and 20 °C of typical lard samples from this feeding experiment (Figs 13.2 and 13.3). With increasing degree of saturation, the low-melting fractions (peak 1 and 2) decline and a very high-melting fraction (peak 5) appears. SFC of lard with less than 30 % SFA is already at 0 °C below 50 % and therefore much too soft for meat products (Gläser et al., 2004). The oxidative stability, however, was quite high, suggesting that antioxidants in the olive oil (Baldioli et al., 1996) might have been transferred to the adipose tissue (Wenk et al., 2000). The firmness of pig adipose tissue can be increased by feeding saturated fats, but even larger effects can be achieved with trans fatty acids. Lard of pigs fed partially hydrogenated canola oil (D in Figs 13.2 and 13.3; Table 13.4) developed melting characteristics close to tallow. When the pigs were fed diets supplemented with 6 % of pure high-oleic sunflower oil (HO) or HO plus increasing amounts of partially hydrogenated canola oil (HR; 1.85 %, 3.70 %, 5.55 %), containing high levels of 18:1 trans fatty acid isomers, 18:1 trans fatty acids and cis-9, trans-11 CLA increased linearly in backfat and firmness was boosted up to eight-fold while the proportion of PUFA even slightly increased (Gläser et al., 2002b). This clearly indicates that trans-vaccenic acid is transformed to cis-9, trans-11 CLA in pigs and demonstrates the inhibiting effect of trans fatty acids on the activity of ∆9desaturase.

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Table 13.3 Fatty acid concentration and amount of double bonds in the diet of finishing pigs and in the outer layer of their backfat. Control

Lard

Fatty acid composition of the diet [g FAME1/kg DM] SFA2 3.8 34.5 3.2 35.3 MUFA3 18:1n-9 cis 2.8 29.4 10.0 18.8 PUFA4 18:2n-6 9.1 15.7 18:3n-3 0.80 1.55 Double bonds 82 256 [mmol/kg DM]

Olive oil

Soybean oil

11.6 43.2 41.0 17.1 15.5 1.42 266

9.0 12.3 11.2 29.2 26.2 2.81 249

Fatty acid composition and fat quality traits of the outer backfat layer [g/100 g FAME] SFA2 16:0 18:0 MUFA3 18:1n-9 PUFA 18:2n-6 18:3n-3 Double bonds [mmol/g] Fat score4 Oxidation stability5 Firmness 6 RIC-Box7 [s]

38.8a 23.4a 13.3a 46.6c 39.6c 14.7b 12.1b 0.93b 2.64c 60.8c 4.33a 151a 205c

35.2b 21.3b 11.9b 49.9b 42.1b 15.0b 12.0b 0.94b 2.79bc 64.2b 4.10a 114b 366b

29.8c 19.3c 8.8c 56.2a 50.4a 14.0b 11.8b 0.92b 2.92b

35.0b 21.0b 12.1ab 39.7d 34.3d 25.4a 21.1a 2.00a 3.18a

66.8b 4.59a 56c 610a

70.1a 2.36b 53c 296bc

Note: Superscripts a–d identify the presence or absence of significant differences between least squares means, i.e. common superscripts indicate no significant difference (Scheffé, p < 0.05). 1 Fatty acid methyl esters. 2 Saturated. 3 Monounsaturated fatty acids. 4 A semi-automated measure according to the iodine value. 5 Induction time [h], measured with Rancimat. 6 Firmness of lard at 0 °C, penetration force [g] at 2.5 mm distance. 7 Rapid interesterification control (crystallization time). Source: Gläser et al. 2002 b.

A similar effect on ∆9-desaturase is achieved by a CLA isomer. In a series of feeding experiments with growing-finishing pigs, clear dose–response and exposure time-dependent effects of CLA supplements on lard firmness were observed (Fig. 13.4) and it was shown that the trans-10, cis-12 isomer is the active isomer in this respect (Scheeder et al., 2004). At the same time, both CLA isomers increased linearly in backfat and muscle TAG as well as PL with increased intake. The trans-10, cis-12 isomer, however, was incorporated to a lower level than the cis-9, trans-11 isomer. This raises the possibility of making use of the firmness-enhancing effect of CLA with only

5 ak Pe

ak 3 ak 4 Pe

Pe

ak Pe

Pe

ak

1

2

322 Modifying lipids for use in food

A

Heat flow (W/g)

B

C

Endotherm

D

– 50 – 40 – 30 – 20 – 10 0 10 20 Temperature (°C) A B SFA 29.9 36.1 cis-MUFA 54.6 39.2 trans-MUFA – – PUFA 15.5 24.7

30

40

50

C 38.5 45.8 – 15.6

D 44.8 41.7 2.0 11.5

Fig. 13.2 Melting profiles of lard, differing in fatty acid composition (Gläser et al., 2004; Copyright Society of Chemical Industry. Reproduced with permission granted by John Wiley & Sons Ltd on behalf of the SCI).

Solid fat content (%)

100 A B C D

80

60 40

20

0 – 10

–5

0

5 10 15 Temperature (°C)

20

25

Fig. 13.3 Development of solid fat content of lard samples, differing in fatty acid composition, with increasing temperature (for fatty acid composition of the samples see Fig. 3.2) (Gläser et al., 2004; Copyright Society of Chemical Industry. Reproduced with permission granted by John Wiley & Sons Ltd on behalf of the SCI).

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Table 13.4 Fatty acid composition (g/100 g FAME) and firmness (N) of extracted backfat of pigs fed different amounts of partially hydrogenated canola oil.

16:0 18:0 16:1 18:1 18:1 18:1 18:1 18:1 18:1 18:1 18:2 18:2 18:3 Firmness2

∆9 ∆6–∆8 ∆9 ∆10 ∆11 ∆9 ∆11 ∆13 ∆9, 12 ∆9, 11 ∆9, 12, 15

trans trans trans trans cis cis cis all cis cis, trans all cis

HO

HOHR

HRHO

HR

18.2 8.9c 1.38b –1 – – – 52.8a 2.30b 0.09d 11.0 0.08d 0.75 0.77c

18.2 10.0b 1.47b 1.77c 1.57c 0.88c 0.31c 46.5b 2.21b 0.20c 10.5 0.70c 0.71 1.60c

18.7 10.5b 2.21a 3.61b 3.12b 1.62b 0.62b 38.2c 2.46a 0.38b 10.7 1.31b 0.73 2.91b

19.2 11.8a 2.34a 5.25a 4.46a 2.54a 0.97a 30.2d 2.51a 0.49a 10.4 1.81a 0.72 6.17a

Note: Superscripts a–d identify the presence or absence of significant differences between least squares means, i.e. common superscripts indicate no significant difference (Student-Newman-Keuls, p < 0.05). Abbreviations: HO = 6 % high oleic sunflower oil (HOSO) in the diet, HOHR = 4 % HOSO, 1.85 % partially-hydrogenated canola oil (PHRO), 0.15 % sunflower oil (SO), HRHO = 2 % HOSO, 3.7 % PHRO, 0.3 % SO, HR = 5.55 % PHRO, 0.45 % SO added to a basal diet. 1 Concentration too low to detect and separate individual 18:1 trans isomers, 2 Maximum force needed to drive a 3.5 mm punch 15 mm into the extracted fat at 0 °C Source: Gläser et al., 2002a.

Penetration force (N)

5 4 3 2 1 0

0

200

400

600 800 1000 Total CLA intake (g)

1200

1400

Fig. 13.4 Firmness of lard extracted from pigs fed diets containing 0, 0.25, 0.5 or 0.75 % CLA from 66–106 kg live weight measured as penetration force of a 3.5 mm punch (Scheeder et al., 2004).

little accumulation of the trans-10, cis-12 isomer, which has been judged critically in a recently launched report about health effects of trans fatty acids (AFSSA, 2005). CLA was also reported to improve feed conversion ratio and to decrease carcass fat content and backfat thickness in pigs (Jahreis et al., 2000). Although these effects have not been found in other studies (Bee, 2000; Dugan et al.,

324 Modifying lipids for use in food 2001; Scheeder et al., 2002b, 2003; Corino et al., 2003), CLA might nevertheless be a promising feed additive in pig production, because it may compensate for the softening effect of PUFA. A higher amount of PUFA in the diet and consequently in backfat might, therefore, be acceptable when CLA is fed at the same time. Thus, CLA provides the possibility of moderating the conflict between technological quality and dietary value of pork to a certain extent and gives some opportunity for a higher proportion of valuable n-3 PUFA in pig feed and therefore products (Enser et al., 2000). For instance, the inclusion of extruded linseed in finishing pig diets, corresponding to an additional 4.5 g ALA/kg feed, decreased lard firmness by about 40 %, but led to an increase of n-3 PUFA in cooked pork loin and neck from about 90 to 250 or 170 to 490 mg/100 g edible portion, respectively (Sottnikova et al., 2004). Arachidonic acid (AA, 20:4 n-6) was slightly decreased and the n-6/ n-3 ratio was lowered from about 10.2 to 3.7 and from 8.8 to 2.8 in loin and neck, respectively. The amount of EPA was on a low level and increased from 7.4 to 17.6 and from 9.6 to 22.7 mg/100 g in cooked loin and neck, respectively, while DHA did not change at all. These findings are consistent with other studies (Kouba et al., 2003; Wood et al., 2004), leading to the assumption that fortifying meat by supplementing ALA to the feed is likely to improve EPA and DPA supply but will not increase DHA supply to the same extent. In contrast, feeding ALA to laying hens markedly increases DHA in the eggs (Bourre, 2005). Eggs, therefore, not only contribute to the supply of n3 LC-PUFA, particularly DHA (Meyer et al., 2003), at present but have great potential to increase the share of DHA provided by farm animal products. Another favourable aspect surely is that the susceptible, highly-unsaturated fatty acids are in that case provided in a naturally well-protected package. While the fatty acid composition can be changed greatly by dietary measures in monogastric animals, there seems to be no possibility to modify TAG structure by feeding fat differing in the positional distribution of fatty acids at the glycerol backbone but not in fatty acid composition (Scheeder et al., 2003a). Changing TAG structure at a given fatty acid composition can probably only be achieved technologically by chemical or enzymatic interesterification.

13.7 Technological modifications Animal derived lipids are used to a great extent directly as foods of animal origin, such as milk, cream and cheese or meat and sausages, without technological modification apart from processing steps such as grinding or churning. Rendered animal fats or butter fat as any other fats and oils may, however, undergo the same modification and refining processes as vegetable oils in order to modify the degree of saturation (hydrogenation), remove undesired colour or flavour compounds (bleaching and deodorization), achieve desired texture, plasticity and melting behaviour (plastication,

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interesterification), separate the fat into fractions with specific characteristics (fractionation) or manufacture special products (powders). These processes are described in detail elsewhere in this book and will only briefly be addressed here, with special regard to animal derived lipids. Fats derived from ruminants are seldom hydrogenated because they are generally already high in saturated fatty acids. Mild hydrogenation of tallow may be applied to reduce highly unsaturated tri- and tetraenoic fatty acids to prevent development of flavour active oxidation products (Dugan, 1987). In lard, fatty acid composition and, therefore, melting properties can vary appreciably due to genetic and dietary effects as described above. Hydrogenation can help to obtain a product with consistent characteristics. Lard can also be completely hydrogenated and blended with unhydrogenated lard to achieve the desired physical characteristics (Dugan, 1987). Interesterification (also known as randomization, ester interchange and transeserification) is applied to randomize the distribution of fatty acids in the triacylglycerols. This is most relevant to lard, in which palmitic acid is typically esterified predominantly to the central sn-2 position of the triacylglycerols. Positional randomization changes the crystal habit of lard from β to β′, which is the preferred polymorphic form for fats used as shortenings. Interesterification of a blend of hard fat (e.g. tallow) and oil can be applied as an alternative to hydrogenation to produce plastic fats (Love, 1996). Interesterification can also be applied to butter fat, achieving a significant change in the triacylglycerol composition, but during the process the typical flavour will be lost and commercial interest in using this process for milk fat is obviously low (Hettinga, 1996). The flavour of milk fat is commonly perceived as highly desirable and is a most important property making milk fat an attractive food ingredient. The physical properties of milk fat, however, often fail to meet the technological functionality required for applications where its use is desired because of the flavour. Milk fat melts over a very wide range from about –30 to 40 °C, but the rather steep melting curve from 0 to 20 °C with a high solid fat content at refrigeration temperatures makes cold butter too firm to spread easily while it is not firm and plastic enough at higher temperatures to be used as shortening in pastry. Several physical modification processes such as tempering, texturization and fractionation can be applied to improve melting behaviour and solid fat content as desired (Kaylegian et al., 1993): air or nitrogen is incorporated into so-called whipped butter to improve the spreadability. With this method the volume is increased by about one third. A thermal treatment to cream before churning was invented by the Alnarp Dairy industry in Sweden giving a name to the processes of temperature profiling of the cream (Alnarping) to decrease the firmness of butter produced in this way. With mechanical treatments (texturizing or working) the crystal network and primary crystal structure of the butter is disrupted and a new structure is formed. It is, however, important to let butter crystallize completely before applying the

326 Modifying lipids for use in food mechanical treatment, and a severe disadvantage of this method is that the new structure is rather weak and the butter will lose its properties when subjected to temperature fluctuations. Blending of high- and low-melting fractions of anhydrous milk fat is another method to achieve milk fat with specific melting properties (Kaylegian and Lindsay, 1992). Fractionation can be achieved by separating triacylglycerols according to their melting points (dry fractionation) or by their solubility in solvents. The crystals formed at different temperatures during a thermally controlled process are then physically separated, e.g. by filtration or centrifugation. Solvent fractionation can be more efficient than dry fractionation, also leading to a better separation of fractions. The use of solvents, however, is often undesired or even prohibited. An interesting approach to fractionation of anhydrous milk fat has recently been reported, using plant oils as solvent (Wright et al., 2005). Although interesting products may be drawn from such a process, the use for fractionation purposes may be questioned, because of an unsatisfactory separation of the solids from the liquid phase. More detailed reviews about these methods and further examples are given by Hettinga (1996) and Kaylegian et al. (1993). Another pragmatic approach to combining desirable characteristics of milk fat with the supposedly beneficial dietetic value of vegetable oils is blending these items to a spread. The International Dairy Federation introduced ‘Guidelines for Fat Spreads’ (IDF Standard 166:1993) to provide a framework for more specific definitions and standards. This standard suggested the use of terms such as Blend, Blended spread, Low fat blended spread, depending on the fat content, for mixed fat products containing 15–80 % milk fat of total fat. Butter–vegetable fat spreads were invented and first introduced in Sweden but are now established fat spreads in various countries (Mann, 1997). For example, it has been reported that butter–vegetable oil blends accounted for 17 % of total spreads used in 1996 in Finland (Lampi et al., 1997). Quite an effort has been undertaken to develop technologies for reducing cholesterol in milk fat. Spreads made from ‘designer fats’ like cholesterolreduced anhydrous milk fat were shown to be acceptable for people used to consuming margarine, but were less liked by butter eaters (Michicich et al., 1999). The potential and drawbacks of various approaches, such as vacuum steam distillation, short path molecular distillation, absorption, solvent and supercritical fluid extraction and enzymatic methods, are described and reviewed by Boudreau and Arul (1993). Industrial application of such methods, however, seems to be scarce, and most of the techniques for reducing cholesterol became irrelevant with growing scientific evidence that dietary cholesterol hardly influences serum cholesterol and – probably more important – when regulations in the USA restricted advertisements for low-cholesterol products to food with less than 2 g SFA per serving (Hettinga, 1996). Cholesterol may, however, become relevant again when discussing the use of animal fats as (deep) frying fats, because of the potential formation of

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cholesterol oxidation products (COP) (Zhang et al., 1991) and their potential role in atherogenesis (Leonarduzzi et al., 2002) and carcinogenesis (Linseisen and Wolfram, 1999).

13.8 Modified animal fats: the relevance of fortifying functional fatty acids in animal lipids It is widely accepted that decreasing the n-6/n-3 ratio in the human diet by a higher intake of n-3 PUFA is advantageous for health, and this is considered in nutritional guidelines (DACH, 2000). EPA and DHA in particular exert beneficial effects. A relevant question is, how far terrestrial animal products contribute to the supply with n-3 LC-PUFA and if strategies to enhance n-3 LC-PUFA content in animal products can substantially improve the supply. A recent survey on the contribution of various food sources to the dietary intake of n-3 LC-PUFA revealed that, under Australian conditions, where daily n-3 LC-PUFA intake of adults is around 150–220 mg, meat and meat products contribute about 20 % of EPA and 12 % DHA of the total intake (Meyer et al., 2003). Eggs provide another 6 % of n-3 LC-PUFA, mainly DHA. We estimated from food disappearance data in Switzerland a similar average daily intake of about 85 mg EPA and 110 mg DHA of which 10 and 9 %, respectively, were contributed by meat. According to these calculations, milk and milk products contribute about one third of the EPA consumption but less than 3 % of DHA. Eggs provide only a little EPA but about 6 % of DHA consumption. To attain a higher n-3 content in animal products by feeding ALA-rich feed-stuffs to farm animals could be one way to achieve a more preferable n6/n-3 ratio in the human diet, even without changing nutritional habits. This approach would be particularly promising with monogastric animals, because the n-6/n-3 ratio in their meat is generally on a high level, due to the high amount of concentrates fed and the high proportion of linoleic acid (18:2n6) in grains (Raes et al., 2004b). The depletion of the natural marine resources and the potentially high mercury contamination of sea fish, which may counteract the beneficial effects of its n-3 fatty acids (Guallar et al., 2002), make alternative n-3 LC-PUFA sources even more attractive. It may also be argued that fish oil should be directly applied to humans (e.g. as capsules) instead of being fed to animals, where it can also exert undesired effects such as increased oxidative damage and/or undesired flavour effects. Making use of the animals’ ability to convert ALA to n-3 LC-PUFA will surely improve the n-6/n-3 ratio and the amount of EPA and DPA in meat but, as outlined above, the amount of DHA will hardly be affected. Nevertheless, it was shown that a feeding strategy, using extruded linseed as feed supplement, to produce n-3 fortified animal products, effectively increased the n-3 fatty acids in blood plasma lipids of the consumers and brought the overall n-6/n-3

328 Modifying lipids for use in food ratio close to the recommended value (Weill et al., 2002). In France, there is already a label programme existing, which has successfully implemented this strategy (http://www.bleu-blanc-coeur.com/prodcat.htm). In ruminants, increased ALA supply is likely to also increase CLA in the products, which may be rated positive, because only the naturally occurring isomers will be enhanced. CLA could also easily be increased in meat of monogastric animals by supplementing synthetic isomers to the feed. However, because of the lack of confidence in the safety of certain CLA isomers, fortification of animal products by supplementing synthetic CLA mixtures cannot be recommended (AFSSA, 2005). A future field of application could nevertheless be supplementation to finishing pigs’ diets in order to improve the firmness of backfat and lard. Compensating in that way for the softening effect of PUFA could allow for a higher amount of beneficial n-3 PUFA in the diet and, consequently, in the meat products. CLA, therefore, provides the potential to moderate the conflict between technological quality and dietary value of pork to a certain extent.

