Die Nahrung: Jahrgang 21, Heft 8 [Reprint 2021 ed.] 9783112567340, 9783112567333

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HEFT 8 • 1977 • 21. JAHRGANG Seite 653—756 NAHRAR 21 (8) 653-756 (1977)

G/i/mue Jowcrmme

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c/eiÄnü&^ie H

> w Begründet von A. Scheunert und K. Täufel H

Von 1957 - 1 9 7 0 Herausgeber K. Täufel t

Q

Herausgegeben vom: Zentralinstitut für Ernährung in Potsdam-Rehbrücke der Akademie der Wissenschaften der DDR (Direktor: Prof. Dr. liabil. H. Haenel)


KeHHH. Coo6m. 8. O KpaTKOcpoHHOM cnocoöe BapbKH H nanpaBJieHHOM pacnpejjejieHHH raKcauiiaHcxfieppaTa(II) MejKHy pacTBopoM h ocanKOM HOBHH criocoS n o j i y q e m i H iiGponnjibHoro cyßcTpaTa H 3 n a T O K H , xoporno o n p a B j i a j i n p H nepepaöoTKe pa3JiHHHbix B H H O B naTOKH. noaroTOBj i e H H b i f l co B c e M H j i o ß a B K a M i i H a x o j i o n y p a c T B o p n a T O K H B a p n T C H T O j i b K O 5—20 M H H y r . Cnocoß BapbKH B03M0>KeH B HeftTpaJlbHOH H CpaBHHTeJlbHO K H C J I O Ä cpene. IlOCJie BapbKH OnncbiBaeTCH

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(rii)

B a p b K H M E T O « p e r y j i n p o B a H H H HOJIM r u ( j

ocaHKe. ( P y n K i i H H H 3 H a T C H i i e 3 T H X $ p a K U H i i n p n JIHMOHHOKHCJIOM

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TexHHHecKHx h npoH3BoncTBeHHbix MacTpaßax. Literatur [1] LEOPOLD, H., U. Z. VALTR, Nahrung 13, 11 (1969). [2] LEOPOLD, H . , D . CAISOVÄ U. J . BASTL, N a h r u n g 13, 681 (1969).

H

6po-

peryjinpoBaHHH

NPOH3BEFLEHHBIX B

nojiy-

663

Kaliumhexacyanoferrat(II) bei Melasselösungen

[ 3 ] L E O P O L D , H . , M . B U R I A N , V . C E R N Y U. J . P A S E K , T s c h e c h o s l o w . P a t . A n m e l d u n g P V 6 6 1 6 — 7 6 .

[4] LEOPOLD, H . , M . BURIAN, V . C E R N Y U. J . PASEK, T s c h e c h o s l o w . P a t . A n m e l d u n g P V 6 7 5 0 — 76.

[5] LEOPOLD, H . , U. M. BURIAN, N a h r u n g 2 1 , 627 (1977).

[6] VORLÄNDER, D., Z. analyt. Chem. 77, 324 (1929). [7] CHRASTIL, J., U. T . WILSON, B i o c h e m . 63, 202 (1975). [8] LEOPOLD, H . , U. Z . VALTR, T s c h e c h o s l o w . P a t . 9 1 768 (1959).

[9] CLARK, D. S., Ind. & Engin Chem. Prod. Res. Devel. 1, 59 (1962). [10] MINC, E . S., L . F . IVANOVA, E . I. GORBATAJA, O. M . K A N D E L , S o v j e t . P a t . 455 992

[11] SCHWARZKOPF, F., Chem. Zentralhalle 83 (II/4), 1536 (1912). D o z . D r . I n g . H . LEOPOLD, I n g , M . BURIAN, I n g . V . C E R N V u n d J. PASEK,

Werk „Julius Fucik", 331 51 Kaznejov b. Pilsen, CSSR Eingegangen 7. 12. 1976

Lachema,

(1975).

Die Nahrung

21

8

1977

665 — 672

D e p a r t m e n t of D a i r y and F o o d Technology, College of Agriculture, University of B a g h d a d , Iraq

Proliferation of and t o x i n formation b y Staphylococcus

aureus in skimmed

and acidified milk as related to different heat treatments and incubation temperatures A B D U L AMIR A .

AL-TALIBI

T h i s s t u d y w a s carried out in an a t t e m p t to minimize t h e t o t a l population of St. aureus in s k i m m e d and acidified milk and henceforth to avoid the endotoxin formation in milk and milk p r o d u c t s prior to manufacturing. D i f f e r e n t h e a t t r e a t m e n t s and incubation temperatures were used for normal and acidified milk. T h e following results were obtained: 1. T h e a c t i v a t e d cells received a lethal effect. T h e s u r v i v i n g cells g r e a t l y depended upon incubation temperatures. T h e r e c o v e r y w a s most suitable a t low .temperatures and it w a s relativ e l y disturbed a t higher incubation temperatures. 2. T h e lag phase continued for a long t i m e prior to cell division. 3. T h e proliferation g a v e sigmoidal curves a t all heat treatments. 4. A gradual decline w a s found in N-values. H o w e v e r , compared w i t h those of t h e control, a high n u m b e r of cell divisions t o o k place a t low incubation temperatures (25 and 30 °C). T h e rate of proliferation w a s low in all t r e a t m e n t cases. 5. T h e coagulase positive reactions showed great variations in time intervals. L o w degree of firmness of blood p l a s m a coagulation w a s obtained a t incubation temperatures of 25, 30 and 45 °C, whereas t h e highest degree w a s obtained a t 37 °C. 6. A l l a b o v e results were almost identical w h e n experiments were performed on b o t h acidified and normal milk w i t h a p H 6.8.

Introduction Most milk produced in rural areas in Iraq at the present time is either home-prosessed or delivered to receiving stations where it is picked up for commercial processing. Milk which is used in making cheese in rural areas is not heat-treated provious to manufacturing because farmers utilize microorganisms present in raw milk (especially those forming lactic acid) as a means of changing the level of ions with the help of rennin used in coagulation of milk casein. As a result, other organisms such as St. aureus become an important source of toxicity in the products ( S T E Y N , 1969), hence many cases of severe diarrhoea have been reported by consumers. Milk which is delivered to receiving stations is also known to be contaminated since it usually sits for long periods of time before it is picked up. The high incidence of contamination of raw milk with St. aureus is due to: 1. Lack of suitable method of treating milk directly after milking. 2. Delay in delivering raw milk to the receiving stations which leads to spoilage in milk and its products.

Die Nahrung

21

8

1977

665 — 672

D e p a r t m e n t of D a i r y and F o o d Technology, College of Agriculture, University of B a g h d a d , Iraq

Proliferation of and t o x i n formation b y Staphylococcus

aureus in skimmed

and acidified milk as related to different heat treatments and incubation temperatures A B D U L AMIR A .

AL-TALIBI

T h i s s t u d y w a s carried out in an a t t e m p t to minimize t h e t o t a l population of St. aureus in s k i m m e d and acidified milk and henceforth to avoid the endotoxin formation in milk and milk p r o d u c t s prior to manufacturing. D i f f e r e n t h e a t t r e a t m e n t s and incubation temperatures were used for normal and acidified milk. T h e following results were obtained: 1. T h e a c t i v a t e d cells received a lethal effect. T h e s u r v i v i n g cells g r e a t l y depended upon incubation temperatures. T h e r e c o v e r y w a s most suitable a t low .temperatures and it w a s relativ e l y disturbed a t higher incubation temperatures. 2. T h e lag phase continued for a long t i m e prior to cell division. 3. T h e proliferation g a v e sigmoidal curves a t all heat treatments. 4. A gradual decline w a s found in N-values. H o w e v e r , compared w i t h those of t h e control, a high n u m b e r of cell divisions t o o k place a t low incubation temperatures (25 and 30 °C). T h e rate of proliferation w a s low in all t r e a t m e n t cases. 5. T h e coagulase positive reactions showed great variations in time intervals. L o w degree of firmness of blood p l a s m a coagulation w a s obtained a t incubation temperatures of 25, 30 and 45 °C, whereas t h e highest degree w a s obtained a t 37 °C. 6. A l l a b o v e results were almost identical w h e n experiments were performed on b o t h acidified and normal milk w i t h a p H 6.8.