13.9 Future trends The current change of view concerning the role of fat and carbohydrates in nutrition may exert some impact on livestock production strategies. Labelling programmes providing primary products (milk, meat, eggs) with an improved fat composition in terms of dietetic value already exist and are likely to expand. The incentive to further develop feeding strategies in order to improve the nutritional value of milk and animal fats by modifying the fatty acid composition to meet nutritional recommendations might become stronger in the light of current trends to low-carb diets of the Atkins type. Improved milk or animal fat might also be used for manufacturing of processed products like cheese and sausages and perhaps regain fields of application lost to hydrogenated plant oils. The discussion about trans fatty acids will go on. There is some evidence that the naturally (in ruminant products) occurring trans MUFA might be less harmful (i) because of the potential to desaturate trans vaccenic acid and/or (ii) because the intake from ruminant products generally is too low to exert negative effects and/or (iii) milk and animal fat might contain other protective or beneficial compounds. Regarding the rather critical reviews about synthetic CLA, it seems unlikely that CLA fortifying strategies will gain ground. However, when used appropriately, CLA might become a valuable tool to control fat composition in pork production, opening possibilities to cope with the demand for firm and at the same time healthy lard.

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Sources of further information and advice

Several valuable reviews are already cited in the text and listed below; however, the excellent review about milk fat and human nutrition should be particularly highlighted (Parodi, 2004). A very useful review on nutritional control of milk fat composition with detailed but concise physiological background information has been given by Chilliard et al. (2000). Concerning CLA, an enormous body of scientific literature is available together with several reviews and books. Most of the relevant literature might be cited in the recent review of Wahle et al. (2004). The proceedings of a Symposium by the British Society of Animal Science about ‘Milk Composition’ provide not only reviews and research papers about genetic and nutritional improvement of milk composition but also a useful introductory review about consumer requirements and future trends (BSAS, 2000). More general information about fats, however, with special emphasis on fats of animal origin is provided by the Weston A. Price Foundation. Although the point of view might be somewhat biased, the critical reader will find interesting and entertaining hints and background information (http:// www.westonaprice.org/knowyourfats/).

13.11

References

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334 Modifying lipids for use in food SAEKI K, MATSUMOTO K, KINOSHITA M, SUZUKI I, TASAKA Y, KANO K, TAGUCHI Y, MIKAMI K,

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(1999), Docosahexaneoic acid: A dietary factor essential for individuals with dyslexia, attention deficit disorder and dyspraxia?, in Tyman J H P, Lipids in Health and Nutrition, Cambridge, The Royal Society of Chemistry, 102–114. SUTTON J D (1989), Altering milk composition by feeding, J Dairy Sci, 72, 2801–2814. VLAEMINCK B, DUFOUR C, VAN VUUREN A M, CABRITA A R J, DEWHURST R J, DEMEYER D and FIEVEZ V (2005), Use of odd and branched-chain fatty acids in rumen contents and milk as a potential microbial marker, J Dairy Sci, 88(3), 1031–1042. WACHIRA A M, SINCLAIR L A, WILKINSON R G, HALETT K, ENSER M and WOOD J D (2000), Rumen biohydrogenation of n-3 polyunsaturated fatty acids and their effects on microbial efficiency and nutrient digestibility in sheep, J Agric Sci, 135(4), 419–428. WAHLE K W J, HEYS S D and ROTONDO D (2004), Conjugated linoleic acids: are they beneficial or detrimental to health?, Prog Lipid Res, 43(6), 553–587. WALKER G P, DUNSHEA F R and DOYLE P T (2004), Effects of nutrition and management on the production and composition of milk fat and protein: a review, Aust J Agric Res, 55, 1009–1028. WARNANTS N, VAN OECKEL M J and BOUCQUE C V (1996), Incorporation of dietary polyunsaturated fatty acids in pork tissues and its implications for the quality of the end products, Meat Sci, 44(1–2), 125–144. WEGGEMANS R M, RUDRUM M and TRAUTWEIN E A (2004), Intake of ruminant versus industrial trans fatty acids and risk of coronary heart disease – what is the evidence?, Eur J Lipid Sci Technol, 106, 390–397. WEILL P, SCHMITT B, CHESNEAU G, DANIEL N, SAFRAOU F and LEGRAND P (2002), Effects of introducing linseed in livestock diet on blood fatty acid composition of consumers of animal products, Ann Nutr Metab, 46(5), 182–191. WENK C, LEONHARDT M and SCHEEDER M R L (2000), Monogastric nutrition and potential for improving muscle quality, in Decker E A, Faustman C and Lopez-Bote, C J, Antioxidants in Muscle Foods – Nutritional Strategies to Improve Quality, 1st edn, New York, Wiley Interscience, 199–227. WHITTINGTON F M, PRESCOTT N J, WOOD J D and ENSER M (1986), The effect of dietary linoleic acid on the firmness of backfat in pigs of 85 kg live weight, J Sci Food Agric, 37(8), 753–761. WOLFF R L, COMBE N A, DESTAILLATS F, BOUÉ C, PRECHT D and MOLKENTIN J (2000), Follow-up of the ∆4 to ∆16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995–1999. Analytical and nutritional implications, Lipids, 35(8), 815–825. WOOD J D, RICHARDSON R I, NUTE G R, FISHER A V, CAMPO M M, KASAPIDOU E, SHEARD P R and ENSER M (2004), Effects of fatty acids on meat quality: a review, Meat Sci, 66, 21–32. WRIGHT A J, BATTE H D and MARANGONI A G (2005), Effects of canola oil dilution on anhydrous milk fat crystallization and fractionation behavior, J Dairy Sci, 88(6), 1955–1965. ZHANG W B, ADDIS P B and KRICK T P (1991), Quantification of 5α-cholestane-3β,5,6β-triol and other cholesterol oxidation products in fast food French fried potatoes, J Food Sci, 56, 716–718. STORDY B J

14 PUFA production from marine sources for use in food G. G. Haraldsson, University of Iceland, Iceland and B. Hjaltason, EPAX AS, Iceland

14.1

Introduction

There is an increasing demand for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrates from the health food industry as food supplements and functional food and from the pharmaceutical industry for drugs. The n-3 polyunsaturated fatty acid (PUFA) concentrates may be roughly divided into three classes. First, ethyl esters of various enrichment levels of EPA or DHA or both, that have been developed into health supplements and drugs (Haraldsson and Hjaltason, 2001). Ethyl esters may also be used as starting material for glycerol derived lipids enriched with these fatty acids. Second, n-3 PUFA concentrates in the natural triacylglycerol (TAG) form or of high TAG content that are available in various enrichment levels where their fatty acid distribution in defined positions of the glycerol backbone is not of much concern (Haraldsson, 2000; Haraldsson and Hjaltason, 2001). Finally, structured TAG containing EPA or DHA located at the mid-position with medium-chain fatty acids (MCFA) at the end-positions of the glycerol moiety are currently the most sophisticated concentration form (Haraldsson, 2005). This chapter is intended to describe the best procedures for concentrating EPA and DHA in fish oil to high levels by physical means and by enzymatic methods involving lipases. Most emphasis will be placed on enzymatic methods to prepare TAG-based concentrates and structured TAG constituting EPA and DHA. Lipases are ideally suited as biocatalysts for esterification and transesterification processes involving the highly labile n-3 PUFA because of their efficiency and the mild conditions under which they act (Haraldsson and Hjaltason, 1992; Haraldsson, 2000). Based on their fatty acid selectivity,

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lipases have been widely used to enrich n-3 PUFA in fish oil by kinetic resolution in hydrolysis, transesterification and esterification reactions (Haraldsson and Hjaltason, 2001). They may be used to concentrate EPA together with DHA or they may offer discrimination between EPA and DHA to concentrate EPA or DHA individually. Since they can tolerate water-free organic media extremely well, they may also be exploited to introduce EPA and DHA into natural TAG. Finally, lipases, owing to their regioselectivity, are perfectly suited as biocatalysts for preparing structured TAG comprising n-3 PUFA at the mid-position and MCFA at the end-positions (Haraldsson, 2005).

14.2 Concentration of n-3 PUFA by non-enzymatic methods Numerous methods are available for concentrating EPA and DHA in fish oils or for separating and purifying EPA and DHA (Ackman, 1988; Breivik et al., 1997; Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001). These methods are summarized in Table 14.1. Usually a combination of fractionation techniques is needed to obtain EPA and DHA in highly purified form. This relates to the complexity of marine oils which often contain more than 50 different fatty acids linked as esters into triacylglycerols (Haraldsson and Hjaltason, 2001). Winterization is a simple cooling that is used to separate the saturated TAG from residual oil in certain selected types of fish oils by precipitation (Ackman, 1986; Breivik and Dahl, Table 14.1

Methods to concentrate EPA and DHA in fish oils.

Fat type

Level of concentration (%)

Triacylglycerols Winterization Organic solvent crystallization

30 35–40

Free acids or monoesters Counter-current fractionation or crystallization Short-path distillation Supercritical fluid carbon dioxide extraction Lipase kinetic resolution Urea complexation

50 50 50–60 50–75 70–80

Separation of EPA and DHA as free acids or monoesters Lipase kinetic resolution HPLC Silver-ion chromatography Corey’s iodolactonization

> > > >

90 95 95 95

Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, HPLC = high-performance liquid chromatography.

338 Modifying lipids for use in food 1992). This results in a slight enrichment in PUFA, up to 30 % levels. Crystallization of TAG from organic solvents at lower temperatures to freeze out more saturated TAG from fish oils has also been used to concentrate fish oils up to 35–40 % levels (Ackman, 1986; Breivik and Dahl, 1992). It is difficult to obtain higher levels on the natural TAG form and, in order to accomplish further concentration, the fatty acids need to be released from the TAG as free acids or monoesters. After the fatty acids are set free, various methods are available (Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001). Short path distillation may easily be used to obtain enrichment levels up to approximately 50 % (Ackman, 1986; Breivik and Dahl, 1992; Shahidi and Wanasundara, 1998). Comparable concentration levels may be obtained by simple counter-current fractionation or by crystallization of free acids or esters (Brown and Kolb, 1955; Ackman, 1986; Breivik and Dahl, 1992; Shahidi and Wanasundara, 1998). The increased polarity of the long-chain n-3 PUFA gives them higher solubility in more polar solvents compared to saturated and monounsaturated fatty acids. Various extraction methods (Medina et al., 1998; Shahidi and Wanasundara, 1998; Haraldsson and Hjaltason, 2001) have been reported for the separation and concentration of long-chain n-3 PUFA up to high levels, including supercritical fluid carbon dioxide extraction (Krukonis, 1984; Mishra et al., 1993; Walker et al., 1999) and silver nitrate solution extraction (Kubota et al., 1997; Ozawa et al., 2001). Urea complexation is a simple and efficient technique to increase the enrichment of free acids or monoesters up to the 70–80 % levels with high EPA and DHA recovery (Haagsma et al., 1982; Ratnayake et al., 1988; Medina et al., 1998; Shahidi and Wanasundara, 1998; Wanasundara and Shahidi, 1999). The saturated or monounsaturated fatty acids complex easily with urea crystals that can accommodate aliphatic long straight-chain fatty acids and crystallize at appropriate reduced temperatures. The presence of several double bonds in long-chain PUFA alters their shape, rendering these molecules more bulky and causing these PUFA to resist complexing with urea. The drawback of this method, however, is the large amount of solvents, chemicals and by-products involved. This method has been scaled up to a multi-tonne production scale in combination with short path distillation by Norsk Hydro/Pronova Biocare in Norway to produce a concentrate of 85 % EPA plus DHA (Breivik and Dahl, 1992; Breivik et al., 1997). A combination of urea complexation and a subsequent supercritical fluid carbon dioxide extraction resulted in concentrates of EPA and DHA in purities exceeding 90 % (Nilsson et al., 1988). Further enrichment almost to 100 % purity level (≥ 95 %) is performed by chromatographic methods. As a result of their higher polarity, long-chain n3 PUFA can be easily separated by high-performance liquid chromatography (HPLC). Reversed-phase HPLC has been particularly useful to isolate longchain n-3 PUFA, and eventually highly pure n-3 PUFA were produced on a relatively large scale by that method (Tokiwa et al., 1981; Beebe et al.,

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1988). Silver ion resin as an absorbent was observed to be more effective than normal chromatography and the purity of the long-chain n-3 PUFA could be improved (Teshima et al., 1978; Adlof and Emken, 1985; GuilGuerrero and Belarbi, 2001). The iodolactonization method, first introduced by Corey and coworkers (Wright et al., 1987), was modified by Russian scientists to allow a further HPLC purification of the DHA iodolactone to accomplish highly pure DHA (Gaiday et al., 1991). Although chromatographic methods can be used to produce highly pure EPA and DHA, large amounts of organic solvents are needed and the production capacity is relatively low. Also, tedious methods involving several repeated HPLC steps are often required.

14.3 Concentration of n-3 PUFA by lipase Owing to their fatty acid selectivity and discrimination against n-3 PUFA, lipases can be used as an alternative means to concentrate EPA and DHA in fish oils (Medina et al., 1998; Haraldsson and Hjaltason, 2001). Compared with more traditional physical and chemical methods, the lipase-catalyzed methods offer numerous advantages. First, the catalytic efficiency of lipases is high, so a relatively low amount of lipase is needed for production on a large scale, and multiple re-use is possible with immobilized lipase. Second, the selectivity of lipase against PUFA is crucial for certain applications, and they may also discriminate between EPA and DHA. Third, the mild conditions of lipase-catalyzed reactions in terms of temperature, pH and pressure are very important when the highly labile long-chain n-3 PUFA are involved. Fourth, lipases retain their high activity under virtually water-free conditions remarkably well, shifting the thermodynamic equilibrium to favor esterification over hydrolysis. This makes them ideally suited to catalyze various esterification and transesterification reactions and for synthesis of highly pure TAG. Finally, as the lipase-catalyzed esterification can be conducted under solvent-free conditions, the bulkiness of the process and investment cost will be considerably reduced, and operators can work in a safer environment. As indicated in Table 14.1 lipases may be used to concentrate EPA and/or DHA up to the 50–70 % levels and also to separate and purify these acids to a large extent. Today there are approximately 70 preparations of lipases commercially available from animal, plant and microbial sources, with the last being most abundant (Bornscheuer and Kazlauskas, 1999). Numerous reports on lipase screening for marine oil fatty acid selectivity have revealed that there are significant variations among lipases depending on their origin and source (Hoshino et al., 1990; Tanaka et al., 1992; Zuyi and Ward, 1993; Shimada et al., 1994, 1997a; McNeill et al., 1996; Haraldsson et al., 1997; Shimada et al., 1997b; Wanasundara and Shahidi, 1998a; Halldorsson et al., 2004). Generally there is a clear-cut preference for the saturated and less unsaturated fatty acids. Many lipases display very low activity towards fish

340 Modifying lipids for use in food oils, and only a few are suitable for biotransformations involving marine oils and n-3 PUFA, all of microbial type. The lipases that have been successfully applied for enrichment of the n-3 PUFA are listed in Table 14.2. They include the Aspergillus niger, Candida rugosa (formerly named Candida cylindracea), Geotrichum candidum, Pseudomonas cepacia (supplied by Amano in Japan as Lipase PS, formerly classified as Pseudomonas fluorescens), Pseudomonas fluorescens (Amano’s Lipase AK, formerly termed Pseudomonas sp.), Rhizomucor miehei (supplied by Novozyme A/S in Denmark immobilized as Lipozyme RM IM, formerly named Mucor miehei), Rhizopus delemar and Rhizopus oryzae lipases. The lipases displaying sufficient activity may be divided into two categories (Haraldsson and Hjaltason, 2001). The first category comprises lipases discriminating against n-3 PUFA including both EPA and DHA offering potential to concentrate EPA and DHA together. The lipases belonging to this category include those from Geotrichum candidum, Pseudomonas fluorescens and Pseudomonas cepacia. The Aspergillus niger lipase also appears to belong to this class, although it may act too slowly to be of practical value (Hoshino et al., 1990; Halldorsson et al., 2004). Lipases offering a strong discrimination between EPA and DHA in favor of EPA belong to the second class. They include the Candida rugosa, Rhizomucor miehei and Rhizopus delemar lipases. The bulk of the prefered fatty acids including EPA devoid of DHA may then subsequently be used as a source for further EPA concentration (Haraldsson and Kristinsson, 1998). The immobilized Candida antarctica lipase (Novozym 435 from Novozyme A/S in Denmark) warrants a special comment. This lipase displays relatively high activity for both EPA and DHA and is of no value to concentrate EPA or DHA. However, it is ideally suited for various biotransformations involving n-3 PUFA of Table 14.2

Lipases successfully used for enriching n-3 PUFA in fish oil.

Lipase

Type concentrate

References

Aspergillus niger Candida rugosa Geotrichum candidum Pseudomonas cepacia Pseudomonas fluorescens Rhizomucor miehei

EPA + DHA EPA + EPA + EPA + DHA EPA DHA DHA

[1, 2] [1–8] [3, 7, 9] [1, 10–12] [1, 10, 11] [13–18] [13, 15] [4, 19, 20] [1, 6]

Rhizopus delemar Rhizopus oryzae

DHA DHA DHA DHA

References for [1]: Halldorsson et al., 2004; [2]: Hoshino et al., 1990; [3]: Shimada et al., 1994; [4]: Shimada et al., 1997a; [5]: Tanaka et al., 1992; [6]: Wanasundara and Shahidi, 1998a; [7]: McNeill et al., 1996; [8]: Wanasundara and Shahidi, 1998b; [9]: Shimada et al., 1995; [10]: Zuyi and Ward, 1993; [11]: Haraldsson et al., 1997; [12]: Maehr et al., 1994; [13]: Haraldsson and Kristinsson, 1998; [14]: Shimada et al., 1998; [15]: Takagi, 1989; [16]: Langholz et al., 1989; [17]: Hills et al., 1990; [18]: Halldorsson et al., 2003b; [19]: Shimada et al., 1997b; [20]: Shimada et al., 1997c. Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid.

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great importance. It has been used in esterifying glycerol with n-3 PUFA concentrates including pure EPA and DHA (Haraldsson et al., 1995). The Candida antarctica lipase has also been observed to display excellent regioselectivity toward the outer positions of the glycerol moiety in transesterification reactions of triacylglycerols (Irimescu et al., 2001a, 2002) and glycerol (Halldorsson et al., 2003a) and is a key lipase in the synthesis of positionally labeled symmetrically structured TAG of the MLM type. It is of great interest that crude hydrolytic enzyme mixtures from fish intestine display reversed fatty acid selectivity. Lie and Lambertsen demonstrated that the hydrolytic enzyme mixture from cod displayed preference for n-3 PUFA over saturated and monounsaturated fatty acids in fish oil hydrolysis (Lie and Lambertsen, 1985). Gellesvik isolated a bile-salt dependent lipase from cod intestines that likewise displayed preference for the longchain n-3 PUFA (Gellesvik, 1991). More recently, Halldorsson et al. reported similar reversed fatty acid selectivity for hydrolytic enzyme mixture from both salmon and rainbow trout intestines in fish oil TAG (Halldorsson et al., 2004) and astaxanthin diester (Halldorsson and Haraldsson, 2004) hydrolysis.

14.3.1 Lipase hydrolysis Lipase hydrolysis of a TAG oil results in the formation of selected free acids and a mixture of monoacylglycerols (MAG), diacylglycerols (DAG), TAG and glycerol depending on the degree of conversion. The higher selectivity of lipase towards the saturated and monounsaturated fatty acids results in their removal from the glycerol backbone of the oils. The less preferred long-chain n-3 PUFA remain in residual acylglycerol molecules. They may be released subsequently by traditional chemical or enzymatic hydrolysis or alcoholysis reactions into the corresponding free fatty acid (FFA) or monoester concentrates for further concentration (Breivik et al., 1997). Usually such lipase-promoted hydrolysis is performed on an oil-to-water (or buffered water) ratio between 1:1 and 1:1.5 as based on weight. The lipase-catalyzed fish oil hydrolysis reaction is illustrated in Fig. 14.1. Yamane and coworkers examined several lipases for selective hydrolysis of cod liver oil and sardine oil to concentrate EPA and DHA (Hoshino et al., 1990). The reactions were conducted O Fish oil

O

TAG

Lipase H2O O

SMFA

O OH

PUFA

O

MAG/DAG/TAG

Fig. 14.1 A simplified fish oil hydrolysis by lipase to afford saturated and monounsaturated free fatty acids (SMFA) and an acylglycerol mixture constituting triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) enriched with n-3 PUFA, EPA and DHA or only DHA, depending on the lipase fatty acid selectivity.

342 Modifying lipids for use in food at 20 °C. With Candida rugosa and Aspergillus niger lipases, acylglycerols containing more than a two-fold increase in the n-3 PUFA content compared to the original fish oils were produced. Both lipases afforded concentration levels up to 50 %. Tanaka et al. described the use of the Candida rugosa lipase to concentrate DHA in fish oil constituting 13 % EPA and 9 % DHA by partial hydrolysis at 37 °C (Tanaka et al., 1992). With the Candida rugosa lipase at 70 % conversion, the DHA content in the acylglycerol product mixture was three times higher than in the original fish oil, whereas the EPA content was reduced to 70 % of that in the original oil. When DHA-rich tuna oil comprising 6 % EPA and 25 % DHA was hydrolyzed with that lipase, acylglycerols of 53 % DHA content were obtained (see Fig. 14.2) with the EPA content (4 %) remaining close to that in the original oil. The Candida rugosa lipase was observed to be ineffective with TAG comprising DHA (Tanaka et al., 1993). Maehr et al. used Pseudomonas cepacia lipase on fish oils comprising 30 % EPA + DHA to produce acylglycerols close to the 50 % EPA + DHA concentration level in 23–50 % weight recovery yields (Maehr et al., 1994). Concentration levels of 70 % were obtained at a higher conversion, but in much lower recovery of only 14–21 %. McNeill and coworkers reported on fish oil hydrolysis employing Candida rugosa and Geotrichum candidum lipases (McNeill et al., 1996; Moore and McNeill, 1996). At 60 % conversion the n-3 content had increased from 30 % in the initial oil to 45 % in the residual glyceride mixture that was enriched with both EPA and DHA. At 80 % conversion a DHA-enriched concentrate with an EPA-to-DHA ratio of 1:5 was afforded (7 % EPA and 40 % DHA) by the Candida rugosa lipase. Shimada and coworkers used the Geotrichum candidum lipase to concentrate EPA together with DHA in tuna oil containing 8 % EPA and 30 % DHA by selective hydrolysis (Shimada et al., 1994). The reaction was conducted at 30 °C and after 16 hours 34 % conversion was obtained resulting in production of glycerol esters of 10 % EPA and 39 % DHA content. A second hydrolysis resulted in acylglycerols comprising 11 % EPA and 47 % DHA in 82 % recovery of these fatty acids in 55 % yield. PUFA-rich TAG, especially those containing DHA, accumulate in the acylglycerol product indicating that TAGcontaining DHA are resistant to Geotrichum candidum lipase (Shimada et al., 1995). This reluctance of many lipases to act on DHA-enriched TAG is a major impediment to the synthesis of positionally labeled symmetrically structured TAG of the MLM type (Irimescu et al., 2001a).

Tuna oil TAG

CRL H2O

O

O MAG/DAG/TAG

SMFA/EPA

OH

DHA

O (53 % (DHA)

Fig. 14.2 Tuna oil hydrolysis by Candida rugosa lipase (CRL) by Tanaka et al. (1992) (for abbreviations see Fig. 14.1).

PUFA production from marine sources for use in food

343

Wanasundara and Shahidi investigated a number of lipases in their hydrolysis of seal blubber and menhaden oils to generate n-3 PUFA concentrates (Wanasundara and Shahidi, 1998a). The reactions were conducted at 35 °C using buffered water. The highest concentration levels of n-3 PUFA were obtained with the Candida rugosa lipase, 44 % for both oils. Wanasundara and Shahidi have optimized their n-3 fatty acid concentration using the Candida rugosa lipase (Wanasundara and Shahidi, 1998b). Enrichment levels of close to 54 % were obtained for both oil types.

14.3.2 Lipase alcoholysis Alcoholysis of fish oil TAG by a short-chain monohydric alcohol may be regarded as a simple modification of the hydrolysis process. In ethanolysis, where ethanol is used instead of water, the product is ethyl esters of the bulk of the more saturated fatty acids in the original fish oil instead of free fatty acids. As with hydrolysis, the residual acylglycerol mixture becomes enriched with EPA and DHA (illustrated in Fig. 14.3) or only DHA, depending on the nature of the lipase fatty acid selectivity. There are two reports on fish oil enrichment of EPA together with DHA by the TAG ethanolysis approach (Zuyi and Ward, 1993; Haraldsson et al., 1997). Zuyi and Ward investigated numerous lipases in alcoholysis of cod liver oil TAG using various primary and secondary short-chain alcohols (Zuyi and Ward, 1993). Their aim was to concentrate both EPA and DHA, and the best results were obtained with the Pseudomonas fluorescens lipase with isopropanol and ethanol. The reactions were conducted in the alcohol as a solvent at 30 °C in the presence of 5 % water by weight. The high water content resulted in high levels of free fatty acids, but the residual acylglycerol mixture comprised EPA + DHA levels close to 50 %. Pseudomonas lipases belong to the category of lipases that discriminate between the bulk of saturated and monounsaturated fatty acids and n-3 PUFA in fish oil as was confirmed by Haraldsson and coworkers (Haraldsson et al., 1997). They observed that two commercially available Pseudomonas cepacia and Pseudomonas fluorescens lipases afford concentrates of approximately 50 % EPA plus DHA in high recoveries, 80 and 90 % for DHA and EPA, respectively, and are highly efficient. The reactions were conducted on sardine oil containing 15 % EPA and 9 % DHA at room temperature, without a solvent, and with only a two-fold stoichiometric amount of ethanol. The O Fish oil TAG

PFL Ethanol

O MAG/DAG/TAG

SMFA

O

EPA/DHA

O

(50 % EPA + DHA)

Fig. 14.3 Fish oil ethanolysis by Pseudomonas fluorescens lipase (PFL) by Haraldsson et al. (1997) to produce saturated and monounsaturated ethyl esters and residual acylglycerol mixture enriched with EPA and DHA.

344 Modifying lipids for use in food reaction is demonstrated in Fig. 14.3. This resulted in a considerable reduction in bulk of the process, and the ethyl esters produced were distilled off by short path distillation from the residual acylglycerol mixture of EPA and DHA. The short path distillation was beneficial in that monoacylglycerols containing shorter-chain fatty acids were distilled off, whereas ethyl esters of EPA and DHA remained in the residue. This resulted in further increases in the EPA and DHA levels in the residual acylglycerol mixture after the distillation (Breivik et al., 1997). Ten per cent dosage of lipase as powder based on the weight of fish oil was used, but the activity of the lipase had already dropped significantly after the first run. Immobilization solved the productivity problem and far less lipase was needed, and it could also be reused more than ten times without any deterioration of the activity and results. These factors render this method highly feasible from an industrialization point of view (Breivik and Haraldsson, 1994; Breivik et al., 1997). Furthermore, the immobilized Candida antarctica lipase converted the acylglycerol mixture remaining after short path distillation into the corresponding ethyl ester concentrate by ethanolysis for further concentration. Only a two-fold stoichiometric amount of ethanol at room temperature was needed. Shimada and coworkers observed lipase to be highly sensitive to the amount of ethanol and the type of acylglycerols to be ethanolyzed (Watanabe et al., 1999). There are also reports on DHA enrichment in fish oil by lipase-catalyzed alcoholysis reactions. Haraldsson and Kristinsson obtained good separation of DHA by ethanolysis of tuna oil TAG comprising 6 % EPA and 23 % DHA under the ethanolysis conditions described above with immobilized Rhizomucor miehei lipase (Haraldsson and Kristinsson, 1998). Seventy per cent conversion into ethyl esters was obtained after 48 hours with the residual acylglycerol mixture containing 54 % DHA (and 6 % EPA) in 78 % recovery. The separation and the performance of the lipase, however, were dramatically improved after a modification based on esterification of free acids with ethanol as will be described later in this chapter (Table 14.3). Shimada and coworkers reported on the alcoholysis of fish oil monoesters with medium-chain fatty alcohols (Shimada et al., 1997c). They used Rhizopus delemar lipase immobilized on a ceramic carrier to effect a selective alcoholysis of tuna oil ethyl esters with lauryl alcohol to enrich DHA. The alcoholysis was conducted at 30 °C using a 1:3 molar ratio of tuna oil ethyl esters to Table 14.3 Comparison between ethanolysis of tuna oil TAG and esterification of tuna oil FFA using the immobilized Rhizomucor miehei lipase. Substrate

Reaction

Conversion (%)

Time (h)

DHA conc. (%)

DHA recov. (%)

Tuna TAG Tuna FFA

Ethanolysis Esterification

70 70

48 11

54 77

78 78

Abbreviations: DHA = docosahexaenoic acid, FFA = free fatty acid, TAG = triacylglycerol.

PUFA production from marine sources for use in food

345

lauryl alcohol. After 50 hours the DHA content in the residual ethyl esters increased from 23 to 52 mol% with 90 % DHA recovery. When ethyl esters comprising 60 % DHA were subjected to the alcoholysis reaction DHA was enriched to 83 %, with DHA recovery above 90 %. The productivity of the immobilized lipase was very high as indicated by studies showing that after nearly 50 runs, replacing the reaction mixture with fresh substrates every 24 hours, there was only a 15 % decrease in extent of alcoholysis. The problem with the Rhizopus delemar lipase was that the initial EPA composition was also elevated. When replacing that lipase with immobilized Rhizomucor miehei lipase after the first enrichment step, higher enrichment levels of DHA were obtained as well as a considerable decrease in the EPA content of the residual ethyl esters under similar conditions (Shimada et al., 1998). In a productivity study, a drop of only 17 % was observed after 100 24 hour reaction cycles. In an experiment involving ethyl esters constituting 60 % DHA, the DHA content of the residual ethyl esters was raised to 93 % with 74 % DHA recovery with EPA falling from about 9 to 3 %. That methodology offered higher concentration levels of DHA with the Rhizomucor miehei lipase, but lower yields of DHA in the residual ethyl ester fraction. These examples demonstrate the potential of lipase to separate and enrich DHA to high purity levels by a multi-step enzymatic treatment involving one or more lipase types.

14.3.3 Lipase esterification There are several reports on selective esterification of fish oil FFA with simple monohydric alcohols using the Rhizomucor miehei lipase to strongly discriminate between EPA and DHA, usually in hexane as a solvent. In one of the first reports, Takagi used methanol and a free fatty acid concentrate of EPA and DHA obtained by urea precipitation of Japanese sardine oil, comprising 30–40 % EPA and 25–30 % DHA (Takagi, 1989). Methyl ester product enriched with EPA (> 50 %) and a residual free fatty acid concentrate enriched with DHA (about 50 %) were obtained in yields of 60 and 40 %, respectively. Schmidtsdorff and coworkers investigated the fatty acid selectivity of the immobilized Rhizomucor miehei lipase toward fish oil fatty acids, also with methanol, to obtain residual free fatty acids comprising 48 % DHA (Langholz et al., 1989). Similar results were reported by Hills et al. on cod liver oil FFA with n-butanol using the same lipase (Hills et al., 1990). In attempts to separate EPA and DHA in fish oils by the immobilized Rhizomucor miehei lipase, Haraldsson and Kristinsson demonstrated that direct esterification of free fatty acids released from fish oil was much more efficient than ethanolysis of fish oil TAG (Haraldsson and Kristinsson, 1998). When tuna oil containing 6 % EPA and 23 % DHA was transesterified with ethanol under the same conditions as described above for the Pseudomonas lipases, 70 % conversion into ethyl esters was observed after 48 hours. The residual acylglycerol mixture comprised 54 % DHA and 6 % EPA by mol,

346 Modifying lipids for use in food with 78 % DHA recovery into the acylglycerol mixture and 75 % EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were esterified with a two-fold stoichiometric amount of ethanol 70 % conversion was obtained after only 11 hours. The residual free fatty acids comprised 77 % DHA and only 2 % EPA by mol. The recovery of both DHA in the residual free fatty acid fraction and EPA in the ethyl ester product remained very high, 78 and 89 %, respectively. Table 14.3 compares the ethanolysis results of tuna oil TAG and esterification of tuna oil FFA with ethanol using the Rhizomucor miehei lipase. The reaction is illustrated in Fig. 14.4. A modification using free fatty acids with glycerol instead of ethanol under similar esterification conditions gave good results with the same lipase as reported by Haraldsson and co-workers (Halldorsson et al., 2003b). Haraldsson and Kristinsson also demonstrated by their ethanol-based procedure that a free fatty acid concentrate of 77 % EPA and 10 % DHA could be freed from DHA in a single step and purified to higher than 90 % EPA content at 50 % conversion with 60 % recovery of EPA (Haraldsson and Kristinsson, 1998). Shimada and coworkers used a highly efficient two-step esterification of free fatty acids from tuna oil to purify DHA up to nearly 90 % purity levels (89 % by weight) with high DHA recovery (71 %) based on the initial oil using the Rhizopus delemar lipase with lauryl alcohol as the acyl acceptor (Shimada et al., 1997a). The reaction was conducted at 30 °C using a 2:1 molar ratio of lauryl alcohol to tuna oil FFA without a solvent in the presence of 20 % water based on weight of substrates. In the first step, the DHA content rose from 23 to 73 % in very high DHA recovery (84 %) at 72 % conversion obtained after 20 hours. It is noteworthy that despite the high water content the lauryl esters were not susceptible for hydrolysis. This relates to lauryl esters being very poor substrates or non-substrates to lipase. The water content was observed to have profound effects on the DHA enrichment levels with the highest value obtained at 20 % water content. It is of interest that with this lipase the shorter-chain alcohols including ethanol and glycerol gave poor results with low or no fatty acid selectivity observed. After the second enzymatic treatment the EPA content remained very low (< 2 %). The reaction is illustrated in Fig. 14.5. Shimada and coworkers have also reported on a two-step enzymatic method to purify DHA from tuna oil consisting of a lipase-promoted tuna oil hydrolysis and a subsequent selective esterification of the resulting FFA (Shimada O Tuna

OH

RML Ethanol

O Tuna/EPA

O O

DHA

OH

(77 % DHA)

Fig. 14.4 Esterification of tuna oil FFA with ethanol by immobilized Rhizomucor miehei lipase (RML) by Haraldsson and Kristinsson (1998).

PUFA production from marine sources for use in food O Tuna

OH

RDL Lauryl alcohol

RDL Lauryl alcohol

O Tuna/EPA

347

O O

DHA OH (73 % DHA)

O DHA

OH

(89 % DHA)

Fig. 14.5

A two-step enzymatic esterification of tuna oil FFA with lauryl alcohol by Rhizopus delemar lipase (RDL) by Shimada et al. (1997a).

et al., 1997b). Pseudomonas fluorescens lipase was exploited to hydrolyze tuna oil at 48 °C using a 1:1 ratio of tuna oil to water by weight. At 79 % conversion 83 % of the DHA was recovered in the FFA fraction. The free fatty acid product obtained from the enzymatic hydrolysis was subjected to a selective esterification with lauryl alcohol catalyzed by the Rhizopus delemar lipase under the above-described conditions. The DHA content in the residual FFA fraction rose from 24 to 72 % by weight in 83 % recovery (69 % overall). After a second selective esterification treatment, the DHA content was elevated to higher than 91 % in 80 % recovery (60 % overall). The examples from the groups of Haraldsson and Shimada demonstrate that enrichment levels well beyond those obtained by urea crystallization can be obtained highly efficiently by lipases. The fact that an immobilized lipase can be re-used tens of times without much deterioration in performance suggests that the application of lipase in the field of marine oils is a highly feasible choice from an industrial point of view. Based on these results, there are reasons to believe that in terms of purifying EPA and DHA, lipase can be used as a powerful alternative to traditional separation techniques up to the chromatography levels by stepwise lipase reactions. Figure 14.6 illustrates how lipases may be introduced complementary to traditional separation techniques to separate and purify EPA and DHA to various purity levels (Haraldsson and Hjaltason, 2001).