Introduction Most milk produced in rural areas in Iraq at the present time is either home-prosessed or delivered to receiving stations where it is picked up for commercial processing. Milk which is used in making cheese in rural areas is not heat-treated provious to manufacturing because farmers utilize microorganisms present in raw milk (especially those forming lactic acid) as a means of changing the level of ions with the help of rennin used in coagulation of milk casein. As a result, other organisms such as St. aureus become an important source of toxicity in the products ( S T E Y N , 1969), hence many cases of severe diarrhoea have been reported by consumers. Milk which is delivered to receiving stations is also known to be contaminated since it usually sits for long periods of time before it is picked up. The high incidence of contamination of raw milk with St. aureus is due to: 1. Lack of suitable method of treating milk directly after milking. 2. Delay in delivering raw milk to the receiving stations which leads to spoilage in milk and its products.

666

AL-TALIBI

3. Lack of sanitary measures in stables which often lead to bovine mastitis and hence to milk contamination. F O R B E S (1969) reported that the passage of St. aureus through bovine teat canal in residual milk is rare, hence most contamination would be from the outside. This work is concerned with the effects of different heat tratments on St. aureus in relation to different incubation temperatures. The purpose of this work is to find out: 1. 2. 3. 4.

The exact heat treatments which are lethal to this organism. The incubation temperatures at which the recovery of the cells would be disturbed. The temperatures at which the endotoxin formation is blocked. The level of acidity associated with both heat treatment andincubation temperatures which serve as a preservative without changing the chemical make-up of milk.

j.

Test Organism

Materials and Methods A strain of St. aureus was obtained from the Veterinary Laboratory in Abu-Ghraib. I t was isolated from cows infected with bovine mastitis. Its pathogenicity was tested on blood agar medium and was found to be beta-hemolysis reaction. A coagulase positive reaction was also found with blood plasma within i h of incubation at 37 °C (Difco-manual, 1948). In addition it gave positive results when applied to the tests reported b y BERGEY (1957). This organism was reactivated on nutrient agar slant at 37 °C for 24 h and was used to inoculate 150 ml of sterilized nutrient broth and used as a main activated culture in this work. 2. Preparation of Skimmed Milk Whole milk was centrifuged to eliminate the fat content. 200 ml of skimmed milk were placed in each of a triplicate of Erlenmeyer flasks and then sterilized at 90 °C for 20 min for three sequential days (DEMETER, 1952). The pH of skimmed milk was adjusted to 6.8. Then it was inoculated with 0.2 ml of activated cells at 37 °C and heat-treated by placing the flasks in a rotary water bath at 50, 55, 6o, and 65 °C respectively. The organisms were kept at above temperatures for 30 min. After these treatments, each flask triplicate was incubated at 25, 30, 37, and 45 °C for recovery and proliferation of the treated cells. 3. Preparation of Acidified Skimmed Milk The p H of triplicate of flasks of 200 ml skimmed milk were reduced to 6.4, 6.2, 6.0, 5.8, 5.4, 5.2, and 5.0 by adding a standard lactic acid solution. Each triplicate was treated as previously and then subjected to the heat treatment. 4. Proliferation of St. aureus After the heat treatment, each triplicate of flasks of skimmed and acidified milk were incubated at 25, 30, 37, and 45 °C for recovery and proliferation of the cells. 1 ml was taken every xo h from each treatment. A series of dilutions was made according to the method of ROBERT KOCH. From each dilution 1 ml is taken and introduced into sterilized plates using nutrient agar as a medium. All plates were incubated at the above incubation temperatures for 5 days. The numbers of colonies were compared with those not subjected to heat treatments (control). The proliferation of surviving cells followed the fundamental law of biological reaction which was established b y JANOSCHEK (1957), this law summerizes the rate of proliferation time which is equal to i/X; one* representing the maximum rate of proliferation at 63.5% and equal to unity in the logarithmic phase and (T) representing the time of proliferation at the previous point. When the experimental points of proliferation were applied sigmoidal curves were obtained. 5. Qualitative test of endotoxin A sample was taken from each flask triplicate every 10 h for the determination of endotoxin formation. This sample was heated at 70 °C for 30 min in order to kill the cells and release the endo-

Staphylococcus

667

aureus i n m i l k

toxin. The casein of the sample was precipitated by adding a few drops of standard lactic acid solution, i ml of whey was taken and added to I ml of rehydrated coagulase plasma (imported from sycco, Sylvana, Millburn, N.J., U.S.A.) for staphylococcal-coagulase determination. The test tubes were incubated at 37 °C for 1 h, within this time the coagulation of blood plasma must take place. Negative results were designated b y ( - ) and positive results by (+), ( + + ) , (+ + +) according to the degree of firmness of coagulation.

Results and Discussion The inoculated and activated cells were affected b y the heat treatments depending on the incubation temperatures as can be seen in Table, i . Table 1 Percentage of dead and surviving cells at different heat treatments and incubation temperatures Incubation temps. [°C] 25

3°

37

45

Heat treatments [°C]

Dead cells

Surviving cells

[%]

[%]

5° 55

22.0

78.0

3 . 7 • IO 5

45-0

55-0

4.0 • IO 5

Inoculated cells [ml- 1 ]

60

71.0

29.0

4.1 • IO 5

65 50 55

88.0

12.0

3.8 • IO 5

33-3 5°i

66.7

3.0 • IO 5

49-9

3 . 7 • IO 5

60

82.0

18.0

90.0

10.0

3-5 • 10 5

65 5° 55

55-2

44.8

3 . 9 • IO 5

70.0

30.0

60

895

10.5

4.0 • IO 5 4.0 • IO 5 4.0 • IO 5

3 . 6 • IO 5

65

100

5° 55

85

15

92

8

4.0 • IO 5

60

97

2

4.4 • IO 5

65

100

—

4 . 2 • IO 5

3 . 9 • IO 6

It is seen from Table i that a gradual decline in cell number is obtained as the heat treatments increased. More specifically at 60 and 65 °C (at all incubation temperatures) the most lethal effect took place. This is due to the fact that the net effect of increasing temperatures is to speed up the chemical reaction. This physical phenomenon occurs not only in the synthesis of protoplasm and in the generation of energy, but also in the destruction of cell protein (OGINSKY et al., 1959). The amount of surviving cells depended on the range of incubation temperatures, where incubation temperatures of 25 and 30 °C and heat treatments of 50 and 55 °C were the most suitable for recovery, incubation temperatures of 37 and 45 °C and heat treatments of 60 and 65 °C were the least suitable ones. B y determining cell-population every 10 h and applying the fundamental law of biological reaction (JANOSCHEK, 1957) it was noticed that the time interval for growth and recovery in comparison to the control experiments depends on incubation temperatures, whereas the lag phase took a long time prior to cell division as seen in Table 2. A t certain incubation temperatures the surviving cells grow very slowly as they were affected b y the heat treatment and hencefore their enzymatic processes were

668

AL-Talibi

Lag

Table 2 phase times for St. aureus at various heat treatments and incubation temperatures Incubation temperatures [°C] 25

Heat treatments [°C] Control 50 55 60

37

45

L a g phase [h] 3

2

2

2

8

12 18 28

10 18 32

38 52 60

42

—

—

25 27 47

65

30

operating at very low rate (OGINSKY et al. 1959) comparing these reactions with the control which was not subjected to the heat treatments. The variations in the lag phase times affected the level of proliferation rate and the value of N (which represents the total population at the end of proliferation (120 h) and equals to 100%) especially at heat treatments of 60 and 65 °C and at incubation temperatures of 37 and 45 °C (Figures 1 — 4 respectively). The data of these figures are summerized in Table 3.

A b b . I.

A b b . 2.

Fig. i . Proliferation of St. aureus in skimmed milk at 25 °C (1 = Control; 2 = Heat treatment 50 °C; 3 = Heat treatment 55 °C; 4 = Heat treatment 60 °C; 5 = Heat treatment 65 °C) Fig. 2. Proliferation of St. aureus in skimmed milk at 30 °C (Explanations see Fig. 1)

Time[h] A b b . 3.

Time[h] A b b . 4.