14.4 TAG concentrates of n-3 PUFA Preparation of TAG up to the 30–35 % EPA + DHA concentration level can be brought about directly on fish oils without splitting the fat by a careful selection of fish oils and simple methods such as winterization, molecular distillation and solvent crystallization (Ackman, 1988; Breivik and Dahl, 1992). A good example of such a concentrate is MaxEPA™ containing 18 % EPA and 12 % DHA. This was the first dietary n-3 supplement in the natural TAG form to be introduced to the market in the early 1980s by Seven Seas in the UK (A History of British Cod Liver Oils, 1994). It was widely used for various clinical studies for over 15 years. Another example is a cod liver oil

348 Modifying lipids for use in food Fish oil triacylglycerols (20–30 % EPA + DHA) Ethanolysis or hydrolysis Ethyl esters or free fatty acids (20–30 % EPA + DHA)

Lipase

Short-path distillation

Lipase

Ethyl esters or free fatty acids (50 % EPA + DHA)

Urea inclusion Ethyl esters or free fatty acids (80–85 % EPA + DHA) HPLC

> 95 % EPA

> 95 % DHA

Fig. 14.6 A flow diagram illustrating how lipase may be used complementary to traditional separation techniques to separate and purify EPA and DHA in fish oil to various purity levels.

concentrate comprising 16–17 % each of EPA and DHA introduced to the market in Iceland in the late 1980s by Lysi Ltd. It was produced by solvent crystallization of refined cod liver oil using acetone-water as a solvent (Haraldsson and Gudbjarnason 1986, unpublished results) and remained on that local market for a few years. TAG concentration beyond that level requires splitting of the TAG into free acids or monoesters, concentration of EPA and DHA by the various physical methods and combination of methods described in Section 14.2 above and reintroduction of such free acid or monoester concentrates into TAG concentrates (Haraldsson, 2000; Haraldsson and Hjaltason, 2001). Although the resynthesis of pure TAG highly enriched with EPA and DHA is not easily carried out by traditional chemical esterification methods, there have been several producers of such EPA- and DHA-enriched TAG by such methods. Usually the esterification reactions involved are incomplete and result in products comprising only 50–70 % TAG, largely contaminated with MAG and DAG and some residual monoesters, usually ethyl esters. There is not much public information available about the details of the

PUFA production from marine sources for use in food

349

methods used, but there are strong indications of triacetin being used as a source of the glycerol backbone with ethyl ester concentrates as the source of the n-3 PUFA by transesterification. Sodium ethoxide or sodium methoxide are used as catalysts to bring about the transesterification reaction. The volatile co-produced ethyl acetate is then simply distilled off during or after the reaction (see Fig. 14.7). Another approach is based on glycerolysis of ethyl ester n-3 PUFA concentrates by reacting glycerol with the monoesters in the presence of sodium alkoxides as a catalyst. In this case the volatile coproduct is ethanol (see Fig. 14.8). In the late 1980s lipases were introduced to the n-3 field to solve these problems of producing TAG highly enriched with EPA and DHA. Haraldsson and coworkers were the first to report the use of lipase for the preparation of such TAG (Haraldsson et al., 1989; Haraldsson and Almarsson, 1991). They used immobilized Rhizomucor miehei lipase as a biocatalyst to bring about transesterification reactions of cod liver oil with EPA and DHA concentrates. Both acidolysis and interesterification reactions were conducted without a solvent, using 10 % dosage of lipase based on weight of fat at 60–65 °C and a three-fold excess of free acids or monoesters based on the number of mol equivalents of esters present in the fish oil TAG. TAG of high purity comprising 60–65 % EPA + DHA and well over 70 % total n-3 PUFA content were produced. This is illustrated in Fig. 14.9. Yamane and coworkers have also reported on a similar solvent-free methodology to enrich cod liver oil TAG up to similar levels by lipasecatalyzed acidolysis using the Rhizomucor miehei lipase and a two-stage acidolysis approach (Yamane et al., 1992, 1993). Adachi et al. reported a similar acidolysis of sardine oil by Pseudomonas sp. lipase in organic solvents (Adachi et al., 1993). The yield of TAG and enrichment levels of EPA and O PUFA

OAc

O

OAc PUFA

O

O

PUFA O

O–Na+

O

O

OAc

Fig. 14.7

AcO

O PUFA

Transesterification of triacetin with PUFA ethyl esters using sodium ethoxide as a catalyst to produce TAG comprising PUFA. O

OH

PUFA O

OH PUFA OH

Fig. 14.8

O Na

O

O

PUFA O O

O O

HO PUFA

Glycerolysis of PUFA ethyl esters using sodium ethoxide as a catalyst to produce TAG comprising PUFA.

350 Modifying lipids for use in food O CLO

O

O O

CLO O O

O

O PUFA

RML

PUFA*

O O

PUFA* O

OR

CLO

O R

O

O PUFA*

OR

PUFA*

– H (acidolysis) – Et (interesterification)

Fig. 14.9 Enrichment of cod liver oil (CLO) with PUFA by acidolysis (R=H) or interesterification (R=Et) reactions. CLO refers to cod liver oil fatty acid composition, but PUFA* to equilibrium composition.

DHA were strongly dependent on the water content. There are numerous reports describing treatment of various types of TAG oils with n-3 PUFA concentrates of both fish and single-cell origin in lipase-catalyzed transesterification reactions. They include incorporation of n-3 fatty acids into vegetable oils (Li and Ward, 1993a; Huang and Akoh, 1994), melon seed oil (Huang et al., 1994), trilinolein (Akoh et al., 1995), evening primrose oil (Akoh et al., 1996), borage oil (Akoh and Moussata, 1998; Ju et al., 1998; Senanayake and Shahidi, 1999), palm stearin (Osorio et al., 2001) and various TAG of medium-chain fatty acids (Lee and Akoh, 1996, 1998), trilaurin, tricaprin and tricaprylin. It appears that the most efficient lipases used acted preferably at the C-1 and C-3 positions to provide positionally labeled structured TAG. High incorporation levels of the n-3 fatty acids were obtained into these positions, although high (but lower) levels of n-3 fatty acids were also incorporated into the mid-position, depending on the reaction time. High levels of n-6 and n-3 fatty acids were produced with n-6-enriched oils such as borage and evening primrose oils. Most of these reactions were conducted in organic solvents and only a few without a solvent, but there are also reports on such reactions under supercritical carbon dioxide conditions (Shishikura et al., 1994). The fish oil TAG transesterification approach was obstructed by the excessive amounts of n-3 PUFA concentrates needed to obtain high enrichment levels of EPA and DHA into fish oil (Haraldsson, 2000). Haraldsson and coworkers developed a method to produce TAG with composition identical to that of the concentrate being used to avoid the above-mentioned limitations (Haraldsson et al., 1991, 1993, 1995). This procedure is based on a direct esterification of stoichiometric amount of free fatty acids with glycerol, and it enabled synthesis of TAG homogeneous with EPA or DHA, i.e. 100 % EPA or DHA. The immobilized Candida antarctica lipase was observed to offer superiority over the Rhizomucor miehei lipase in esterifying glycerol with free fatty acids of varying n-3 PUFA content (Haraldsson et al., 1991). That lipase was highly efficient in generating TAG of both 100 % EPA and DHA content using only stoichiometric amount of pure EPA and DHA (Haraldsson et al., 1995). No solvent was required and the reaction was performed at 65 °C under vacuum with 10 % dosage of the immobilized

PUFA production from marine sources for use in food

351

lipase based on substrate weight. The reaction is displayed in Fig. 14.10 for EPA. TAG homogeneous with both EPA and DHA of excellent purity were accomplished in virtually quantitative yields. The same methodology was used by Kosugi and Azuma to prepare nearly pure TAG (96 %) of EPA, DHA and arachidonic acid under similar conditions using the Candida antarctica lipase (Kosugi and Azuma, 1994). There are also reports on a similar direct esterification of glycerol with n-3 PUFA concentrates where the reaction was conducted in an organic solvent (Li and Ward, 1993b; Cerdan et al., 1998; He and Shahidi, 1998). TAG concentrate preparation by direct esterification of n-3 PUFA-enriched partial acylglycerols obtained from Candida rugosa lipase promoted hydrolysis of fish oil with n-3 PUFA as free acids has also been reported. Such an acylglycerol mixture obtained at 70 % hydrolysis level of tuna oil containing 4 % EPA and 53 % DHA was treated by Tanaka et al. with n-3 PUFA containing 23 % EPA and 57 % DHA to obtain TAG of higher than 90 % purity (Tanaka et al., 1994). The reaction was conducted at 50 °C with a three-fold molar excess of n-3 PUFA using 40 wt % molecular sieves as a dehydrating agent and an immobilized Chromobacterium viscosum lipase. Similar results were obtained by McNeill and coworkers in their treatment of an acylglycerol mixture from fish oil hydrolysis with stoichiometric amounts of DHA-enriched fatty acids (McNeill et al., 1996; Moore and McNeill, 1996). TAG of 95 % purity were obtained with both the immobilized Rhizomucor miehei and Candida antarctica lipases with continuous removal of water using vacuum at 55 °C. There is no doubt that the Candida antarctica lipase offers superiority over other lipases in terms of TAG synthesis involving n-3 PUFA. That lipase is highly efficient, tolerating the n-3 PUFA very well, and highly pure TAG were produced under the proper conditions with little or no contamination by MAG or DAG. No solvent is needed and only stoichiometric amounts of substrates. That lipase is suitable for the production of pure TAG of whatever desired composition identical to that of the starting free acids as was demonstrated by Haraldsson and coworkers (Haraldsson et al., 1991). This lipase is, therefore, highly feasible for industrialization (Haraldsson and Hjaltason, 2001), and there is little doubt that this is the future lipase for production of highly pure TAG comprising high levels of n-3 PUFA.

O OH

O

OH

EPA CAL

3 EPA OH

O O

EPA O

3 H2O

O

OH O

EPA

Fig. 14.10 Direct esterification of glycerol with pure EPA to prepare structured TAG homogeneous with EPA by immobilized Candida antarctica lipase (CAL).

352 Modifying lipids for use in food

14.5 Positionally labeled structured TAG derived from fish oil Structured TAG comprising certain types of fatty acids at the outer-positions and different fatty acids at the mid-position of the glycerol backbone have gained increasing attention as dietary and health supplements. Of particular interest from human nutritional point of view are structured TAG possessing biologically active long-chain PUFA located at the mid-position with MCFA at the end-positions (Miura et al., 1999). The MCFA located at the endpositions undergo a rapid hydrolysis by pancreatic lipase, are absorbed into the intestines and are rapidly carried into the liver where they are consumed as a quick source of energy. The remaining 2-monoacylglycerols (2-MAG), on the other hand, become a source of essential fatty acids after being absorbed through the intestinal wall (Christensen et al., 1995). They are accumulated as TAG in the adipose tissues or as phospholipids in the cell membranes from where they can be released upon demand for their desired biological functions. Various approaches have been designed to undertake the preparation of positionally labeled MLM structured TAG comprising n-3 PUFA and MCFA. The simplest method is to treat fish oil, of which the mid-position usually constitutes a significantly higher n-3 PUFA composition than the end-positions (Christie, 1986; Hölmer, 1989), with a regioselective lipase. The lipase acts preferably or exclusively at the outer-positions by promoting fatty acid exchange reactions with MCFA as free acids (acidolysis) or monoesters (interesterification). Shimada and coworkers reported on the production of such structured TAG enriched with DHA at the mid-position by exchanging fatty acids at the end-positions of tuna oil for caprylic acid (CA) using an immobilized 1,3regioselective Rhizopus delemar lipase (Shimada et al., 1996, 2000). The reaction was conducted at 30 °C using approximately a six-fold molar excess of CA based on the fish oil TAG. After 40 hours the incorporation level of CA into the fish oil had reached a steady state and remained at 43 mol%. The immobilized lipase could be used 15 times (over 30 days) without a significant decrease in the CA content. Regiospecific analysis indicated that the regioselectivity of the lipase was very high and that the extent of acyl migration was very low. The fatty acid composition of the mid-position was observed to hardly change during the reaction, whereas the fatty acid composition of the end-positions changed dramatically, apart from DHA being resistant to the lipase action. The reaction is shown in Fig. 14.11. Similar acidolysis was reported by Jennings and Akoh to incorporate capric acid into TAG highly enriched with EPA (41 %) and DHA (33 %) with and without organic solvent at 55 °C (Jennings and Akoh, 1999). The highest capric acid incorporation levels of 65 % were obtained in hexane with a molar ratio of 1:8 between the TAG and capric acid, but 56 % under solventfree conditions with a 1:6 molar ratio. Analysis of the mid-position suggests

PUFA production from marine sources for use in food O FO

O O

CA

O O O

O

O

PUFA

OH

CA

O O

PUFA O O

RDL FO

353

O

CA

Fig. 14.11 Production of fish oil derived positionally labeled structured TAG containing caprylic acid (CA) and PUFA by fish oil acidolysis using immobilized Rhizopus delamar lipase (RDL).

that some acyl-migration was taking place during this reaction by the presence of capric acid at that position. Yamane and coworkers enriched single-cell oil (SCO) of high DHA (35 %) and docosapentaenoic acid (DPA; 10 %) content with CA under acidolysis conditions using Rhizomucor miehei and Pseudomonas fluorescens lipases (Iwasaki et al., 1999). Both lipases required extended reaction time of several days and high ratios of CA to SCO TAG. Much higher incorporation levels were obtained for the Pseudomonas lipase with the final CA content of the TAG reaching 65 mol% after 168 hours at 18.8 CA/SCO molar ratio at 30 °C. Xu and coworkers studied the effects of water content and reaction time on production of such positionally labeled TAG of the MLM type under pilot batch conditions using the immobilized Rhizomucor miehei lipase on fish oil and capric acid (substrate ratio 6:1 FFA/TAG in mol) under solvent-free conditions at 60 °C (Xu et al., 1998). After 30 hours over 65 % incorporation of the MCFA had taken place into the end-positions together with 12 % acylmigration levels into the mid-position during the reaction. Distillation under vacuum was used to separate the structured TAG and free acid products when further acyl-migration was noticed to take place. They also reported the use of a packed-bed reactor with the same lipase as a biocatalyst to treat menhaden oil under acidolysis with caprylic acid (Xu et al., 2000). The task to generate such fish oil derived structured TAG was addressed differently by Bornscheuer and coworkers in their two-step strategy (Schmid et al., 1998; Soumanou et al., 1998). In the first step, 2-MAG enriched with n-3 PUFA were generated from fish oil TAG by lipase-catalyzed ethanolysis using a 1,3-regioselective lipase in organic solvent. The resulting 2-MAG were subsequently esterified in a second enzymatic step. This strategy worked well for less unsaturated TAG, but when fish oils containing n-3 PUFA were used less favorable results were accomplished. This relates to the low yield of the 2-MAG intermediate as a result of low activity of the lipases toward TAG comprising EPA and especially DHA, but also to complications in isolating and purifying the n-3 PUFA-enriched 2-MAG. The first step was improved when lipases that are not usually considered to be 1,3-regioselective, Pseudomonas fluorescens and Candida antarctica lipases, were used in the ethanolysis reaction (Irimescu et al., 2001b; Wongsakul et al., 2003). A

354 Modifying lipids for use in food further n-3 PUFA enrichment of the 2-MAG from tuna oil was effected by low-temperature crystallization by freezing out the saturated 2-MAG (Wongsakul et al., 2003). Yamane and coworkers followed the methodology of Bornscheuer and coworkers in producing structured TAG enriched with DHA at the midposition and CA residues at the end-positions (Irimescu et al., 2001b). They exploited the immobilized Candida antarctica lipase in a highly efficient and regioselective ethanolysis of bonito oil TAG to yield 93 % of the 2-MAG with 44 % DHA content in only two hours at 35 °C. The subsequent re-esterification was conducted directly on the crude reaction mixture in the presence of the ethyl esters produced from the end-positions of the original oil, after filtering off the enzyme and stripping off the excessive ethanol. The reaction was conducted for only one hour under vacuum at 35 °C using the immobilized Rhizomucor miehei lipase and excessive amount of ethyl caprylate (7–8-fold stoichiometric excess). Structured TAG comprising well over 40 % DHA at the mid-position and well over 90 % CA content at the endpositions were obtained. Their approach is illustrated in Fig. 14.12. This method offers various advantages over other reported methods. It is fast, the regioselectivity is high and acyl-migration is kept at a minimum. High yields of the 2-MAG intermediates are obtained with the original fatty acid composition in that position largely preserved. However, an efficient separation, presumably by short-path distillation and purification of the final product, needs to be demonstrated.

14.6 MLM type structured TAG comprising pure n-3 PUFA and MCFA Positionally labeled symmetrically structured TAG of the MLM type comprising pure homogeneous PUFA such as EPA or DHA and MCFA of absolute regioisomeric and chemical purity are ideally suited as libraries of pure compounds for various purposes. For example, they may be exploited to compare the effects of individual fatty acids by biological screening, as standards for analysis, fine chemicals and as potential drugs. Figure 14.13 O FO

O

O

PUFA

O OH CAL

O O O

FO

OH

O

OH

O

O

PUFA CA

O RML

CA

O

PUFA

O O O O

CA

Fig. 14.12 Production of fish oil derived positionally labeled structured TAG containing caprylic acid (CA) and PUFA by fish oil ethanolysis using immobilized Candida antarctica lipase (CAL) and re-esterification of the resulting 2-MAG with CA by Rhizomucor miehei lipase (RML) (FO = fish oil fatty acids located at the end-positions of the glycerol moiety of the fish oil TAG).

PUFA production from marine sources for use in food

355

O O O O O O

O O O O O O

Fig. 14.13 The structure of homogeneous positionally labeled symmetrically structured TAG of the MLM type containing capric acid and EPA (top) and caprylic acid and DHA (bottom).

shows the structure of homogeneous positionally labeled symmetrically structured TAG of the MLM type comprising capric acid and EPA (top) and CA and DHA (bottom). Such structured TAG require total synthesis from glycerol with a full regioselectivity control. There are two alternative approaches: a fully enzymatic approach involving two or three enzymatic steps and a two-step chemoenzymatic approach. In the fully enzymatic approach, the first step involves the synthesis of homogeneous TAG containing the PUFA that is intended to be accommodated at the mid-position. In a two-step process this is followed by transesterification with an ethyl ester of MCFA by a 1,3-regioselective lipase (see Fig. 14.14). An alternative three-step procedure requires a 2-MAG homogeneous with the PUFA that is achieved by alcoholysis of the homogeneous TAG by a 1,3regioselective lipase. The pure MCFA is then introduced to the end-positions by lipase in the third step (see Fig. 14.15). In the first step of the chemoenzymatic approach a 1,3-regioselective lipase is exploited to prepare 1,3-DAG of a pure MCFA. This is followed by a subsequent chemical coupling reaction of pure EPA or DHA into the free mid-position (see Fig. 14.16 below). Both approaches have their drawbacks and limitations. The fully enzymatic approach is obstructed by the need of a three-fold excess of the pure EPA or DHA. Also, large excesses of MCFA are often required in order to reach satisfactory results in terms of yields and purity of the final structured TAG. The main advantage is that the overall process constitutes environmentally friendly processes, where no toxic and hazardous chemicals or organic solvents are involved. This approach is usually hampered by extreme difficulties in affording products of absolute regioisomeric and chemical purity that may need tedious purification processes involving organic solvents. Provided that a strict regiocontrol is maintained using a fully regioselective

356 Modifying lipids for use in food O

O

O

OH OH

EPA

OH

O

EPA

CA

O O

CAL OH

O

O

EPA

CA

O

EPA

O O

O O

RML O

EPA

O

CA

Fig. 14.14 Synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and EPA by a fully enzymatic two-step approach using immobilized Candida antarctica lipase (CAL) for direct esterification and Rhizomucor miehei lipase (RML) for interesterification. O O DHA

O

DHA

O

OH

OH

O O

O

CA

CAL

O

O

O

DHA

O OH

CA

DHA

O O O

RML

DHA

O

OH

CA

Fig. 14.15 Synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and DHA by a fully enzymatic three-step approach using immobilized Candida antarctica lipase (CAL) for ethanolysis of TAG homogeneous with DHA and Rhizomucor miehei lipase (RML) for a subsequent esterification of the resulting 2-MAG. (The first step to produce the homogeneous TAG is not included.) O

O OH OH

CA

OH

RML

CA

O

O O

EPA OH DCC

OH O

O CA

O

EPA O O

OH O

CA

O

CA

Fig. 14.16 Chemoenzymatic synthesis of MLM type symmetrically structured TAG containing caprylic acid (CA) and EPA by immobilized Rhizomucor miehei lipase (RML) and dicyclohexylcarbodiimide coupling agent (DCC) by Yamane’s approach.

lipase and a suitable coupling agent, the chemoenzymatic approach has the advantages of offering chemically and regioisomerically pure products where only stoichiometric amounts of the pure fatty acids are needed. The drawbacks relate to use of chemicals and organic solvents, but that is widely practised in pharmaceutical synthesis and can be justified when the advantages in terms of purity are borne in mind. When homogeneous products of that purity are involved, analytical methods such as high-field 1H and 13C nuclear magnetic resonance (NMR) spectroscopy are ideally suited to monitor the regioisomeric purity and regioselectivity control.

14.6.1 MLM type structured TAG by fully enzymatic approach The first attempts involving a fully enzymatic process to produce regioisomerically pure structured TAG of the homogeneous MLM type were

PUFA production from marine sources for use in food

357

reported by Yamane’s group (Irimescu et al., 2000, 2001c). Their two-step approach was based on a lipase-promoted preparation of TAG containing pure EPA (EEE) and a subsequent interesterification with ethyl caprylate by a 1,3-regioselective lipase. This process is illustrated in Fig. 14.14. Immobilized Candida antarctica lipase was used to esterify glycerol with pure EPA to afford the homogeneous EEE in 90 % conversion yield. The crude reaction product mixture was then subjected to the second enzymatic step without purification, apart from removal of the lipase. That step was also conducted without a solvent using immobilized 1,3-regioselective Rhizomucor miehei lipase and a hundred-fold molar excess (50-fold stoichiometric amount) of ethyl caprylate (Yamane, 2000). The final reaction product mixture constituted 89 % of the intended CEC (where C is caprylic acid and E is EPA), but no attempts were made to isolate and purify the desired product from the bulk of the reaction product mixture constituting only 3 wt % of the desired structured TAG. Shimada and coworkers made an effort to simplify Yamane’s approach somewhat by treating TAG homogeneous with EPA in three successive acidolysis reactions using 15 mol parts of CA (Kawashima et al., 2001). The reaction was conducted for 48 hours each time at 30 °C using their immobilized Rhizopus delemar lipase. After the three successive acidolysis reactions, the CA content of the TAG product reached 66 mol%. The product was still a mixture constituting 86 wt % of the desired CEC structured TAG, but it was contaminated with 2 % of the undesired regioisomeric CCE. No attempts were made by the groups of Yamane and Shimada to isolate and purify the desired product or to fully characterize it, and the required purification by fractional multi-step molecular distillation and preparative column chromatography is expected to be tedious. The main impediment in the fully enzymatic synthesis of MLM-type structured TAG comprising MCFA such as CA and DHA (CDC) is the very low activity of 1,3-regioselective lipase on TAG containing DHA located at the end-positions. Such TAG are very resistant to lipase action, and DHA remains in place at the end-positions causing the final product to constitute a mixture of regioisomers. Yamane and coworkers managed to solve this by a three-step modification based on a highly 1,3-regioselective lipase-catalyzed ethanolysis of the homogeneous DHA TAG and a subsequent lipase-promoted esterification of the resulting 2-MAG with a different lipase (Irimescu et al., 2001a). The immobilized Candida antarctica lipase was observed to display excellent regioselectivity in ethanolysis of both tridocosahexaenoylglycerol (DDD) and trieicosapentaenoylglycerol (EEE) at 35 °C using a 33-fold stoichiometric excess of ethanol to TAG. For DHA a mixture was afforded comprising 93 % of the desired 2-MAG together with 2 % of starting TAG and 5 % of the 1,2-DAG intermediate. There was no noticeable formation of any of the undesired regioisomers such as 1,3-DAG or 1(3)-MAG during this step. The crude product mixture with the co-produced DHA or EPA ethyl esters

358 Modifying lipids for use in food present was introduced to the third enzymatic re-esterification reaction. This was transesterification with a 20-fold molar excess of ethyl caprylate using the immobilized Rhizomucor miehei lipase as biocatalyst under reduced pressure at 35 °C for one to five hours. The process is shown in Fig. 14.15. The product contains not only the structured TAG contaminated with other acylglycerols that need to be removed, but also two molar equivalents of EPA or DHA ethyl esters and excessive amounts of ethyl caprylate that need to be separated. Therefore, the desired structured TAG product constituted less than 15 % by weight in the crude reaction product mixture. As before, the structured TAG were not isolated nor were they fully characterized. Again, it is evident that tedious chromatography procedures will be needed for purifying these positionally homogeneously labeled structured TAG obtained from the fully enzymatic approach, demanding organic solvents. Yamane’s method has recently been modified and significantly improved by Hou and coworkers (Irimescu et al., 2002). They managed to improve the regiocontrol and yields in the second step by lowering the temperature to 25 °C and carefully controlling the ratio of the substrates to a molar ratio of 77:1 between ethanol and the homogeneous TAG of DHA and various other PUFA including EPA. For DHA the final acylglycerol reaction mixture constituted 97 % of the desired 2-MAG (together with 3 % of the corresponding 1,2-DAG) after seven hours in 93 % recovery yield as based on initial TAG but, evidently, some glycerol was formed during the reaction (varying from 3–20 % depending on type of PUFA). For the corresponding EPA synthesis, the purity of the 2-MAG was 98 % in 75 % recovery yield; 98 % pure 2-MAG of DHA was obtained from the reaction mixture after purification by selective extraction in 87 % yield. This treatment required the use of organic solvents (acetonitrile, hexane and chloroform) to remove excessive amounts of the PUFA, ethanol and the co-produced glycerol. The subsequent reesterification step was dramatically improved when the purified 2-MAG was directly esterified with a stoichiometric amount of CA at 25 °C with the immobilized Rhizomucor miehei lipase under vacuum. The desired regioisomerically pure DHA structured TAG adduct was obtained after eight hours in 96 % purity. Again, isolated yields of the purified structured TAG from these very successful processes were not reported and need to be demonstrated together with a full characterization of these products and their 2-MAG intermediates.

14.6.2 MLM type structured TAG by chemoenzymatic approach The chemoenzymatic approach is based on lipase regioselectivity to produce symmetric 1,3-DAG of a pure MCFA as a key intermediate. A subsequent chemical introduction of a long-chain PUFA into the mid-position results in a symmetrically structured TAG of the MLM type. The basis for the enzymatic part of the chemoenzymatic approach was laid by Schneider and coworkers in their lipase-promoted 1,3-DAG generation (Berger et al., 1992; Aha et al.,

PUFA production from marine sources for use in food

359

2000). A whole range of regioisomerically pure 1,3-DAG of medium- and long-chain saturated fatty acids was obtained in good yields (74–85 %) from glycerol adsorbed on silica gel using the immobilized Rhizomucor miehei lipase in organic solvents. Vinyl esters were superior to free acids and methyl esters as acyl donors in terms of reaction rate and product yields. The regioselectivity, however, was by no means perfect since there were clear indications of acyl-migration taking place under these conditions. Yamane and coworkers were the first to report a chemoenzymatic synthesis of an MLM type structured TAG containing pure EPA and caprylic acid (Rosu et al., 1999). In the first step, regioisomerically pure 1,3-DAG was prepared by modification of the procedure of Schneider and coworkers using Lipozyme as the biocatalyst. Yields comparable to those of Schneider and coworkers were obtained (75–80 %). Purified 1,3-DAG (95 %) was then subjected to the subsequent chemical esterification step, where dicyclohexylcarbodiimide (DCC) was used as a chemical coupling agent to introduce pure EPA into the mid-position of the 1,3-DAG in the presence of 4-dimethylaminopyridine (DMAP). This resulted in TAG of 98 % purity, but in only 42 % yield, and the product turned out not to be regioisomerically pure. The synthetic approach is illustrated in Fig. 14.16. Haraldsson and coworkers managed to improve dramatically on the chemical coupling step by replacing DCC with 1-(3-dimethylaminopropyl)-3ethylcarbodiimide (EDCI) as a coupling medium in the presence of DMAP. This was demonstrated in their synthesis of a structured TAG comprising pure stearic acid (Haraldsson et al., 2000) and caprylic, capric and lauric acid (Halldorsson et al., 2001) residues at the end-positions with pure EPA or DHA at the mid-position by the chemoenzymatic approach. The enzymatic step was based on the procedure of Schneider and coworkers (Berger et al., 1992; Aha et al., 2000) in ether to afford the regioisomerically pure 1,3DAG adducts in moderate to good yields after purification by crystallization. Pure EPA and DHA were then introduced to the mid-position using EDCI as a coupling agent in the presence of DMAP in dichloromethane in excellent yields (≥ 90 %), after purification by treatment on silica. Recently, Haraldsson and coworkers reported a modification of their chemoenzymatic approach towards synthesis of structured TAG of the above MLM type (Halldorsson et al., 2003a). A dramatic improvement of the regiocontrol and yields of the enzymatic step were described. This is based on a rapid, irreversible transesterification of glycerol using vinyl esters of the MCFA and the immobilized Candida antarctica lipase in dichloromethane at 0–4 °C. The Candida antarctica lipase acted exclusively at the glycerol end-positions and no acyl-migration took place. The yields (90–92 %; see Table 14.4) are based on pure material after re-crystallization. In the subsequent coupling reaction EDCI was used as a chemical coupling agent to introduce EPA and DHA into the mid-position of the 1,3-DAG adducts. The reaction was conducted at room temperature in dichloromethane in the presence of 30–50 % DMAP (as based on mol) using an exact

360 Modifying lipids for use in food Table 14.4 Yields and type of products and 1,3-DAG intermediates (1a,b,c) from the chemoenzymatic synthesis of MLM type structured TAG comprising pure MCFA and EPA (2a,b,c) or DHA (3a,b,c). Compound

MCFA

PUFA

Yield

1a b c 2a b c 3a b c

–C7H15 –C9H19 –C11H23 –C7H15 –C9H19 –C11H23 –C7H15 –C9H19 –C11H23

– – – EPA EPA EPA DHA DHA DHA

90 92 90 90 93 92 90 94 95

% % % % % % % % %

Abbreviations: DHA = docosahexaenoic acid, EPA = eicosapentaenoic acid, MCFA = medium-chain fatty acid, PUFA = polyunsaturated fatty acid.

stoichiometric amount of EPA or DHA as based on the 1,3-DAG adduct. The reactions were completed in 12–15 hours. Chemically and regioisomerically pure structured TAG were afforded as colorless and slightly yellowish oils, respectively, for the EPA and DHA adducts, in yields of 90–95 % (see Table 14.4) after chromatographic treatment on silica gel. No sign of any acyl-migration side-reaction was observed to take place during the coupling reaction. Their approach is demonstrated in Fig. 14.17. A whole range of structured MLM type TAG was synthesized by this methodology ranging from C8 to C16 saturated fatty acids. All C8–C12 products and intermediates are listed in Table 14.4 and all were obtained in excellent yields. The yields are based on isolated and purified material and all compounds were fully characterized and their regioisomeric and chemical purity established by modern methods. High-resolution 1H and 13C NMR spectroscopy were of great use to monitor the regiocontrol of both reactions and to establish the regioisomeric purity of all compounds involved.

14.7 Industrial aspects and future trends Lipase has been in use in the fats and oils field since the 1980s with the use of lipase in the n-3 PUFA area dating back before 1990. Despite the existence of a number of feasible processes, it has taken a long time for lipase to become accepted and commercialized within the n-3 PUFA industry. There is, however, little doubt that several Japanese industrial companies are using lipase for producing n-3 PUFA concentrates on an industrial scale both through kinetic resolution processes and a subsequent esterification into TAG. It is by no means easy to get detailed information on this from the literature. Due to regulatory issues in Japan, chemically concentrated ethyl esters and

PUFA production from marine sources for use in food

OH

OH

MCFA

O

CAL OH

O

O

O MCFA

O

O O

MCFA OH O O

PUFA

OH

O

PUFA O O

EDCI/DMAP MCFA

361

O

MCFA

Fig. 14.17 Chemoenzymatic synthesis of MLM type symmetrically structured TAG comprising MCFA (C8–C12) and PUFA (EPA or DHA) by immobilized Candida antarctica lipase (CAL) and EDCI.

acylglycerols are not appproved for food or dietary supplement application. Therefore products for such application in Japan must be enzymatically concentrated. There are also companies in Europe and presumably North America that have started industrial-scale production of n-3 PUFA concentrates as TAG by the lipase technology. There is little doubt that this will develop further in the near future. Table 14.5 lists some of the main n-3 PUFA concentrate producers with indications of their main products in terms of composition and type (ethyl esters vs TAG). The names and homepages of the companies are provided. The concentrates are divided into three levels of EPA and DHA, 30–70 %, 70–90 % and 90–100 %, as well as ethyl esters and TAG (or acylglycerols). The table shows that a number of companies produce the 30–70 % and 70–90 % concentration levels both as ethyl esters and TAG. Some of these products are highly enriched with EPA, others with DHA and still others with EPA and DHA. In Europe TAG concentrates are dominant in the market while ethyl esters are widely used in USA. There appears, however, to be a trend in the USA that the market is moving from ethyl esters to TAG although this is a slow process. The highest purity category includes virtually pure EPA and DHA. Only one company supplies such purity levels as TAG, i.e. Chemport in Korea. A few Japanese suppliers provide such products as ethyl esters. Most of the Japanese companies making the highly concentrated n-3 ethyl esters are using the final product as an active pharmaceutical ingredient (API) and the bulk oil is not commercially available. Omacor® is a pharmaceutical ethyl ester product from Pronova Biocare in Norway that contains 460 mg EPA and 380 mg DHA per 1000 mg. This has a marketing license in EU and USA as well as other countries as a prescription drug. Likewise, Mochita in Japan has a highly concentrated EPA (higher than 96 %) approved as a pharmaceutical drug in Japan. It is anticipated that structured TAG will also be developed further where lipase will be utilized. There are already several such products on the marked. Marinol™ D-40 is a concentrated enzymatically produced fish oil from Lipid Nutrition (a lipid division of Loders Croklaan: www.lipidnutrition.com). It has a total of 40 % DHA in the form of glyceride. Marinol™ C-38 is a similar product comprising both EPA and DHA, total of 38 %. All of the marinol products can be used in dietary supplements and find application in

Table 14.5

Producers of EPA and DHA n-3 PUFA concentrates.

Company

Country

Web page

PUFA concentrates (%) 30–70

Pronova Biocare Ocean Nutrition Croda Chemport KD Pharma Napro Pharma Arjuna Naturals Maruha Nissui Lipid Nutrition Sepu DSM Nutr. Prod. Sanmark Bizen NOF Tama Biochemical Harima Chemicals

Norway Canada UK Korea Germany Norway India Japan Japan Malaysia Korea USA/Holl China Japan Japan Japan Japan

Abbreviation: PUFA = polyunsaturated fatty acid.

epax.com ocean-nutrition.com croda.com/europe/hc chemport.co.kr kd-pharma.de napro-pharma.no arjunanatural.com maruha.co.jp nissui.co.jp lipidnutrition.com sepufc.com nutraaccess.com sanmarkltd.com bizen.co.jp nof.co.jp tama-bc.co.jp harima.co.jp

70–90

90–100

EE

TG

EE

TG

x x x x x x x

x x x x

x x x x x

x x x x

x x x x x

x

x x x

x x x

EE

TG

x x

x

x x

x

x x

x

PUFA production from marine sources for use in food

363

functional food, medical food, enteral food and infant nutrition. Japanese producers of such structured TAG include Nissui and NOF. There are already strong indications that n-3 PUFA-enriched phospholipids will become important in the near future. Purified marine phospholipids highly enriched with DHA, but lower in EPA, were observed to play decisive roles in aquaculture involving the production of halibut juveniles from larvae (Hjaltason et al., 2005a,b). Krill oil from Antarctic krill has already been marketed as a source of DHA in the form of a mixture of marine phospholipids and marine TAG by Neptune Technologies and Bioresources Inc. in Canada (www.neptunebiotech.com) (Sampalis, 2005). A French company is also producing purified marine phospholipids on a relatively small scale (Phosphotech: www.phosphotech.com). The Japanese companies NOF and Bizen are also suppliers of such purified marine type phospholipids.