Fig. 3. Proliferation of St. aureus in skimmed milk at 37 °C (Explanations see Fig. 1) Fig. 4. Proliferation of St. aureus in skimmed milk at 45 °C (Explanations see Fig. 1)

669

Staphylococcus aureus in milk

Table 3 Total population of St. aureus and the rate of proliferation a t different heat treatments and incubation temperatures during an incubation time of 120 h Incubation temperatures

Heat treatment [°C]

25 °C

37 °C

30 °C

45 "C

NV(M)

RP

NV(M)

RP

NV(M)

120 460 380 250 150

0.0200 0.0166 0.0135 0.0120 0.0114

150 560 420 300 250

0-0375 0.0154 0.0135 0.0122 0.0118

240 210 160 120

0-0334 0.0182 0.0145 0.0127

300 3 • io5 4 • IO 5 8- io4

0.0323 0.0123 0.0120 0.0112

—

—

—

-

Control 50 55 60 65

RP

NV(M)

RP

NV(M) = N - v a l u e in millions; R P = R a t e of proliferation

In this Table a large recovery of cells took place in total population at incubation temperatures of 25 and 30 °C in comparison with the control (which received no heat treatments), but a gradual decrease in value of N was noticed as the heat treatments increased. On the other hand the rate of proliferation decreased at all incubation and heat treatments, whereas at incubation temperatures of 37 and 45 °C and at all heat treatments there was a noticeable decrease in value of N and also in the rate of proliferation compared with control values; because the recovery of the cells was too slow and the heat processes were lethal factors for the cells, where at heat treatment of 65 °C there was no proliferation to be mentioned. Table 4 The coagulase reactions of blood plasma w i t h bacterial suspension a t different h e a t treatments a n d incubation temperatures Incubation temperature 25 °c

Time

m

60 90 120

[h]

H e a t t r e a t m e n t [°C]

50 30 40

Incubation temperature 30 °C

55

+ + + + +

60

+ + + +

—

+ + +

50

65 —

20

—

30

—

+ +

H e a t treatment [°C]

70 80

90 120

+ + + ++ ++ +++

Incubation temperature H e a t t r e a t m e n t [°C]

50 20

30 40

50 80 100 no 120 46

+ + ++ ++ +++ +++ +++ +++

60

55 . -

+ + ++ ++ +++ +++ +++

Die Nahrung, 21. Jhg., Heft 8

—

.

—

—

—

+

+ ++ ++

—

+ + ++ ++ +++

—

+ ++ •

+

+

Time

45 °C

M

H e a t treatment [°C]

50

65

—

+

65

— — —

+ +

Incubation temperature

37 °C M

60

55

— — — — —

50 100 no 120

60

55 —

—

+ +

—

+ +

65 —

—

—

—

—

—

—

670

AL-TALIBI

The reaction of blood plasma with whey bacterial suspension showed variations in time interval for the coagulase positive tests depending mostly upon the number active and recovered cells and also on both heat processes and incubation temperatures as shown in Table 4. According to the coagulase reactions of blood plasma it has been noticed that at 25 C C incubation temperature the firmness was too low and first appeared after 30 h and continued so till the end of proliferation, in spite of the fact that N-value at this temperature was very high. It seems that the cells were highly inactive. A t 30 °C incubation temperature, the firmness began to be higher within 80 h and more so in 120 h but weak at 60 and 65 °C heat treatments. The incubation temperatures 30 and 37 °C are regarded as very critical because the toxicity was too high and showed a greater degree of firmness in 80 h and alo at heat treatment 60 °C which first appeared in 70 and 50 h respectively and no sign of toxicity at heat treatment of 65 °C. Whereas at 45 °C incubation temperature and in spite of a low N-value the first sign of toxicity appeared in n o h and no toxicity in 60 and 65 °C heat treatment. The firmness of blood plasma indicates the amount of endotoxin released after killing the cells at 70 °C for 30 min. MUELLER (1969) has quantitatively determined the toxicity of St. aureus at about 25 mg/g of nutrient. In this work the toxicity was determined qualitatively as the number of active cells/ml depending upon incubation and heat processes as seen in Table 5. Table 5 Number of cells required to cause the toxicity in milk Incubation temps. [°C] 25 3°

37 45

Degree of firmness

+

+ ++ +++ ++ +++ +

Number of cells/ml required 1 —500- 10 6 1 — 15 IO6 1 6 - 120 IO6 121 — 4 5 ° - IO6 1 — 3 ° - IO6 6 3 1 - 1 4 9 . IO 1 5 0 - 3 5 ° - IO6

4

IO5

HAMMER et al. (1957) and AL-TALIBI (1972) proved that the toxin is heat resistant up to 100 °C for 1 h and there is not any denaturation of toxin composition and the effect of toxin is not removed b y heat treatment. The H + -concentrations at p H 6.4, 6.2, and 6.0 associated with heat treatments showed almost the same reactions concerning the proliferation of St. aureus and also the firmness of toxicity as the data were compared with those in Tables 3 and 4. AL-TALIBI (1972) proved that this organism is high acidity tolerant down to p H 3.6 in synthetic media, because at this point of actual acidity the cells could show proliferation and also a low level of firmness.of toxicity. AL-SHAIKHLY (1968) proved that high level of acidity will affect the growth of exotoxinproducing organism. In skimmed milk having a p H ranging from 5.8 down to 5.0, the casein content precipitated as a result of high temperatures and high level of actual acidity, therefore these flasks were discarded from this experiment.

Staphylococcus

aureus in milk

671

Conclusion I n o r d e r t o a v o i d t o x i c i t y a n d a g r e a t p o p u l a t i o n of St. aureus

in r a w m i l k , it is

a d v i c e d t h a t t h e d a t a of T a b l e 6 s h o u l d b e u s e d p r i o r t o m a n u f a c t u r i n g or u s i n g it as a l i q u i d and/or d e l i v e r y t o t h e r e c e i v i n g s t a t i o n s . Table 6 Heat treatment [°C] 50 55 60

65

Maximum waiting time [h] respectively

Incubation temperature [°C] —

25 25 25

1

30 30 3°

37 37 37

45 45 45 45

—

—

100

20

20

100

40

30

40

120

60

80

50

120

30

According to these d a t a m a x i m u m population were not obtained except at heat t r e a t m e n t of 6o a n d 65 °C a n d i n c u b a t i o n t e m p e r a t u r e of 45 °C. I t is s u g g e s t e d in t h i s s t u d y n o t t o a c i d i f y t h e m i l k a t a n y l e v e l of p H in o r d e r t o a v o i d t h e t o x i c i t y or t o m i n i m i z e t h e m a x i m u m p o p u l a t i o n . Zusammenfassung Abdul Amir A. Al-Talibi: Vermehrung von und Toxinbildung durch Staphylococcus aureus in entrahmter und angesäuerter Milch bei verschiedenen Hitzebehandlungsarten und Inkubationstemperaturen Die Arbeit behandelt das Problem der Keimzahlverringerung von St. aureus in entrahmter und angesäuerter Milch und damit die Vermeidung der Endotoxinbildung in Milch und Milchprodukten vor der Verarbeitung. Unterschiedliche Hitzebehandlungen und verschiedene Inkubationstemperaturen wurden für beide Sorten Milch benutzt. Folgende Ergebnisse werden festgestellt: 1. Die aktivierten Zellen bekommen einen lethalen Effekt, und die Anzahl der tiberlebenden Zellen ist meist abhängig von den Inkubationstemperaturen. Die niedrigen Temperaturen sind für die Widergewinnung der Zellen besser geeignet, während die hohen Temperaturen relativ störend wirken. 2. Die lag-Phase dauert lange, bis die Zellteilung einsetzt. 3. Die Vermehrung ergibt sigmoide Kurven bei allen Hitzebehandlungen. 4. Die N-Werte zeigen eine allmähliche Abnahme. Im Vergleich zu denen der Kontrolle findet jedoch eine große Zahl von Zellteilungen bei niedrigen Temperaturen (25 und 30 °C) statt. Die Geschwindigkeit der Vermehrung ist in allen Fällen langsam. 5. Die Coagulaserpositiven Reaktionen zeigen große Variationen in Zeitintervallen. Die Festigkeit der Blutplasmagerinnung ist gering bei Inkubationstemperaturen von 25, 30 und 45 °C, dagegen hoch bei 37 °C. 6. Alle erwähnten Resultate sind bei Durchführung der Experimente mit entrahmter und angesäuerter Milch sowie einem pH-Wert von 6,8 annähernd homolog. Pe3ioHe A S f l y j i b Amhp A . A j i b - T a J l H ß n ; Pa3MH0?KeHne Staphylococcus aureus Vi 0Öpa30BaHHe HMH TOKCHHOB B 06e3?KlipeHH0M H nOUKHCJieHHOM MOJIOKe npH pa3JIHHHBIX CnOCOÖOB TenjioBOH oöpaßoTKH h TeMnepaTyp iiHKySauiiM PaöoTa n o c B H m e H a npobJieMe yMeHbineHHH KOJiimecTBa St. aureus b c h h t o m h noRKUcJieHHOM MOJIOKe H, TeM CaMHM, H3ÖeraHHIO 06pa30BaHHH 3HAOTOKCHHOB B MOJIOKe II MOJiOHHbix nponyKTax nepen nepepaßoTKe. BbiJiH Hcn0Jib30BaHbi pa3JiHiHbie cnocoöbi 46»

672

AL-TALIBI

TenjiOBOß o 6 p a 6 o T K H H pa3JiimHbie T e M n e p a T y p b i n H K y S a q m i . n o j i y n e H b i c n e n y i o m M e pe3yjibTaTH. 1 . AKTHBHpOBaHHbie KJieTKH npHOÖpeTaiOT JieTajIbHblH 3$(j»eKT, H KOJIHMeCTBO nepejKMBaeMBix KJieTOK 3aBiicnT n a m e B c e r o OT T e M n e p a T y p b i H i m y ß a a M H . H i i 3 K n e T e M n e p a T y p b i 6 o j i e e npHroHHbi «Jin o ö p a T H o r o n o j i y q e m i j i KJICTOK, B TO BpeMH KaK BbicoKHe T e M n e p a T y p b i cpaBHHTejibHO MemaiOT. 2 . Jl3r-(J»a3a npoHOjijKaeTCH n o HacTynjieHHH p a s n e n e H H H KJieTKH. 3 . Pa3MHOHieHne n p o n c x o n H T n o cnrMOiinajibHOH KpbiBoft n p a B c e x ycjiOBHHX T e M n e p a TypHOH o 6 p a 6 o T K H . 4 . B e j n u n r a a H nocTeneHHO CHHHiaeTCH. n o cpaBHeHHio c KOHTpojieM npn H H 3 K O Ö TeMnep a T y p e ( 2 5 H 30°) HMeeT MecTO Q o j i b i n o e KOJiimecTBO p a 3 n e j i e H H f t KJieTOK. C K o p o c T b pa3MH0»KeHHH BO B c e x c j i y i a n x MefljieHHa. 5 . nojiOHtHTejibHbie p e a i f u n n K o a r y j i a s h i rnnpoKO B a p b H p y i o T B n p o M e j K y T K a x BPEMEHH. IlpOiHOCTb CBepTblBaHHH CblBOpOTKH KpOBH n p H T e M n e p a T y p e HHKySailHH B 2 5 , 3 0 M 4 5 ° C HH3KaH, a n p n 3 7 ° C BbiconaH. 6 . B e e ynoMHHyTbie p e 3 y j i b T a T b i npoBegeHHH onbiTOB Ha CHHTOM H noHKHCJieHHOM MOJIOKC npHMepHO OHHHaKOBO h noKa3biBaioT B e j m m r a y p H 6 , 8 .

References AL-SHAIKHY, J. S., and Y . Z. ISHAC, T h e Iraqi J. Agric. Sei. 3 (2) 62 — 72 (1958). AL-TALIBI, A. A., A S t u d y on the Proliferation of St. aureus in Skim Milk at Different Temperatures and the E f f e c t of Various NaCl Concentrations and p H - R a n g e s on this Proliferation in Synthetic Media. Vol. I, IST Sei. Conference Sei. Res. Foundation, Baghdad, March25 —30, p. 1 1 7 — 1 2 4 (1972). BERGEY'S Manual of Determinative Bacteriology (1975), 7 t h Ed., Baillivere, Tindall and C o x L t d . , London. DEMETER, K . J., Bakteriologische Untersuchungsmethoden der Milchwirtschaft, E u g e n Ulmer, S t u t t g a r t 1952. Difco Manual Microbiological and Clinical L a b o r a t o r y Procedures, 8 t h Ed., Detroit I, Michigan, U . S . A . 1948. FORBES, D., J. D a i r y Res. 35, 399 (1968). HAMMER, B . W., and J. B . FREDERICK, D a i r y Bacteriology, 4 t h Ed., New Y o r k , N . Y . , John W i l e y and Sons, Inc. 1957. JANOSCHEK, A., Stat. Vierteljahrschrift 10 (1/2), 204 — 211 (1957). MUELLER, F. J., Alimenta 8 (4), 9 3 — 9 5 (1969). NOEVAK, E . K . , and T . DEAK, A c t a Microbiol. A c a d . Sei. Hung. 17, 1 — 1 2 (1970). OGINSKY, E . L., and W . W . UMBREIT, A n Introduction to Bacterial Physiology, 2 n d Ed., W . H . Freeman and Co., San Francisco and London 1959. SXEYN, D. G., Vitalstoffe Zivilisationskrankheiten 14 (2), 66 — 69, (3), 123 — 124 (1969). Dr. A . A m i r AL-TALIBI, University of B a g h d a d , College of Agriculture, A b u Ghraib, Iraq Eingegangen 13. 1. 1977

Die Nahrung

21

8

1977

673-684

Dairy and Food Industries Department, Faculty of Agriculture, Al-Azhar University, Cairo, A.R. E g y p t

Chemical composition and organoleptic properties of R a s cheese, made under different technological conditions A . F . M. EL-ERIAN, H . A . HASHEM a n d A . M. A B O - E L - H E B A

The present studies have been performed with a view to the modification and shortening of the process of ripening. To shorten the duration of ripening and to increase the development of aroma and flavour substances, a rennet paste, lipase and cheese hydrolysate, alone or in combination, were added. To accelerate fat hydrolysis, either the milk or the cream was homogenized prior to processing. The water, salt, fat, total acid, total nitrogen, soluble nitrogen and volatile fatty acid contents as well as the pH value and the formol number were determined in fresh cheese and throughout the ripening process (after 15, 30, 60, 90, 120 and 180 days). Furthermore, the products were organoleptically evaluated. The results obtained with the various experimental variants are discussed. Cheese ripening is a complicated process. It involves several biochemical changes including the fermentation of lactose, the degradation of the proteins, and the hydrolysis of fat. All these processes result in the gradual change in the cheese curd from toughness to mellowness and in the development of the aroma and taste which together constitute the typical cheese flavour. Earlier workers have shown that the breakdown of casein during the ripening proceeds under the influence of rennet enzymes, bacterial enzymes and the enzymes present in the original milk. As a result of these processes a complex mixture of polypeptides (proteoses, peptones), amino acids, amines and ammonia is formed. Cheese fat is also subject to the effect of lipolytic enzymes either from the original milk or produced by the cheese flora. Ras cheese was produced originally in Greece where it is known as „Cephalotyre" cheese. It is also a close type to Caskawal cheese. In Egypt the names ,,Romy" and ,,Ras" are commonly used. This cheese is the most popular type of the hard cheese varieties known in Egypt. Its popularity is mainly due to its manufacturing simplicities and its characteristic pungent flavour that suits the Egyptian taste.

This work was designed to obtain more basic information as to the chemistry of the ripening process of Ras cheese. Some trials were included to enhance the cheese ripening. In this respect use was made of the following enzymes and their mixtures: Rennet paste, lipase and a cheese hydrolyzate. Homogenization of the milk or the cream was included in the study as a trial to intensify the fat hydrolysis. Different trials were carried out, including the combination of the different used factors mentioned above. The following abbreviations are used throughout the whole study: (C) (R) (RH)

= Control experiment where Ras cheese was made out of heat treated Buffaloes'milk in the usual way without any additions or modifications. = Ras cheese made as mentioned above in (C) except for using a Rennet paste instead of the usual rennet. = Ras cheese from homogenized milk with Rennet paste as a rennet substitute.