14.8

References

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ACKMAN R G

364 Modifying lipids for use in food BREIVIK H, HARALDSSON G G

and KRISTINSSON B (1997), Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid, J Am Oil Chem Soc, 74, 1425–1429. BROWN L B and KOLB D X (1955), Application of low temperature crystallization in the separation of the fatty acids and their compounds, Prog Chem Fats Lipids, 3, 57–94. CERDAN L E, MEDINA A R, GIMENEZ A G, GONZALEZ M J I and GRIMA E M (1998), Synthesis of polyunsaturated fatty acid-enriched triglycerides by lipase-catalyzed esterification, J Am Oil Chem Soc, 75, 1329–1337. CHRISTENSEN M S, HÖY C-E, BECKER C C and REDGRAVE T G (1995), Intestinal absorption and lymphatic transport of eicosapentaenoic (EPA), docosahexaenoic (DHA), and decanoic acids: dependence on intramolecular triacylglycerol structure, Am J Clin Nutr, 61, 56– 61. CHRISTIE W W (1986), The positional distributions of fatty acids in triglycerides, in Hamilton R J and Rossell J B, Analysis of Oils and Fats, London, Elsevier Science, 313–339. GAIDAY N V, IMBS A B, KUKLEV D V and LATYSHEV N A (1991), Separation of natural polyunsaturated fatty acids by means of iodolactonization, J Am Oil Chem Soc, 68, 230–233. GELLESVIK D R (1991), Fatty acid specificity of bile salt-dependent lipase: Enzyme recognition and super-substrate effects, Biochim Biophys Acta, 1086, 167–172. GUIL - GUERRERO J L and BELARBI E - H (2001), Purification process for cod liver oil polyunsaturated fatty acids, J Am Oil Chem Soc, 78, 477–484. HAAGSMA N, VON GENT C M, LUTEN J B, DE JONG R W and DOORN E (1982), Preparation of an n-3 fatty acid concentrate from cod liver oil, J Am Oil Chem Soc, 59, 117–118. HALLDORSSON A and HARALDSSON G G (2004), Fatty acid selectivity of lipase towards astaxanthin diesters, J Am Oil Chem Soc, 81, 347–353. HALLDORSSON A, MAGNUSSON C D and HARALDSSON G G (2001), Chemoenzymatic synthesis of structured triacylglycerols, Tetrahedron Lett, 42, 7675–7677. HALLDORSSON A, MAGNUSSON C D and HARALDSSON G G (2003a), Chemoenzymatic synthesis of structured triacylglycerols by highly regioselective acylation, Tetrahedron, 59, 9101– 9109. HALLDORSSON A, KRISTINSSON B, GLYNN C and HARALDSSON G G (2003b), Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol, J Am Oil Chem Soc, 80, 915–921. HALLDORSSON A, KRISTINSSON B and HARALDSSON G G (2004), Lipase selectivity toward fatty acids commonly found in fish oil, Eur J Lipid Sci Technol, 106, 79–87. HARALDSSON G G, HÖSKULDSSON P A, SIGURDSSON S TH, THORSTEINSSON F and GUDBJARNASON S (1989), The preparation of triglycerides highly enriched with ω-3 polyunsaturated fatty acids via lipase catalyzed interesterification, Tetrahedron Lett, 30, 1671–1674. HARALDSSON G G (2000), Enrichment of lipids with EPA and DHA by lipase, in Bornscheuer U T, Enzymes in Lipid Modification, Weinheim, Wiley-VCF, 170–189. HARALDSSON G G (2005), Structured triacylglycerols comprising omega-3 polyunsaturated fatty acids, in Hou C T, Handbook of Industrial Biocatalysis, Boca Raton, FL, CRC Press, Inc., Taylor and Francis Group, 18-1–18-21. HARALDSSON G G and ALMARSSON Ö (1991), Studies on the positional specificity of lipase from Mucor miehei during interesterification reactions of cod liver oil with n-3 polyunsaturated fatty acid and ethyl ester concentrates, Acta Chemica Scandinavica, 45, 723–730. HARALDSSON G G and HJALTASON B (1992), Using biotechnology to modify marine lipids, INFORM, 3, 626–629. HARALDSSON G G and HJALTASON B (2001), Fish oils as sources of important polyunsaturated fatty acids, in Gunstone F D, Structured and Modified Lipids, New York, Marcel Dekker, Inc., 313–350. HARALDSSON G G and KRISTINSSON B (1998), Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase, J Am Oil Chem Soc, 75, 1551–1556.

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365

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15 Production, separation and modification of phospholipids for use in food J. Pokorný, Institute of Chemical Technology, Prague, Czech Republic

15.1

Introduction

15.1.1 Chemical structure of phospholipids Phospholipids are natural products containing fatty acids and phosphoric acid in their molecule. They also contain an alcohol; in phospholipids important for food purposes this almost exclusively glycerol (they are called glycerophospholipids). The 1- and 2-positions of the glycerol molecule are esterified with fatty acids and the 3-position with phosphoric acid. Saturated fatty acids, frequently palmitic acid, are preferentially bound at position 1, while position 2 is preferentially esterified with polyenoic fatty acids, most often linoleic acid. In the literature, phospholipids are often abbreviated as follows: PC = phosphatidylcholines; PE = phosphatidylethanolamines; PI = phosphatidylinositols; PS = phosphatidylserines; PA = phosphatidic acids; PG = phosphatidylglycerols; PDG = phosphatidyldiacylglycerols; SP = sphingolipids. In addition to the hydroxyl group of phosphoric acid esterified with a diacylglycerol residue, another hydroxyl group of phosphoric acid is esterified with choline, ethanolamine, serine, inositol, glycerol or diacylglycerol (Fig. 15.1). In many scientific texts, they are called by their group name, i.g. phosphatidylcholine. However, the phospholipid classes are not chemically individual as they contain different fatty acids; therefore, they may each be considered as a group of structurally related compounds. Nevertheless, they are usually called by their class name, e.g. ‘phosphatidylcholine’ means a group of all the phosphatidylcholines present. Some physiologically important phospholipids do not contain bound glycerol, but an acyl group is bound as an amide to the primary amino group of a long-chain aminoalcohol, such as

370 Modifying lipids for use in food O

O

R1

O

R1

O

R2

O

R2

O

O O

O

OH

P

O O

O

N

O

P O

O Phosphatidic acids

Phosphatidylcholines

O

O

R1

O

R2

O

1

O

R2

O

R O O

O

NH3

O

P

OH O O

O

P

OH O

O

O Phosphatidylethanolamines

Phosphatidylglycerols O

O R1

O

R2

O

O O

O

P

OH OH O HO

R1

O

R2

O

OH OH

O O

O

P O

O

O Phosphatidylinositols

NH3

O

OH

Phosphatidylserines OH

O O

R

NH

P

O

N

O

O Sphingomyelins O O HO

R O

O

P

O

X

O Lysophosphatidyl derivatives

Fig. 15.1

Chemical structures of some important phospholipids (R – the respective fatty acid residues; X – residues bound to phosphatidic acid).

sphingosine or related compounds. Sialic acid or neuraminic acid may also be present. They are bound in mycophospholipids, containing also bound sugars.

Production, separation and modification of phospholipids 371 15.1.2 Sources of phospholipids Phospholipids are present in small amounts (up to 1 % dry weight) in all plant or animal tissues because phospholipids form the boundary layers (membranes) of all cells and sub-cellular particles. They are consumed with food but, for industrial purposes, only two sources are of importance, i.e. egg yolk and crude soybean oil. Neural tissues, such as brain, are also rich in phospholipids, but they are less readily available for industrial production. The phospholipid content in soybeans is not much different from the content in other oilseeds, but most oilseeds contain about 40 % oil, while soybeans contain only about 20 %. Therefore, crude oil produced by extraction of crushed soybeans contains about 3 % phospholipids. Other crude oils are not so rich in phospholipids as they are more diluted with co-extracted neutral lipids. Therefore, the yield of phospholipids from crude soybean oil is much higher than is case with other crude oils, and the product has an advantageous composition (Table 15.1). Phospholipid concentrates obtained by plant-scale operations are called lecithins. They are not pure phospholipids as they always contain 30–40 % triacylglycerols and other lipidic components. In older literature, lecithin often means phosphatidylcholines.

15.1.3 Industrial production of phospholipids Egg yolk is rich in lipids (about 31–36 % dry weight). The lipid fraction consists of triacylglycerols, of which 3 % are phospholipids and 5 % is cholesterol. The lipid fraction can be obtained from the dried raw material. As egg lecithin is a relatively expensive ingredient in food products, it is produced only for special preparations where the price is not so critical, such as in the pharmaceutical industry. Soybean lecithin is much more important than egg lecithin as an ingredient in the food industry, as it can be easily obtained as a by-product of soybean oil production. Soybeans are crushed and treated with steam. Proteins present in soybean meal lipoproteins are denaturated at high temperature, and Table 15.1 Composition of the most important phospholipid classes in egg yolk, soybean and other important sources. Phospholipid class

Egg yolk

Cows’ milk

Bovine brain

Rapeseed

Sunflower seed

Soybean

LP PA PC PE PI PS SP

3–8 trace 66 – 82 8 – 24 carbohydrate > fat; and there is additional research that appears to confirm this phenomenon (Johnstone et al., 1996; Blundell and Macdiarmid, 1997). However, other studies have suggested that this effect is decided almost exclusively by energy density (Raben et al., 2003). Three major areas associated with fat structure have been investigated with regard to their satiating effectiveness (French, 2004). Chain-length and degree of saturation of triacylglycerols have a substantial impact on their physico-chemical properties. Several studies have investigated the effect of these factors on appetite and eating behaviour. Medium-chain triacylglycerols (MCT), hydrolyzed to medium-chain fatty acids, are absorbed more rapidly than long-chain triacylglycerols. Medium-chain fatty acids are directly absorbed into the portal system, whereas long-chain fatty acids and their monoacylglycerols are transported in chylomicrons through the lymphatic system. In addition, MCT are preferentially oxidized and are able to cross the inner mitochondrial membrane without acylcarnitine transferase (Bremer, 1983; Langhans, 1996). MCT are more satiating than long-chain triacylglycerols (TAG). Furthermore, MCT added to a very low-energy diet will speed weight loss and enhance satiety in the first two weeks of weight loss (Krotkiewski, 2001). MCT have been suggested as potential agents for the prevention of obesity (St-Onge and Jones, 2002). Clearly, protein has the greatest effect on satiety, but research published in the last few years suggests that, with an equal caloric density, fat and carbohydrate have comparable effects on satiation, unlike the consensus during the 1990s.

18.1.2 Low-fat foods More than 1000 reduced- or low-fat products were introduced each year during the 1990s (USDHHS, 1990; ADA, 1998; Wylie-Rosett, 2002). The three most popular reduced-fat product categories included fat-free or lowfat milk, (Wylie-Rosett, 2002), salad dressing, sauces or mayonnaise (ADA, 1998) and cheese/dairy products (USDHHS, 1990). A survey conducted for the Calorie Control Council in 1998 (Calorie Control Council, 2004) indicated that these product categories are consumed by approximately one half of those who consumed low-fat products. In addition, consumption of fat-reduced or fat-free margarine/spreads, chip/snack foods, meat products and ice cream/ frozen desserts was reported by more than a third of the consumers that use reduced fat products. Since ~ 88 % of the adult population consumes low- or

446 Modifying lipids for use in food reduced-fat foods and beverages, the large majority of the population has demonstrated substantial interest in foods that are promoted as low in fat (Wylie-Rosett, 2002; Calorie Control Council, 2004).

18.1.3 Health issues In spite of the successful development and commercialization of low-fat food products and widespread acceptance and consumption of these products, as of 2001 obese and overweight adults comprised 58 % of the population in the USA (Mokdad et al., 2003). The obesity problem is not limited to the USA either. In 2000 the World Health Organization reported that more than one billion adults were overweight and over 300 million of these were clinically obese. Throughout the entire world, the problem of obesity is increasing, and not only in industrialized nations but also in urban areas of developing nations (WHO, 2004). A reduction in caloric intake and a simultaneous increase in energy expenditure via regular exercise is the primary solution recommended by knowledgeable nutritionists and medical experts. However, many are not listening to the experts, so the application of appropriate food technologies designed to reduce fat content and caloric density could help ameliorate the severity of the problem. The macronutrient composition of a diet can influence hunger, satiety, food intake, body weight and body composition (Rolls, 1995). Fat, rather than carbohydrates, has been the macronutrient most associated with overeating and obesity. Fat is often consumed in excess because palatability and caloric density of fat are high and satiety is low. Low-fat foods in combination with the appropriate fat substitute can potentially reduce caloric intake by making less palatable low-fat foods more desirable. Fats contribute to the appearance, taste, mouth feel, lubricity, texture and flavor of many food products, provide essential fatty acids and are carriers of fat-soluble vitamins (Akoh, 1995; Artz and Hansen, 1996a,b). The amount and type of fat present in foods determines the characteristics of that food and can affect consumer acceptance. An ideal fat replacer should be completely safe and physiologically inert, and achieve a substantial fat and caloric reduction while maintaining the desired functional and sensory properties of a conventional high-fat product (Grossklaus, 1996). Historically, dietary fats and oils have been considered a primary source of energy without regard to the health effects of their specific complement of fatty acids and sterols (Glueck et al., 1994). In 1995, fats accounted for ~ 38 % of the total calories in the diet of Western populations, particularly in the USA (Akoh, 1995). However, dietary fat intakes greater than 11 % of the total caloric intake only developed after the domestication of mammals and the subsequent selective breeding of genetically fatter animals (Garn, 1997), indicating that a high-fat diet has become the norm only relatively recently in the history of Homo sapiens. Although there are many nutrition recommendations that remain

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controversial, there is a consensus among health and nutrition professionals that North Americans should reduce their dietary fat intake and alter the composition of their dietary fat (Gershoff, 1995). The American Dietetic Association (ADA, 2005) has indicated that the majority of fat replacers, when used in moderation by adults, are safe and useful adjuncts to reducing the fat content of foods and may play a role in decreasing total dietary energy and fat intake. They suggest that it is not a good idea to consume low-fat and low-calorie products in unlimited amounts, particularly if these foods are consumed in place of nutrient dense foods. The moderate use of low-calorie, reduced-fat foods, combined with low total energy intake, could potentially promote dietary intake consistent with the objectives of Healthy People 2010 and the 2005 Dietary Guidelines for Americans. The relationship of dietary fat and cholesterol to coronary heart disease is supported by extensive and consistent clinical, epidemiological, metabolic and animal evidence. Studies strongly indicate that the formation of atherosclerotic lesions in coronary arteries is increased in proportion to the levels of total and low-density lipoprotein (LDL) cholesterol in blood which, in turn, are increased by diets high in total fat (Glueck et al., 1994). Reduction in the relative amounts of high-fat food products in the diet can be an effective means of reducing caloric intake and is consistent with public health goals to reduce the risk of chronic diseases (Borzelleca, 1992, 1996; Degraaf et al., 1996). Thus, dietary fat is one of the major nutrition concerns of Americans. In response to the rising consumer demand for reduced-fat foods, the food industry has developed a multitude of non-fat, low-fat and reduced-fat versions of regular food products (Calorie Control Council, 2004). To generate reducedfat or fat-free products that have the same sensory characteristics as the regular fat version, food manufacturers frequently employ fat substitutes (Miller and Groziak, 1996) made from carbohydrates, protein or fat, or a combination of these components. Many of the carbohydrate- and proteinbased fat substitutes have received GRAS (Generally Recognized As Safe) status from the Food and Drug Administration (FDA) (Artz and Hansen, 1996a). In January 1996, the US Food and Drug Administration approved olestra (currently termed Olean® – Proctor & Gamble Co, USA) for use in savory snacks (Akoh, 1996; Freston et al., 1997; Zorich et al., 1997). This fat substitute is sucrose polyester or sucrose fatty acid polyester whose Code of Federal Regulation (CFR) reference number is CFR 172.867 (US Government, 1997). There are other fat substitutes with the potential to partially replace some, but not all, calories from fat under development or in the market (Calorie Control Council, 2004). Fat substitutes could replace a significant proportion of dietary fat and become macronutrient substitutes (Borzelleca, 1992). Hence, the safety of these materials must be established via extensive safety testing prior to FDA approval and introduction into the food supply (Artz and Hansen, 1996b). Appropriate methods of safety evaluation must be used. Traditional methods for the safety evaluation of macronutrient substitutes are inappropriate, since

448 Modifying lipids for use in food an evaluation of concentrations high enough to provide a 100-fold safety factor is not feasible (Borzelleca, 1996).

18.2 Fat substitute chemistry 18.2.1 Synthesis and composition of fat substitutes This section will address the synthesis and/or preparation of some of the major fat substitutes in use or under development that have potential as fat substitutes or fat replacers. The terms fat substitutes and fat replacers will be used interchangeably in this chapter. While many of the fat substitutes are included in this chapter, the absence of a fat substitute from this chapter implies nothing about its utility, safety or potential. Some of the fat substitutes here discussed are those that contain fatty acids attached to a molecule other than glycerol, such as in olestra. In other examples, such as the esterified propoxylated glycerols, the attachment has been modified to reduce the susceptibility of the compound to fatty acid release via lipase. The other category of fat substitute discussed will be reduced calorie triacylglycerols.

18.2.2 Esterified propoxylated glycerols (EPGs) Fatty acid esterified propoxylated glycerols (EPGs) (ARCO Chemical Company, Newtown Square, PA) were developed for use as frying fat substitutes. Glycerol is propoxylated with propylene oxide to form a polyether triol (Fig. 18.1). Fatty acids are then esterified to the triol (Gillis, 1988; White and Pollard, 1988, 1989a,b,c; Dziezak, 1989; Anon, 1990; Cooper, 1990; Arciszewski, 1991; Duxbury and Meinhold 1991; Hassel, 1993) to R O R

O O R Triacylglycerol

R O

R O

O

O

O

O

O

O O

O

O R

Fatty acid esterified propoxylated glycerol

Fig. 18.1 Triacylglycerol and fatty acid esterified propoxylated glycerol. In this figure, R refers to a fatty acid or acylgroup, connected with an ester bond.

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form a series of oligomers. Figure 18.2 is a capillary chromatogram of a separation of a series of fatty acid esterified propoxylated oligomers containing from three to 13 molecules of propylene oxide per molecule of glycerol. The propoxylated triacylglycerol is similar to natural fats in structure and functionality. Fatty acid EPG is a low-to-non-caloric oil that is heat-stable, only very slightly digestible and non-toxic. Preparation of propoxylated glycerides for use as fat substitutes involved transesterifying propoxylated glycerol with fatty acid esters in a solvent-free system (ARCO Chemical Technology Limited Partnership, Wilmington, DE) (Cooper, 1990) to avoid substances unacceptable in food systems.

18.2.3 Fatty acid partially esterified polysaccharide (PEP) The ARCO Chemical Technology Limited Partnership (Wilmington, DE) has patented a polysaccharide (PEP) that is partially esterified with fatty acids (White, 1990). It is non-absorbable, non-digestible and non-toxic. Suitable oligo/polysaccharide materials include xanthan gum, guar gum, gum arabic, alginates, cellulose hydrolysis products, hydroxypropyl cellulose, starch hydrolysis products (n < 50), karaya gum and pectin. The preferred level of esterification involves one or more hydroxyl groups per saccharide unit with one or more fatty acids.

FID response (mvolts × 10)

18.2.4 Carbohydrate fatty acid esters The carbohydrate-fat combination fat substitutes include those derived from polydextrose, sugar alcohols, altered sugars, starch derivatives, cellulose and gums. They can also be made from components from rice, wheat, corn, oats, tapioca or potato, and they can replace from 50 to 100 % of the fat in foods (Glueck et al., 1994). The synthesis and analysis of carbohydrate fatty acid esters have been

3.80

3.60

3.40

3.20 5.50

6.00 6.50 7.00 Time (minutes × 10)

7.50

Fig. 18.2 Solid fat content separation of fatty acid EPG triacylglycerol oligomers (FID = flame ionization detector).

450 Modifying lipids for use in food reported by several groups (Akoh and Swanson, 1989a,b; Drake et al., 1994; Rios et al., 1994). Carbohydrate fatty acid polyesters with a degree of substitution (DS: number of hydroxyl groups esterified with long-chain fatty acids) of 4–14 are lipophilic, non-digestible, non-absorbable, fat-like molecules with the physical and chemical properties of conventional fats and oils and are referred to as low-calorie fat substitutes (Akoh, 1994a; 1995). Swanson has published work from his laboratory on carbohydrate fatty acid esters synthesized from a variety of carbohydrate sources under a range of catalytic conditions (Akoh and Swanson, 1990; Akoh, 1994a,b; 1995), including glucose, sucrose, raffinose, stachyose and verbascose fatty acid esters (Akoh and Swanson, 1989a). The synthesis of both trehalose octa-oleate and of sorbitol hexa-oleate (Akoh and Swanson, 1989b) have been reported. Oleic acid esters of erythritol, pentaerythritol, adonitol and sorbitol were prepared by transesterification with an excess of methyl oleate to form complete esters (Mattson and Volpenhein, 1972). The esters formed were erythritol tetra-oleate, pentaerythritol tetra-oleate, adonitol penta-oleate and sorbitol hexa-oleate. These esters were not susceptible to in vivo lipolysis by lipolytic enzymes of rat pancreatic juice, suggesting potential application as lowcalorie oils (Mattson and Volpenhein, 1972; Akoh and Swanson, 1989a). Chung et al. (1996) also reported the preparation of a sugar alcohol fatty acid ester made with sorbitol. Enzymatic methods for the synthesis of carbohydrate fatty acid esters have been discussed in detail by Riva (1994). One of the most promising enzymes tested, particularly for fatty acid esterification of the alkylated glycosides, was a lipase from the yeast Candida antarctica, which had been immobilized on macroporous resin beads. Mutua and Akoh (1993) have reported the synthesis of glucose and alkyl glycoside fatty acid esters in organic solvents using Candida antarctica as a catalyst.

18.2.5 Sucrose polyester (SPE) – olestra (Olean®) The most extensively studied and publicized of the low-to-non-caloric fat substitutes are the sucrose fatty acid esters (Fig. 18.3). Typically, sucrose fatty acid esters are prepared from the reaction of sucrose with long-chain OR

OR

OR

OR R = Fatty acid group OR

O O

RO

OR OR

OR

Triacylglycerol

O OR

OR

Olestra

Fig. 18.3 Triacylglycerol and olestra. In this figure, R refers to a fatty acid or acyl group, connected with an ester bond.

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fatty acid methyl esters (Gardner and Sanders, 1990). Depending upon the reaction conditions, anywhere from one to eight fatty acids can be attached. Olestra is the generic name for the mixture of hexa-, hepta- and octa-esters of sucrose formed with long-chain fatty acids. Digestive enzymes do not release the fatty acids, so olestra is non-caloric (Gershoff, 1995). Olestra was approved by the FDA for use in savory snacks in 1996 (Akoh, 1996). A review by Akoh (1995) has described various methods used to prepare sucrose polyesters. SPE have been prepared in 80–90 % yields by reacting sucrose octa-acetate (SOAC) with methyl palmitate. This solventfree method was improved by Volpenhein (1985). Yamamoto and Kinami (1986) reported another method to produce sugar fatty acid esters. The methods of Volpenhein (1985) and Yamamoto and Kinami (1986) required molecular distillation to remove unreacted fatty acid methyl esters. Akoh and Swanson (1990) reported a solid phase extractionbased synthesis procedure with yields between 96.6 and 99.8 % of the purified SPE.

18.2.6 Alkyl glycoside fatty acid esters Alkyl glycoside fatty acid esters are non-ionic, non-toxic, odorless and biodegradable compounds with emulsification properties. Direct esterification of reducing sugars such as glucose and galactose often results in excessive sugar degradation and charring. Therefore, alkylation to form, for example, the methyl glycoside, is necessary to convert reducing sugars with reactive C-1 anomeric centers to non-reducing, less reactive, anomeric C-1 centers (Akoh and Swanson, 1989a,b). The alkyl groups used were fatty acid methyl esters, primarily methyl oleate, although there was one example of peanut oil fatty acid methyl esters. Alkyl glycoside fatty acid esters can be used to replace fat in food products, such as frying oils and Italian salad dressings (Curtice-Burns, Inc., Rochester, NY) (Meyer et al., 1989). Alkyl glycosides can be formed by reacting a reducing saccharide with a monohydric alcohol, such as methanol. The hydroxyl groups of these alkyl glycosides are then esterified to form a lower acyl ester alkyl glycoside. The lower acyl ester alkyl glycoside is then admixed with a fatty acid lower acyl ester and an alkali metal catalyst to form the reaction mixture. Soybean, safflower, corn, peanut and cottonseed oils are preferred since they contain C16–C18 fatty acids that do not volatilize at the temperatures used for interesterification. Albano-Garcia et al. (1980) reported a solventfree synthesis of methyl glucoside esters of coconut fatty acids. Akoh and Swanson (1989a,b) have synthesized novel alkyl glycoside polyesters, such as methyl glucoside polyesters methyl galactoside polyesters, and octyl glucoside polyesters by solvent-free interesterification. To achieve high yields, the alkyl glycosides’ free hydroxyl groups were modified by acetylation prior to interesterification. Glucosides containing 1–50 alkoxy groups can be used as fat substitutes

452 Modifying lipids for use in food at substitution ranges of 10–100 % in low-calorie salad oils, plastic shortenings, cake mixes, icing mixes, mayonnaise, salad dressings and margarines (Procter & Gamble Co., USA) (Ennis et al., 1991).

18.2.7 Reduced calorie fat-based fat replacers The objective for these products is similar to that for the protein- and carbohydrate-based fat substitutes, a substantial reduction in calories, rather than a complete elimination of fat. These compounds cannot be used for frying. Examples for fat-based replacers include caprenin, captrin and salatrim. Caprenin is a reduced calorie triacylglycerol (Procter and Gamble) formed by the esterification with selected fatty acids: caprylic (8:0), capric (10:0) and behenic (22:0). Since behenic acid is only partially absorbed and shortchain acids produce fewer calories than the common C16 and C18 acids, the caprenin contains ~ 5 rather than the normal 9 kcal per gram. Caprenin has functional (melting) properties similar to cocoa butter and is intended to replace some of the cocoa butter in selected confectionery products. It is digested, absorbed and metabolized by the same pathways as other triacylglycerols. Captrin from Stepan Food Ingredients (Anon, 1994) is a randomized triacylglycerol made from linear saturated fatty acids, primarily caprylic and capric. Some of the proposed uses include baked goods, confections, dairy product analogs, snack foods and soft candy. Another fabricated triacylglycerol, similar to caprenin, is salatrim, a triacylglycerol comprising a mixture of long-chain (primarily stearic acid) and short-chain (acetic, propionic and butyric) fatty acids randomly esterified to glycerol (Smith et al., 1994). It contains approximately 5 kcal per gram rather than the 9 kcal/gram contained in regular fats and oils. An entire symposium on Salatrim was published in the February 1994 issue of the Journal of Agriculture and Food Chemistry. The research reported included structural characterization(s) of the oil, an analysis of the oil in food products and an extensive series of papers on the metabolism and toxicology of the oil in various animal and human model systems. Salatrim has the same utility as caprenin as a fat replacer in reduced-fat systems and could be used as a cocoa butter substitute in confectionery products and in baked products and filled dairy products. Caprenin and salatrim are of little use for deep fat frying applications, since release of the smaller molecular weight fatty acids is likely to cause undesirable flavor effects. Salatrim has been prepared by interesterification of saturated long-chain fatty acid (LCFA) triacylglycerols and short-chain fatty acid (SCFA) triacylglycerols (Klemann et al., 1994). The SCFA (triacetin, tripropionin, tributyrin) were reacted with LCFA (hydrogenated canola oil, cotton seed oil and soybean oil) using sodium methoxide as catalyst. An alternative to regular cooking oils, developed and marketed in Japan, has essentially the same calories as regular fats and oils, although the oil is metabolized differently. Diacylglycerol cooking oils have (mainly) a 1,3

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configuration. The taste and texture are similar to triacylglycerol cooking oils. However, 1,3-diacylglycerols are not hydrolyzed to 2-monoacylglycerols in the gut, so absorption and metabolism of 1,3-diacylglycerols differ from that of triacylglycerols. The physiological differences include lower postprandial lipemia and, in comparison to normal triacylglycerol cooking oils, an increased percentage of the fatty acids are oxidized rather than being stored as body fat. Preliminary studies suggest that these differences in the metabolism of diacylglycerols and triacylglycerols can be exploited to reduce the amount of body fat stored from the consumption of cooking oil and other food items with added fats and oils. It has been widely sold in Japan since its introduction in early 1999, and the product is being test-marketed in the USA. Increased consumption of diacylglycerol (DAG) oil may provide an additional means of reducing obesity, while concurrently achieving desirable food product characteristics and maintaining good food product quality (Flickinger and Matsuo, 2003; Katsuragei et al., 2004).

18.2.8 Fat-based systems designed to enhance satiety In 2000 and 2001 it was shown that a fat emulsion (Olibra® – Lipid Technologies Provider AB, Sweden) formulated from palm oil and oat oil fractions can affect the energy and macronutrient intakes in lean, overweight and obese subjects up to 36 hours post-consumption (Burns et al., 2000, 2001). It was suggested that the reason for the increase in satiety and reduction in food consumption was the ‘ileal-brake’ mechanism. There is also evidence that this effect is dose-dependent in lean adults, which is consistent with this mechanism (Burns et al., 2002). This product is now being marketed in USA. In 1998, Safeway began sale of Swedish yoghurt containing Olibra®. Safeway marketed the yoghurt product Skåne Dairy Maval which, since its release in southern Sweden in January, has achieved a 2 % share of the national fruit yoghurt market, despite selling at more than twice the price of rival brands. In 2005 the Swedish firm LTP, Lipid Technologies Provider AB, signed a license and supply agreement in the USA with General Nutrition Corporation (GNC), giving GNC an exclusive right in the USA to LTP’s satiety ingredient Olibra®, for the dietary supplement market.

18.3 Food applications Many factors must be considered when selecting a fat substitute, in addition to the obvious and critical sensory quality questions. Is any thermal processing applied to the product? How severe is the thermal processing (pasteurization versus sterilization)? How pH sensitive is the fat substitute? How long will the product be stored, and are there undesirable textural or flavor changes that occur during long-term storage or during excessively turbulent shipping? Will it be refrigerated? Must it be refrigerated? What home preparation steps

454 Modifying lipids for use in food are involved? Since there are generally water activity changes, is the product microbiologically stable? Are there ‘opportunities’ for abuse in the home, i.e. if opened and left on the counter overnight is food poisoning a possibility?

18.3.1 Baked goods Mono- and diacylglycerol emulsifiers can be used to replace as much as 50 % of the fat in baked goods, since they can be used as a 50/50 emulsifier/ water mixture (Frye and Setser, 1993; Vetter, 1993). Additional emulsifiers used similarly include sodium stearoyl lactylate, sorbitan monostearate and polysorbate 60 at high hydration levels. A hydrated blend of emulsifiers developed specifically for fat replacement includes stearyl monoglyceryl citrate, ethylene glycol monostearate and lactylated monoglycerides. Simplesse™ (CP Kelco Aps, USA) protein hydrolysates, Sta-Slim™ 143 (Tate and Lyle, UK) and sucrose fatty acid esters have been used as fat substitutes in yoghurt, sour cream, cream cheese, cheese spread and frozen dairy desserts. Additional applications are discussed by Frye and Setser (1993), and Sharp (2001) has discussed some of the technical limitations regarding baked goods and reduced-fat formulations. Low-fat shortbread cookies have been prepared using carbohydrate-based fat substitutes (Sanchez et al., 1995). A combination of carbohydrate-based fat substitutes (Litesse® – Danisco Sweeteners, Denmark, N-Flate® – National Starch and Chemical Co, USA, Rice*Trin ® – Weinberg Food, Inc, USA, Stellar™ – A E Staley Manufacturing Co, USA, or Z-trim™ – Fibergels Technologies, USA) and emulsifiers (such as diacetyl-tartaric esters of monoacylglycerols, glycerol monostearate or sodium stearoyl-2-lactylate) were used. The principal effects of fat substitutes on shortbread cookie attributes were an increase in moisture content, greater toughness and reduced specific volume. Zoulias et al. (2002) examined four different types of fat mimetics using cookies. A fat reduction of 35–50 % was achieved with acceptable cookies.

18.3.2 Dairy, frying fats and table spreads Olestra can be used as a partial fat substitute in shortening, margarines and frying oils (Hollenbach and Howard, 1983; Robbins and Rodriguez, 1984; Roberts, 1984). Cheddar cheese has been prepared with milk fat sucrose polyesters (Crites et al., 1997). No significant differences in moisture, pH or whey titratable acidity were observed between the control cheese and cheeses containing milk fat sucrose fatty acid polyester. Cheese containing milk fat sucrose polyester contained fat globules that were smaller and more uniform in size compared to a control cheese with no added sucrose polyester. Bachmann (2001) reviewed various types of cheese analogs that had been developed using fat substitutes. A patent was awarded for the incorporation of the alkyl glycoside fatty

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acid polyester into food products (Curtice-Burns, Inc., USA) (Winter et al., 1990). The fat substitute could replace fats in such products as shortening, margarine, butter, salad and cooking oils, mayonnaise, salad dressing and confectionery coatings or ‘invisible fats’ in such foods as oilseeds, nuts, dairy and animal products. Substitution at 10–100 % is possible, preferably in the range of 33–75 %.

18.3.3 Novel applications One creative use of fat substitutes was the suggestion that sucrose fatty acid esters could be used to inhibit lipoxygenase and thereby improve food quality (Nishiyama et al., 1993). There was an increase in the binding strength of the sucrose fatty acid monoester and soybean lipoxygenase-1 (L-1) as the fatty acid carbon chain-length was increased from eight to 12. Thermodynamic analysis of the binding constants indicated that the binding was hydrophobic in character. Sucrose fatty acid esters can also suppress lipase activity and even have an antibacterial effect in some cases.

18.4

Toxicology

As with any new food ingredient, fat substitutes must be tested on animals before they are tested on humans. Ordinarily in animal toxicological tests, food additives are fed at dietary levels several fold in excess of the concentrations that will occur in foods destined for human consumption (Gershoff, 1995). This is done to provoke potential toxic responses and to establish safety factors. Because the amount of a fat substitute that could occur in the human diet is very large relative to other food additives such as added colors or flavors, feeding the fat substitutes at very high levels could result in spurious results, since this would require reducing a large part of the nutrients in the diet. Munro (1990) has pointed out that responses that ‘at first glance may be considered to be of toxicological significance may on further investigation be the result of dietary nutrient imbalance or physiological perturbation induced by the test material when fed at excessive exposure levels’. Diets with a large component percentage of fat substitutes could become unpalatable with a poor consumption rate leading to poor growth that might be wrongly interpreted as a sign of toxicity. Measurements of growth, of blood and urine chemistry, plus gross and histologic examination of tissues are often made. In addition, when appropriate, carcinogenicity, genotoxicity and reproductive and developmental toxicity testing may also be performed. Even if animal testing proves negative, the FDA recognizes that confirmatory studies in humans are an important part of confirming the safety of macronutrient substitutes (Gershoff, 1995). In toxicological studies, potential effects of fat substitutes that may not be evident in standard toxicological tests also need to be considered, based on

456 Modifying lipids for use in food physiological effects that may be specific to the chemical or physical properties or the mechanism or site of action of the substitute. There is also a need for confirmatory human studies in normal as well as at-risk populations, such as people with diabetes or compromised gastrointestinal (GI) tracts, or abnormalities that could possibly be caused by the fat substitute under consideration. For non-absorbable fat substitutes, effects on GI epithelium, colonic microflora ecology, bile acid physiology, pancreatic function and laxation effects should be considered (Munro, 1990; Vanderveen and Glinsmann, 1992; Glueck et al., 1994; Gershoff, 1995). For fat substitutes that are absorbed, absorption, distribution, metabolism and elimination of the substitute should be considered. The most exhaustively studied fat substitute has been Olestra. Olestra is neither hydrolyzed nor absorbed (Mattson and Volpenhein, 1972; Miller et al., 1995). Olestra is not toxic, carcinogenic, mutagenic or teratogenic and when fed to animals at doses up to 10 % of the total diet, no toxic effects on weight gain, hematology, urinalysis or tissue pathology were noted (Bergholtz, 1992). Since it is not absorbed, the only organ that olestra contacts is the GI tract. It has no significant effect on gastric emptying, total transit time, fecal water or pH of pancreas, fecal bile acids or interohepatic circulation of bile acids. It was reported that for specific GI symptoms (gas, diarrhea, abdominal cramping), there were no significant differences between humans who consumed chips with either olestra or triacylglycerols (Freston et al., 1997; Cheskin et al., 1998). Gut microflora do not metabolize olestra under anaerobic conditions, but during waste treatment, it is degraded aerobically in sludgeamended soils (Haighbaird et al., 1997; Thomson et al., 1998).