Die Nahrung

21

8

1977

673-684

Dairy and Food Industries Department, Faculty of Agriculture, Al-Azhar University, Cairo, A.R. E g y p t

Chemical composition and organoleptic properties of R a s cheese, made under different technological conditions A . F . M. EL-ERIAN, H . A . HASHEM a n d A . M. A B O - E L - H E B A

The present studies have been performed with a view to the modification and shortening of the process of ripening. To shorten the duration of ripening and to increase the development of aroma and flavour substances, a rennet paste, lipase and cheese hydrolysate, alone or in combination, were added. To accelerate fat hydrolysis, either the milk or the cream was homogenized prior to processing. The water, salt, fat, total acid, total nitrogen, soluble nitrogen and volatile fatty acid contents as well as the pH value and the formol number were determined in fresh cheese and throughout the ripening process (after 15, 30, 60, 90, 120 and 180 days). Furthermore, the products were organoleptically evaluated. The results obtained with the various experimental variants are discussed. Cheese ripening is a complicated process. It involves several biochemical changes including the fermentation of lactose, the degradation of the proteins, and the hydrolysis of fat. All these processes result in the gradual change in the cheese curd from toughness to mellowness and in the development of the aroma and taste which together constitute the typical cheese flavour. Earlier workers have shown that the breakdown of casein during the ripening proceeds under the influence of rennet enzymes, bacterial enzymes and the enzymes present in the original milk. As a result of these processes a complex mixture of polypeptides (proteoses, peptones), amino acids, amines and ammonia is formed. Cheese fat is also subject to the effect of lipolytic enzymes either from the original milk or produced by the cheese flora. Ras cheese was produced originally in Greece where it is known as „Cephalotyre" cheese. It is also a close type to Caskawal cheese. In Egypt the names ,,Romy" and ,,Ras" are commonly used. This cheese is the most popular type of the hard cheese varieties known in Egypt. Its popularity is mainly due to its manufacturing simplicities and its characteristic pungent flavour that suits the Egyptian taste.

This work was designed to obtain more basic information as to the chemistry of the ripening process of Ras cheese. Some trials were included to enhance the cheese ripening. In this respect use was made of the following enzymes and their mixtures: Rennet paste, lipase and a cheese hydrolyzate. Homogenization of the milk or the cream was included in the study as a trial to intensify the fat hydrolysis. Different trials were carried out, including the combination of the different used factors mentioned above. The following abbreviations are used throughout the whole study: (C) (R) (RH)

= Control experiment where Ras cheese was made out of heat treated Buffaloes'milk in the usual way without any additions or modifications. = Ras cheese made as mentioned above in (C) except for using a Rennet paste instead of the usual rennet. = Ras cheese from homogenized milk with Rennet paste as a rennet substitute.

674

EL-ERIAN/HASHEM/ABO-EL-HEBA

( R H L ) = R a s cheese made from milk and homogenized cream plus the addition of Rennet paste, commercial lipase and a cheese hydrolyzate. Different cheeses were analysed for its moisture, salt, pH, acidity, total nitrogen, soluble nitrogen, formol number, f a t and total volatile f a t t y acids. Analysis of the cheeses was carried out at the fresh stage and at the ages of 1 5 , 30, 60, 90, 120 and 180 days. Organoleptic properties were tested at the same intervals. The proposed scoring table of N E L S O N et al. [ 1 1 ] was applied.

Materials and Methods Milk Whole Buffaloe's morning milk was obtained from the Mostored Experimental Farm, Faculty of Agriculture, Al-Azhar University. Casein to fat ratio of such milk was standardized to approximately 0.7. The milk was flash pasteurized at 173 ° F and instantly cooled to 90 °F.

Rennet R e n n e t powder was obtained from Chas Phizer & Co., Inc. New York, U.S.A. Rennet paste was prepared from the fourth stomach of a butchered, 2 weeks old, milk fed calf. I t was cut to small cubes and then milled. The milled fourth stomach plus its coagulated milk were soaked in 1 1 1 5 % NaCl brine and left in the refrigerator for one week. The mixture was daily mixed and rotated. The filtrate of the final mixture was used in R a s cheese manufacturing at the ratio of 10 ml/45 kg of milk.

Lipase Commercial lipase, Light and Co., England, was used after being mixed with Rennet paste. F r o m the lipase 4 g were used per 100 kg of milk.

Homogenization Pasteurized Buffaloe's milk was cooled to 150 ° F and homogenized at the pressure of 2000 p.s.i. I n other experiments 2 0 % fat pasteurized cream was homogenized at 2000 p.s.i. and then added to the milk prior to cheese manufacturing.

The hydrolyzate Two samples of R a s cheese of good quality were analysed for its flora. Out of the dominating type fifty strains were isolated and checked for its ability to protein hydrolysis. One typical strain t h a t possessed a very strong protein hydrolysing character was used for the preparation of the hydrolyzate. The following tests were carried out to identify the strain: — — — — — — — — — — — — —

Morphology test for GRAM stained organisms ( + ) . Catalase test ( —). Formation of acid ( + ) and C 0 2 (—) from glucose [1]. Growth at 10 °C ( + ) and 45 °C ( + ) [15]. Growth at pH 9.6 ( + ) [16]. Survival at 63 °C for 30 min ( + ) [17]. Action on litmus milk ( + ) [18]. Production of acid from glycerol under anaerobic conditions ( —) [6]. Ability to tolerate methylene blue, 0 . 1 % in milk [ + ] [13]. Salt tolerance in the presence of 2 ( + ) , 4 ( + ), 5 ( + ), and 6.5 ( + ) % NaCl [10]. Gelatin liquefaction ( + ) [5]. Hydrolysis of arginine ( —) [12]. Carbohydrate oxidation (—) and fermentation ( + ) [8].

The strain was grown in sterilized skim-milk in a shaker for 15 days at 30 °C. Soluble protein was measured throughout the growth. The growth was stopped when the soluble nitrogen level reached its maximum. The milk was freezed at — 20 °C for 24 h and then thawed for 12 h at room temperature. This was repeated four times. Finally the milk was filtered. The filtrate was added to the cheese curd n the vat at the rate of 1 1 per 100 kg of milk after removing the whey.

675

R a s cheese

Cheese manufacture The procedure followed b y ABD-EL-TAWAB et al. [2] for making R a s cheese was adopted. 1 % of lactic acid starter (Str. lactis and Str. thermophilus 1 : 1 ) was added to pasteurized milk at 90 °F, mixed well and left to develop acidity up to 0 . 1 9 % . The rennet was dissolved in cooled 5 % NaCi solution, 25 g of rennet per liter. The prepared rennet was added at the proportion of 100 ml per 100 kg of milk. After coagulation the curd was cut vertically and horizontally with cheese knives and the temperature was raised to 1 1 3 ° F in 1 5 min. Curd was held at t h a t temperature for 30 min and whey was drained to the level of the curd (acidity 0 . 1 4 % ) . Salt was added 2 kg per 1 0 0 1 of milk used and the curd was stirred in the brine solution for 15 min. Finally, the remaining whey was drained completely and the curd was cooled, moulded and pressed with about 160 lbs for the first 2 h. Pressing was continued overnight after increasing the weights to 1000 lbs. Cheese mould was then dried and resalted b y rubbing dry salt on both sides. T h e salting process lasted for 12 days and then t h e cheese was waxed and ripened a t 60 ° F for 6 months. Moisture, salt, total nitrogen, acidity, fat, pH, soluble nitrogen, formol number and total volatile f a t t y acids determinations were carried out according to EL-ERIAN et al. [3].

Results and Dicsussion Moisture In agreement with literature recordings, the moisture content of the cheese decreased when the ripening progressed. This decrease was most remarkable during the early stages of ripening, and it became less pronounced upon aging. Table 1 shows the moisture content of the different cheeses throughout the different ripening stages. In fresh cheese the effect of the homogenization on the cheese yield is very clear. Homogenization of either the milk or the cream caused an increase of the ability of the fresh cheese to hold moisture. This increase ranged between 4.30% in (RHL) cheese and Table 1 Moisture content of the different R a s cheeses throughout different times of the ripening period Moisture content [ % ] a t the age of Trial

fresh

15 days

3° days

39-71

37-77 39-79 45-36 43-37

(C)

41.76

(R)

43.80

42.62

48.11

47-13 44-44 43-U

(RH) (RHL) (RHLH) (C) (R) (RH) (RHL) (RHLH)

46.06 46.18

= = = = =

40.32

60

90

120

180

days

days

days

days

35-99 37-79

34-54 35-88

31.88

27-45

42.81

40.69

41-33 37-98

39-65 36.25

33-55 38.99 37-15 34-56

28.94 36.89

33-59 32.28

Control Rennet paste Rennet paste + homogenized milk Rennet paste + homogenized cream + lipase Rennet paste + homogenized cream + lipase + cheese hydrolyzate.

6-35% in (RH) cheese. This may be explained by the findings of VOITKUS [20] who attributed it to the effect of the increase of the amount of water bound to the surface layer of the fat globule as a result of homogenization. A slight increase can be seen also (Table 1) in fresh cheese made from homogenized milk (RH) compared to cheese made from milk and homogenized cream. The same slight increase was also recognized as an effect of using the rennet paste instead of only rennet enzyme in manufacturing cheese. Cheeses with high fresh moisture content included higher moisture content at

676

El-Erian/Hashem/Abo-El-Heba

the end of the ripening stage of 180 days as seen in Table i . This table also shows that the control Ras cheese has the lowest content of moisture compared to the rest of the trials, their value came to 27.45%. Highest value was found in cheese made from homogenized milk plus the addition of Rennet paste. This value reached 36.89% in 180 days old Ras cheese. The moisture contents of the different trials, in the fully ripened stages, are in the range found b y different authors studying Ras cheese [3, 7, 14, 21]. It can be concluded from Table 1 that homogenization and addition of Rennet paste, lipase or cheese hydrolyzate or its combinations can lead to the production of an acceptable Ras cheese product as far as the final moisture content is concerned. Fat Table 2 shows that the fat content of all the different cheeses apparently increased steadily following the decrease of moisture in the cheese. As can be expected fresh control cheese with lower moisture content had a higher fat content compared with the rest of the trials, which contained higher moisture contents. All the analysed cheeses attained the Egyptian legal standards for the fat/solids ration in all ripening stages. The final results for fat content (Table 2) are in agreement with earlier workers. YOUSSEF [21] gave an average of 48.56% for fat content on dry matter basis with a m a x i m u m of 5 1 . 3 0 % a n d a m i n i m u m of 4 5 . 0 7 % f o r l o c a l R a s C h e e s e .

It was also clear from the authors findings that homogenizing the milk caused a higher loss of fat in the cheese whey, than homogenizing the milk cream. It can be concluded also that the homogenization caused a slight increase in the fat that was lost and drained with the whey. The slight decreases that can be seen in the values of fat/solids ratios could be due to the depletion of the fat of some of the free volatile f a t t y acids during the determination [4]. No clear effects can be concluded on the use of either Rennet paste, lipase or cheese hydrolyzate on the total fat content of the produced cheese. Salt Table 3 shows the salt content and the salt/water ratio on the different analysed cheeses. It is clear that both the salt percentages and the salt/water ratios increased upon cheese aging; this is due to the loss of moisture from the cheese. Control fresh cheese attained the highest content for both NaCl and salt/water ratio, being 3.93% and 9.40% respectively. The lowest contents occurred in the cheese made out of homogenized cream milk plus the addition of Rennet paste, lipase and cheese hydrolyzate (RHLH). The same was true for the final ripened cheeses. The results came to 4.48% and 16.30% for control cheese and 3.68% and 11.40% for (RHLH) cheese for NaCl and salt/water ratio respectively. EL-ERIAN et al. [3] reported the range of 4.75 bis 5.24% for salt content in local Ras cheese while YOUSSEFS values came to 2.81—4.90%. The results of these authors show that the findings of the present authors are in agreement with the average salt content in local made Ras cheese of the Egyptian market. The salt content of Ras cheese is closely related to that of Caskawal cheese (3,7,14,21]. Acidity and pH Table 4 shows the acidity and the p H of the different cheeses throughout different times of the ripening period of 180 days. Acidity increased in all cheeses steadily till the age of 90 days, after which a levelling up occurred in the acidity values till the end of the ripening period. The acidity values varied considerably between the different fresh cheeses. Both (C) and (R) fresh cheeses attained lower acidity values compared to the rest of the trials. The highest acidity content was found in (RHLH) fresh cheese,

677

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681

R a s cheese

were 130 to 150 and 12.3 to 14.5% for both formol number and soluble/total nitrogen ratio, respectively. Table 6 shows that the highest increase in formol number occurred between 60 and 120 days of cheese aging in all trials. The same table shows that the final results for the ratio soluble/total nitrogen of all the trials are in agreement with the findings of ELERIAN et al. [3]. for imported Ras cheese. For locally made Ras cheese he found lower values, ranging between 16.2 and 17.6%. The corresponding values of 14.5% and a formol number of 150 may suggest the early ripening of (RHLH) cheese trial around the age of 120 days. It is clear from Tables 5 and 6 that in the first stages of ripening, soluble nitrogen was formed at a higher rate than amino nitrogen (expressed in formol number). The difference might be explained b y the fact that soluble nitrogen is produced by the action of milk proteases, rennet enzymes and bacterial enzymes, while the formation of amino acids in cheese mainly depends on bacterial enzymes [19]. Table 7 T o t a l volatile f a t t y acids in t h e d i f f e r e n t R a s cheeses t h r o u g h o u t d i f f e r e n t t i m e s of t h e r i p e n i n g period T o t a l volatile f a t t y acids* a t t h e age of Trial

(C)

2.9

(R)

4-7 12.7 11.0 10.0

(RH) (RHL) (RHLH)

15 days

30 days

60 days

90 days

120 days

180 days

4-4 7.0 13-6 13.0 13.0

7-7 9-7 14.9 15.0 16.0

8.7 10.7 20,7 20.0 19.0

9.0

10.3

13-7 20.7 20.0 20.0

14-7 20.7 20.0 21.0

11.0 21.0 20.7 21.0 21.0

* expressed a s m l 0.1 N N a O H / 1 0 g of cheese

Volatile fatty acids Table 7 shows the total volatile fatty acids in the different cheeses throughout the ripening period of 180 days; after that the main three factors controlling the release of the volatile f a t t y acids were: — Homogenization, — Addition of Rennet paste combined with homogenization, — Addition of lipase. Control cheese (C) attained the lowest level of volatile fatty] acids either in the fresh state or in fully ripened cheese. Fresh control cheese reached values from 2.9 to 11.0 in 180 days old cheese, respectively. (RH) cheese exhibited the V.F.A. content of 12.7 in the fresh state, while (RHL) cheese attained the highest value of 24.0 in the fully ripened stage. Addition of cheese hydrolyzate seems to have no clear effect on the production of volatile fatty acids, as seen in (RHLH) cheese. Table 7 shows that the values for total volatile fatty acids in (RH), (RHL) and (RHLH) cheeses at the fresh state and at the age of 15 days are higher than that of the control cheese at the end of 180 days of ripening. It is also clear that the highest increase in total volatile fatty acids in the cheeses occurred between 15 and 60 days of cheese age. The only exception was found in (R) cheese where the highest increase occurred between 120 and 180 days of cheese aging.

682

EL-ERI AN/HASHEM/ABO-EL-HEB A

T h e present results are in agreement w i t h the findings of ISMAIL [9] w h o stated t h e increase in t o t a l volatile f a t t y acids in R a s cheese as a result of the addition of commercial lipase t o milk prior to manufacture. Table 8 Organoleptic properties of the different cheses at different ages of ripening Cheese

Organoleptic test

Trial days

Consistency

(C)

30 60 120 180

Short, crumply with no holes Short, crumply with no holes Short and little chalky Dry, chalky and few cracks

Fresh curdy, clean after taste Flat Acid taste, bitter Cheesy, volatile fatty acids taste, moderate salt

(R)

3° 60 120 180

Short, crumply with no holes Curd flakes, with no holes Curd flakes, few cracks Curd flakes, lot of cracks

Fresh curdy, clean after taste Flat Flat Slightly bitter, volatile fatty acids taste

(RH)

30 60 120 180

Open Open Open, few cracks Open, wide cracks

Acid, bitter, volatile fatty acids taste Cheesy, volatile fatty acids taste Very cheesy, rancid Very sharp, rancid, unclean off flavours

(RHL)

3° 60 120 180

Weak, open Open, few cracks Open, few cracks Open, lot of wide cracks

Acid Rancid, slightly bitter, very cheesy Rancid, bitter, very cheesy Rancid, bitter, unclean, off flavours

(RHLH)