18.5 Future trends Research and development on the heat-stable fat-based fat substitutes slowed substantially after the FDA’s ruling limited fat substitutes to the ‘savory snack’ category. Olestra is still the only heat-stable fat substitute approved for food use in the USA. Frito-Lay has over half the market in the ‘savory snack’ category. Only one fat substitute, olestra (Proctor & Gamble Co.), is used in Frito-Lay products, so the market possibilities for other fat substitutes are very limited. If, or perhaps when, other product categories are approved in the USA, a limited number of other fat substitutes, such as sorbestrin or fatty acid esterified propoxylated glycerol, have a relatively good chance of further development and eventual approval. Other technologies are also being investigated, such as lipase inhibitors and identification of the compounds that control satiety in the brain. These could be marketed in the future, as well. While the best solution to the adult and childhood obesity problem in the USA is clearly an increase in exercise and a reduction in the calorie intake, most consumers seem to prefer other options. Although many companies in the US food industry respond to consumer needs, most respond best to

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consumer demands. Companies continue to produce only the products that consumers purchase. If consumers continue to purchase ‘super-size’ portions, those products and similar products will be provided. It is very likely the market for fat substitutes will remain strong for the foreseeable future.

18.6

References

(1998), Position of American Dietetic Association: fat replacers, J Am Diet Assoc, 98, 463–468. ADA (2005), Position of the American Dietetic Association: fat replacers, J Am Diet Assoc, 105, 266–275. AKOH C C (1994a), Synthesis of carbohydrate fatty acid polyesters, in Akoh C C and Swanson B G, Carbohydrate Polyesters as Fat Substitutes, New York, Marcel Dekker, Inc., 9–35. AKOH C C (1994b), Oxidative stability of fat substitutes and vegetable oils by the oxidative stability index method, J Am Oil Chem Soc, 71, 211–216. AKOH C C (1995), Lipid based fat substitutes, Crit Rev Food Sci Nutr, 35, 405–430. AKOH C C (1996), New developments in low calorie fats and oils substitutes, J Food Lipids, 3, 223–232. AKOH C C and SWANSON B G (1989a), Synthesis and properties of alkyl glycoside and stachyose fatty acid polyesters, J Am Oil Chem Soc, 66, 1295–1301. AKOH C C and SWANSON B G (1989b), Preparation of trehalose and sorbitol fatty acid polyesters by interesterification, J Am Oil Chem Soc, 66, 1581–1587. AKOH C C and SWANSON B G (1990), Optimized synthesis of sucrose polyesters: comparison of physical properties of sucrose polyesters, raffinose polyesters and salad oils, J Food Sci, 55, 236–243. ALBANO-GARCIA E, LORICA R G, PAMA M and DE LEON L (1980), Solventless synthetic methods for methyl glucoside and sorbitol esters of coconut fatty acids, Philip J Coconut Stud, 5, 51–54. ANON (1990), Fat substitute update, Food Technol, 44, 92–94. ANON (1994), Stepan seeks GRAS status for captrin, INFORM, 5, 1167–1168. ARAYA H, HILLS J, ALVINA M and VERA G (2000), Short-term satiety in preschool children: a comparison between high protein meal and a high complex carbohydrate meal, Int J Food Sci Nutr, 51, 119–125. ARCISZEWSKI H (1991), Fat functionality, reduction in baked foods, INFORM, 2, 392–395. ARTZ W E and HANSEN S L (1996a), Current development in fat replacers, in McDonald R E and Min D B, Food Lipids and Health, New York, Marcel Dekker, Inc. 385–415. ARTZ W E and HANSEN S L (1996b), The chemistry and nutrition of nonnutritive fats, in Perkins E G and Erickson M D, Deep Frying, Chemistry, Nutrition and Practical Applications, Champaign, IL, AOCS Press, 210–222. BACHMANN H P (2001), Cheese analogues: a review, Int Dairy J, 11(4–7 Special Issue SI), 505–515. BERGHOLTZ C M (1992), Safety evaluation of olestra, a non-absorbed fatlike fat replacement, Crit Rev Food Sci Nutr, 32, 141–155. BLUNDELL J E and MACDIARMID J I (1997), Fat as a risk factor for overconsumption: satiation, satiety, and patterns of eating, J Am Diet Assoc, 97(suppl), S63–S69. BORZELLECA J F (1992), Macronutrient substitutes: safety evaluation, Regul Toxicol Pharmacol, 16, 253–264. BORZELLECA J F (1996), A proposed model for safety assessment of macronutrient substitutes, Regul Toxicol Pharmacol, 23, S15–S18. BREMER J (1983), Carnitine – metabolism and functions, Physiol Rev, 63, 1420–1479. ADA

458 Modifying lipids for use in food BURNS A A, LIVINGSTON M B E, WELCH R W, DUNNE A, ROBSON P J, LINDMARK L, REID C A, MULLANEY U and ROWLAND I R

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19 Filled and artificial dairy products and altered milk fats E. Hammond, Iowa State University, USA

19.1

Introduction

Milk fat sells for a higher price than most other fats and oils, and nearly all of it is used in foods (Hammond, 2000). Milk fat owes its favored economic value to its subtle flavor, the unique flavors it generates when heated and its melting profile. These characteristics are shaped by rumen fermentation and the mammary gland (Walstra and Jenness, 1984). In the rumen the polyunsaturated fatty acids typical of the animals’ diets are hydrogenated by micro-organisms. In the mammary gland short-chain saturated fatty acids are introduced. These factors give milk fat a unique composition. Like other animal fats, milk fat requires no deodorization to make its delicate flavor acceptable, although milk may acquire undesirable flavors from silage or plants such as garlic or onion in the cows’ diet. Many of the mild flavors unique to good quality milk are inherent in its biosynthesis and others are produced during pasteurization. Heating milk fat can release methyl ketones from 3-keto esters and γ- and δ-lactones from 4- and 5-hydroxy esters. These precursors are present in small amounts in milk fat (Hammond, 1989). These flavors are of particular importance in baked goods traditionally made with milk fat. For other dairy products, particularly ripened cheese and cheeses made from lipase-treated milk, the release of flavorful short-chain free fatty acids is vital to their flavor. The role of these flavor compounds limits the use of milk fat substitutes in many dairy applications. Milk fat progresses from a fairly hard solid at refrigeration temperatures to a liquid at slightly above human body temperature (Walstra and Jenness, 1984). The amount and distribution of fatty acids in the triacylglycerols are vital for this melting profile. For fat in many edible applications, it is important

Filled and artificial dairy products and altered milk fats

463

that the fats melt completely at body temperature, so this characteristic must be repeated in milk fat substitutes in filled dairy products. The hardness of milk fat at refrigeration temperatures is regarded as a disadvantage, especially in the USA where very soft textured bread is the norm (Brunner, 1974; Bobe et al., 2003; Chen et al., 2004). Finding acceptable ways to soften the texture of cold butter has long been a goal of the dairy industry. Conversely, it is relatively easy to control the texture of margarine to be spreadable at refrigeration temperatures. The texture and melting properties of milk fat vary with the breed and the cows’ diets and, thus, these properties typically vary with season of the year (Kurtz, 1974). Cows on pasture generally give softer milk fat than those on dry feeds. Lack of uniformity in foods is always a disadvantage, and this is another advantage of margarines. Milk fat from pasture-fed cows is also yellower, but this change in butter color and in milk fat substitutes can easily be controlled by the addition of natural pigments, such as annatto or βcarotene. Nutritional considerations also affect the consumption of dairy products and their substitutes. Milk fat, like other animal fats, contains significant amounts of cholesterol, and many people are interested in limiting their cholesterol intake (Walstra and Jenness, 1984). There is interest in minimizing the cholesterol content of milk fat, but many of these techniques require isolating the fat from the dairy product. Vegetable oil substitutes for milk fat do not have significant amounts of cholesterol, and plant sterols are sometimes regarded as conferring a nutritional advantage. Milk fat is a poor source of polyunsaturated fatty acids because of rumen hydrogenation, and this particularly affects its use in infant formulas. Milk fat is regarded as one of the most atherogenic fats that is consumed in large amounts in Western countries (Ulbricht and Southgate, 1991). It is rich in saturated fatty acids, especially palmitic, myristic and lauric acids that are considered particularly atherogenic. Milk fat appears to suffer unnecessarily from laws that require labels to state the saturated fat content because it is rich in stearic acid, which is generally regarded as not being atherogenic. Likewise, the saturated fatty acids of chain-lengths four through ten are metabolized differently from longer-chain fatty acids and are regarded as non-atherogenic; however, they are all saturated and are listed as such on labels.

19.2 Filled and imitation dairy products Dairy products made with the substitution of milk fat with other fats are termed ‘filled dairy products’, ‘imitation dairy products’ or ‘dairy product analogs’. Imitation products are generally made without milk protein or milk fat, although products made with sodium caseinate are also termed imitation dairy products because the caseinate is considered a chemically modified

464 Modifying lipids for use in food product (Winkelmann, 1974). The production of filled and imitation products has been driven by scarcity of dairy products and by economics. The economic advantage of a filled product obviously depends on the amount of fat that it contains, so the first instance of a filled product was margarine, which has become sufficiently successful to merit a chapter of its own in this volume (Chapter 17). During the 1960s and 1970s, there was a great upsurge in interest in making milk fat substitutes. Hydrogenation technology had matured to the point where vegetable fats could be tailored to fit a variety of needs. Also at that time, there was a real concern that the dairy industry could not expand enough to meet future demands for dairy products (Jonas, 1975). Statistics on the consumption of filled and imitation milk are scattered, and many of them are dated, but filled and imitation dairy products captured significant proportions of the market milk, ice cream, coffee creamer and whipped topping markets in these decades. In general, they gained most in products where the fat content was relatively high, where the physical properties of milk fat were not ideal, where the flavor difference caused by substituting milk fat was not noticeable or where the instability of the dairy product to off-flavor development was a problem. These market losses caused considerable concern in dairy circles in the USA, and counter measures were considered (Hedrick, 1969). Today in some countries a trend towards natural foods has dampened enthusiasm for filled and imitation dairy products, but in other countries reconstituted milk is flourishing. In 2002 the recombined milk industry was estimated to have had a product turnover worth $5–6 billion (Sanderson, 2004). This industry had grown especially in South and Central America, China and Southeast Asia and the Middle East with major imports in powdered whole milk, skim milk and whey (Davidson, 2004). Now dairy interests often take a more relaxed stance and look on dairy products as ingredients for all kinds of food, including filled ones. Trade in these products is dominated by New Zealand, Australia and the European Union (Sanderson et al., 2004). However, lactose intolerance in Chinese and Southeast Asian populations may limit the consumption of dairy products. In a recent study in Thailand (Sirichakwal and Puwastien, 2005), lactose intolerance was measured by observing digestive symptoms (diarrhea, abdominal cramps, flatulence, etc.) during the 24 hours following the consumption of milk. The results showed no lactose intolerance in children (5–6 years), but the incidence of lactose intolerance increased with the age of subjects to 18 % for adolescents (13– 16 years), 40 % for adults (18–45 years) and 64 % for subjects 45–60 years old. Measurement of lactose maldigestion, by the presence of > 20 ppm of hydrogen in the subjects’ breath within seven hours after consuming dairy products, yielded greater percentages than the intolerance study but similar trends. Reducing the lactose in the products by fermentation or other means greatly reduced the incidence of maldigestion and intolerance.

Filled and artificial dairy products and altered milk fats

465

The 1990s have seen the advent of materials with fat-like mouth feel but zero or reduced caloric content, such as sucrose polyesters, short-chain fatty acid-rich fats, micro-particulate proteins and carbohydrates (Smith, 1995; Lindsay, 1996). Plans to have food labels report the amounts of trans fatty acid they contain are likely to have a significant effect on the acceptance of hydrogenated foods in filled dairy products, and the fats used for these purposes may require significant reformulations. Regulatory agencies usually have been concerned that filled and imitation products are not missing important nutrients that are contained in the traditional product and that the label not be deceptive. However, decisions about the acceptability of the names of products can be affected by the political influence of those with an economic interest in the outcome (Weik, 1969; Winkelmann, 1974). Many of the products used for filled and imitation dairy products have been proprietary products, and information about their constituents, fatty acid compositions, melting points and solids content at various temperatures is not readily available. Frequently these products, produced under a particular proprietary name, are prepared from different starting materials depending on price and availability, so the fatty acid composition can vary from time to time. An example of this may be seen in the data of Horvath et al. (1971) who reported on the fat in several brands of filled milk and coffee whiteners over a six-month period. Also, the materials used can vary with geographical location, and a number of countries have encouraged the use of locallyproduced fat and oil sources in making filled and imitation dairy products (Winkelmann, 1974). Thus, the fatty acid composition of milk fat substitutes can vary considerably. Table 19.1 gives the composition of the fat content and fatty acid composition of a number of filled and imitation dairy products. Often more attention is given to keeping the melting point, stability and solid fat profile of milk fat substitutes within a narrow range. The solid fat index and other properties of a number of fats used as milk fat substitutes that were manufactured by the former Durkee Company are given in Table 19.2. One of the problems with filled products made with hydrogenated fats is that they developed a unique flavor called ‘hardening flavor.’ These are particularly noticeable in products with bland flavors. There are two types of hardening flavor, one generated when a fat is hydrogenated and one that develops in hydrogenated fats that have been deodorized and then allowed to oxidize (Kawada et al., 1966). The former seem to arise from hydrogenation of oxidation products produced during processing, and the second arises from the oxidation of the fatty acids that have been isomerized during hydrogenation (Merker and Brown, 1956). Attempts to identify specific flavor compounds that are responsible for the hardening flavors have led to conflicting results that may reflect differences in hydrogenation conditions (Kawase et al., 1970; Feenstra and Meijboom, 1971; Yasuda et al., 1975).

Table 19.1

Fat percentage and fatty acid percentage composition of some filled and imitation dairy products and fats used to make them. Fatty acid

Product

Fat%

8:0

10:0

12:0

14:0

16:0

18:0

16:1

18:1

18:2

18:3

Fat type

F milk 1 2 3 4 I milk Fl I milk Pd1 2 3 F concentrate I sour cream I creamer 1 2 I creamer Pd I topping Aer I topping Pd I topping gel Polawar® E31 Confao® 5

3.4 3.2 3.5 3.4 3.7 26.4 27.2 22.3 9.2 19.6 9.2 11.2 35.6 21.2 44.7 23.8 100 100

5.3 3.3 – 0.3 6.7 5.3 0.1 – – 5.6 3.7 0.1 4.0 2.1 1.8 2.6 3.5 –

4.1 2.6 – 0.3 5.5 5.3 0.1 – – 5.5 2.9 0.1 4.4 3.0 2.6 5.0 3.0 –

64.1 41.1 0.6 0.3 46.1 42.6 0.6 – – 44.2 70.5 0.7 40.6 38.0 38.4 36.1 41.5 –

12.5 7.9 0.9 – 18.8 18.2 0.8 0.1 – 18.3 10.7 0.2 17.9 15.4 15.2 15.4 12.0 –

5.9 9.9 14.8 9.8 9.6 10.9 12.9 11.0 10.8 10.8 4.6 9.3 11.2 11.4 14.4 12.7 9.0 7.0

4.7 7.2 6.9 6.7 8.1 11.3 9.8 5.5 3.6 12.1 6.4 10.2 19.0 19.7 24.9 18.8 17.5 13.0

– – – – – – 0.8 0.3 0.4 – – – – – – 1.0 – –

3.1 27.3 54.1 82.0 5.2 5.7 62.6 28.7 25.3 3.1 1.2 79.3 2.9 9.1 1.6 5.7 13.5 63.0

0.3 0.7 21.8 0.6 – 0.5 11.5 47.6 51.4 0.3 – 0.2 – 1.1 1.2 1.5 – 6.0

– – 0.9 – – – 1.2 6.8 8.4 – – – 0.3 – – 1.0 – –

PC PCHV V HV PC PC HV V V PC PC HV PC PC PC PC PC Ra

Note: Polawar®, Bears Co., Russia; Confao®, Aarhus/Karlshamn, Sweden. a Also contains 1 % 20:0, 3 % 20:1, 1 % 22:0 and 6 % 22:0. Abbreviations: Aer = aerosol, C = coconut, F = filled, Fl = fluid, H = hydrogenated, I = imitation, P = palmkernel, Pd = powder, R = rapeseed, V = vegetable. Source: Based on the data of Posati et al. (1975) and Lausten (1986).

Table 19.2

Properties of milk fat and some milk fat substitutes. Max

Max

Solid fat index (°C)

Product

Wiley melting pt (°C)

Lovibond color

Iodine value

Form

10

21

27

33

38

43

Paramount® C Paramount® X Kaomel® Kaola Dariplus® R Dariplus® S Hydrol 92 Milk fat Dariplus® L Durkex® 100 Betrkerme

38–39 44–46 35–38 31–34 38–41 36–38 33–36 35

2.0R 2.0R 3.0R 2.5R 2.0R 2.0R 1.0R 8.0R 2.0Ra 2.0Rb 4.5R

3 3 58–63

Flake Flake Flake Plastic Plastic Plastic Plastic Plastic Semi liquid Liquid Flakes

68 69 72 40 55 34 57 33

56 58 63 18 33 18 33 14

40 50 55 8 19 13 8 10

12 27 25