30 60 120 180

Weak, Weak, Weak, Weak,

Bitter, acid Rancid, slightly bitter taste, very cheesy Rancid, bitter, unclean taste Rancid, unclean, off flavours

Organoleptic

Flavour

open open, few cracks few cracks lot of wide cracks

properties

T a b l e 8 shows the organoleptic properties of the different cheeses at the age of 30,60, 120 and 180 days. A s Buffaloes' milk was used for cheese manufacture grading of the cheeses was expected t o be in a lower level if compared to the usually used cow's milk. I t is v e r y clear from Table 8 t h a t in all treatments except the control and Rennet paste addition, the cheeses ripened at an earlier stage. T h e age of 60 d a y s could be recommended for ending the ripening process for ( R H ) , ( R H L ) and ( R H L H ) trials. E x t r a ripening time caused a quick deterioration in the b o d y and f l a v o u r of the cheese. Addition of Rennet paste plus milk cream homogenization can be recommended for quick R a s cheese ripening w i t h high volatile f a t t y acids taste. The addition of lipase will cause an e x t r a rancid f l a v o u r during the early ripening stages. Conclusions T h e most i m p o r t a n t results can be concluded in the following points: — Moisture content of the cheese decreased as the ripening progressed. — Homogenization of either the milk or the cream caused an increase in the ability of the fresh cheese to retain moisture. The increase ranged between 4 . 3 % in ( R H L ) cheese and 6 . 3 5 % in (RH) cheese.

Ras cheese

683

— Cheeses with high initial moisture content included a final higher moisture content at the end of the ripening period of 180 days. — Homogenization and addition of Rennet paste, lipase or cheese hydrolyzate or its combinations lead to the production of acceptable Ras cheese product for its moisture content. — Homogenizing the milk caused a higher loss of fat in the cheese whey than homogenizing the milk cream. Homogenization in general caused a slight increase in the fat that was lost and drained with the whey. — No effect was detected by using either Rennet paste, lipase or cheese hydrolyzate on the total fat content of the different cheese trials throughout the ripening period. — Both the salt percentages and the salt/water ratios increased upon cheese aging and were due to the loss of moisture from the cheese. — Homogenization caused the production of more acid cheese. — Homogenization of cream caused the release of higher acidity values than the homogenization of the whole milk. — Addition of Rennet paste gave a fresh cheese a slightly higher pH than the control cheese which was due to the release of some of the alkaline compounds. — Homogenization resulted in cheeses with a lower pH. Such lowering effect increased by the presence of lipase, and was even mores pronounced when both lipase and cheese hydrolyzate were added. — Homogenization of either the milk or the cream resulted in producing fresh cheese with a low soluble nitrogen content but higher in its formol number than cheeses that were made from unhomogenized milk. — The highest increase in formol number occurred in the period from 60 — 120 days of cheese age in all trials. — During the first stages of ripening soluble nitrogen was formed at a higher rate than amino nitrogen (expressed in formol number). — Total volatile fatty acids in homogenized milk or cream cheeses at the fresh state and at the age of 15 days were higher than that of the control cheese at the end of 180 days of ripening. The highest increase in total volatile fatty acids in the cheeses occurred between 15 and 60 days of cheese age. — The age of 60 days can be recommended as an end of the ripening process for homogenized milk or cream trials. Extra ripening time caused a quick deterioration in the body and flavour of the cheese. Addition of Rennet paste plus milk cream homogenization can be recommended for quick Ras cheese ripening with high volatile fatty acids taste. Zusammenfassung A. F. M. El-Erian, H. A. Hashem und A. M. Abo-El-Heba: Chemische Zusammensetzung und organoleptische Eigenschaften von unter verschiedenen technologischen Bedingungen hergestelltem Ras-Käse Die Untersuchungen wurden im Hinblick auf eine Modifizierung und auf eine Verkürzung des Reifeprozesses durchgeführt. Zur Verkürzung der Reifedauer und zur Erhöhung der Bildung von Aroma- und Geschmacksstoffen wurden eine Lab-Paste, Lipase und Käsehydrolysat einzeln oder in Kombination zugesetzt. Um eine Beschleunigung der Fetthydrolyse zu erreichen, wurde entweder die Milch oder der Rahm vor der Verarbeitung homogenisiert.

684

E L - E R I A N / H ASHEM /A B O - E L - H E B A

I m Frischkäse und während der Käsereifung (nach 15, 30, 60, 90, 120 und 180 Tagen) wurden der Gehalt an Wasser, Salz, Fett, Gesamtsäure, Gesamtstickstoff, löslichem Stickstoff und flüchtigen Fettsäuren bestimmt, der pH-Wert und die Formol-Zahl ermittelt und die Produkte sensorisch überprüft. Die mit den verschiedenen Versuchsvarianten erzielten Ergebnisse werden diskutiert.

Pe3iOMe A . ynHbix r p y n n h 2 MOJieii cyjibiJrHnpyjibHbix r p y n n Ha MOJib HUMepHoro /3-jiaKTorjioßyjiHna oÖHapyjKHBaMTCH 0,4 MOJieii HHcyjib$H«Hbix r p y n n , 2 , 2 MOJieii cyjib(j»rHnpHjibHbix r p y n n 11 1 , 8 MOJieft c y j i b ^ H H H b i x HOHOB Ha MOJib NHMEPHORO / ? - j i a K T o r j i o 6 y j i i i H a .

CyjibliimHbie

110HM npHMO onpenejiHKJTCH KaK c e p o B o n o p o a HJIH H3 pa3HHUH BejiHiHH, nojiyqeHHbix aMnepoMeTpHHecKH-apreHTOMepTHiecKOH THTpauHeii H MCTOHOM SjuiMaHa (peaKmiH oÖMeHa c D T N B ) . JHHcyjib$HHHbie r p y n n B i onpenenHiOTCH cnoMombio D T N B n o c J i e BOccTaHOBjiemiH c HaTpHH rnjipHnOM 6 o p a . IlojiyHeHHbie n o c j i e BoccTaHOBJieHHH c HaTptiii rnapHjiOM 6 o p a cyjibijiriinpMJibHbie r p y n n b i n0BT0pH0 OKHCJIHIOTCH n o 3aKOHy CKopocTH BToporo n o p n f l K a . I l o c j i e BoccTaHOBJieHHH HHcyjib$HHHbix r p y n n HaTpnii rHnpnnoM 6 o p a HapymaeTCH apreHTOMeTpniecKH-aMnepoMeTpimecKoe onpenejieHHC cyjib$rHnpHJibHbix r p y n n c BpamaiomiiMCH n-nacTiiHiaTHM njiaraHOBbiM 3JieKTp0H0M H3-3a n p n cyTCTBHH 6 o p a . Literatur [1] [2] [3] £4]

DANEHY, J . P . , u. W . H . HUNTER, J . org. Chem. 32, 2047 (1967). DANEHY, J. P . , u. J. A . KREUZ, J. A m e r . Chem. Soc. 83, 1109 (1961). DONOVAN, J. W . , u . T . M . WHITE, B i o c h e m i s t r y 10, 32 (1970). PARKER, A . J., u. N . KHARASCH, C h e m . R e v . 59, 583 (1959).

[5] SCHÖBERL, A., u. H. TAUSENT, Proc. int. Wool Textile Res. Conf., Australia 1955, Vol. C, S. 150. 4S*

704

NÖTZOLD/SCHLEGEL/BREITFELD/FREIMUTH

[6] ANDERSON, L. O., U. G. BERG, Biochem. biophysica Acta [Amsterdam] 192, 534 (1969). [7] SOKOLOVSKI, M . , T . SADEH U. A . PATCHORNIK, J . A m e r . C h e m . S o c . 86, 1 2 1 2 (1964).

[8] STRICKS, W. J . , U. J . M. KOLTHOFF, Analyt. Chem. 25, 1050 (1953). [9] GAWRON, O., U. G. ODSTRCHEL, J . Amer. chem. Soc. 89, 3263 (1967). [ 1 0 ] KHARASCH, N . , U. R . S . SWIDLER, J . org. C h e m . 1 9 , 1 7 0 4 ( 1 9 5 4 ) .

[ 1 1 ] BOHAK, Z., J . biol. Chem. 239, 287S (1964). [12]

B O H A K , Z . , z i t . b e i S P A N D E , T . F . , B . W I T K O P , Y . D E G A N I U. A . P A T C H O R N I K , A d v a n c e s

Protein

Chem. 24, 97 (1970). [ 1 3 ] F R E I M U T H , U . , B . S C H L E G E L , E . G A H N E R U. H . N Ö T Z O L D , N a h r u n g 1 8 , K 5

(1974).

[ 1 4 ] ZAHN, H . , U. L . LUMPER, H O P P E - S E Y L E R ' S Z . p h y s i o l . C h e m . 3 4 9 , 7 7 ( 1 9 6 8 ) .

[ 1 5 ] ROBBINS, F . M . , U. J . A . FIORITI, N a t u r e 200, 5 7 7 ( 1 9 6 3 ) .

[16] TANIGUCHI, N., Analyt. Biochem. (New York) 40, 200 (1971). [ 1 7 ] ASCHAFFENBURG, K . , U. J . D R E W R Y , B i o c h e m . J . 65, 2 7 3 ( 1 9 5 7 ) .

[18] ELLMAN, G. L., Arch. Biochem. Biophysics 82, 70 (1959). [19]

NÖTZOLD, H . , B . S C H L E G E L , I . T I N I U S U. U . F R E I M U T H , Z . C h e m . 1 2 , 2 4

(1972).

[20] ROBYT, J . F., R . J . ACKERMAN U. C. G. CHITTENDEN, Arch. Biochem. Biophysics 147, 262 (1971). [21] ACKERMAN, R . J . , U. J . F. ROBYT, Analyt. Biochem. (New York) 50, 656 (1972). [22] MOORE, S . , R . D . COLE, H . G . GRUNDLACH U. W . H . STEIN, P r o c . 4 t h I n t e r n . C o n g r . B i o c h e m . ,

Wien 1958. [23] GÖTZE, B., Diplomarbeit Technische Universität Dresden, 1972. [24] ELLMAN, G. L., Arch. Biochem. Biophysics 74, 443 (1958). [25] MAN, MING, U. R . G. BRYANT, Analyt. Biochem. (New York) 57, 429 (1974). [26] BUTTERWORTH, P . H . W . , H . BAUM U. J . PORTER, A r c h . B i o c h e m . B i o p h y s i c s 1 1 8 , 7 1 6 ( 1 9 6 7 ) .

[27] ANDERSSON, L.-O., Arch. Biochem. Biophysics 1 3 3 , 277 (1969). [28] KIRKPATRICK, A . , U. J . A . MCLAREN, A n a l y t . B i o c h e m . ( N e w Y o r k ) 56, 1 3 7 ( 1 9 7 3 ) .

[29] MROWETZ, G., U. H. KLOSTERMEYER, Z. Lebensmittel-Unters, u. -Forsch. 149, r34 (1972). Prof. Dr. rer. nat. habil. U. FREIMUTH, Wissenschaftsbereich Lebensmittelchemie der Sektion Chemie der Technischen Universität Dresden, DDR-8027 Dresden, Mommsenstr. 13 Eingegangen 27. 12. 1976

Die Nahrung

21

8

1977

705 — 710

Zentralinstitut für Ernährung in Potsdam-Rehbrücke (Direktor: Prof. Dr. H. HAENEL), Forschungszentrum für Molekularbiologie und Medizin, Akademie der Wissenschaften der D D R und Forschungszentrum für Nahrungs- und Ernährungshygiene des Instituts für Hygiene und Epidemiologie in Prag, CSSR (Leiter: Prof. Dr. A. WOLF)

Beitrag zur Migration und Toxikologie von 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)5-chlorbenztriazol W . - J . UHDE u n d J. HORÀCEK

Untersuchungen zum Einsatz eines UV-Absorbers auf Hydroxybenztriazol-Basis für Bedarfsgegenstände aus Niederdruck- und Hochdruck-Polyäthylen sowie Polypropylen zeigen, daß bei wäßrigen und sauren Lebensmitteln und bei alkoholischen Genußmitteln mit niedrigem Alkoholgehalt nur eine geringe Migration stattfindet. Sonnenblumenöl, n-Heptan (als eine fettsimulierende Testlösung) und 5o%iges Äthanol als Testlösungen ergeben höhere Migrationswerte. Zur Bestimmung des UV-Absorbers werden gaschromatographische, polarographische und dünnschichtchromatographische Methoden eingesetzt. Aufgetretene Bedenken über eine mögliche photosensibilisierende Wirkung von 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)5-chlorbenztriazol werden experimentell überprüft und erweisen sich als unbegründet. Gegen den Einsatz von 0,3% des UV-Absorbers zur Stabilisierung von Polyolefinen für die Lebensmittelverpackung gibt es für die meisten Lebensmittel keine gesundheitlichen Bedenken. Im Falle fetthaltiger und stark alkoholischer Lebensmittel muß eine Zulassung jedoch von den Anwendungsbedingungen abhängig gemacht werden. Für die Stabilisierung von Polyolefinen gegen UV-Licht wird empfohlen, bei Polypropylen 0,3 bis 0,6% und bei Polyäthylen 0,2 bis 0,4% 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)-5-chlorbenztriazol 1 einzusetzen [1]. Infolge seiner geringen Flüchtigkeit bei hohen Temperaturen und seiner guten Beständigkeit gegen thermische Zersetzung kann es ohne nennenswerte Verluste und Zersetzung bei der Verarbeitung eingesetzt werden. Neben der eigentlichen Erhöhung der Lichtschutzstabilisierung des Plastmaterials selbst kann die Anwesenheit des UV-Absorbers auch auf das verpackte Lebensmittel hinsichtlich der Haltbarkeit einen günstigen Einfluß ausüben. Die LD 5 0 per os für Mäuse und Ratten ist mit > 5000 mg/kg Körpergewicht der Versuchstiere sehr hoch. Die einmal täglich über 4 Wochen vorgenommene Applikation einer 5%igen Emulsion des Präparates in wäßrigem Gummiarabikum ergab keine lokale Irritation und keine resorptive Giftwirkung. Auch Untersuchungen zur subchronischen Toxizität (90-Tage-Versuch) verliefen befriedigend [12]. Auf eine mögliche photosensibilisierende Wirkung ist von JANECKOVA u. a. [14] hingewiesen worden. Auf Grund der ausgedehnten toxikologischen Prüfungen kann der UV-Absorber in den geprüften Konzentrationen als nicht toxische Substanz angesehen werden. Als tolerierbarer Migrationswert werden i o p p m vorgeschlagen [13].

Im Rahmen der hier vorliegenden Arbeit soll nachgewiesen werden, ob dieser in Polyolefinen vorhandene UV-Absorber vom Benztriazol-Typ von LebensmittelModellflüssigkeiten herausgelöst wird. Zu diesem Zweck mußten zunächst geeignete 1

Tinuvin 326, Handelsname der C I B A - G E I G Y AG, Basel (Schweiz)

Die Nahrung

21

8

1977

705 — 710

Zentralinstitut für Ernährung in Potsdam-Rehbrücke (Direktor: Prof. Dr. H. HAENEL), Forschungszentrum für Molekularbiologie und Medizin, Akademie der Wissenschaften der D D R und Forschungszentrum für Nahrungs- und Ernährungshygiene des Instituts für Hygiene und Epidemiologie in Prag, CSSR (Leiter: Prof. Dr. A. WOLF)

Beitrag zur Migration und Toxikologie von 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)5-chlorbenztriazol W . - J . UHDE u n d J. HORÀCEK

Untersuchungen zum Einsatz eines UV-Absorbers auf Hydroxybenztriazol-Basis für Bedarfsgegenstände aus Niederdruck- und Hochdruck-Polyäthylen sowie Polypropylen zeigen, daß bei wäßrigen und sauren Lebensmitteln und bei alkoholischen Genußmitteln mit niedrigem Alkoholgehalt nur eine geringe Migration stattfindet. Sonnenblumenöl, n-Heptan (als eine fettsimulierende Testlösung) und 5o%iges Äthanol als Testlösungen ergeben höhere Migrationswerte. Zur Bestimmung des UV-Absorbers werden gaschromatographische, polarographische und dünnschichtchromatographische Methoden eingesetzt. Aufgetretene Bedenken über eine mögliche photosensibilisierende Wirkung von 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)5-chlorbenztriazol werden experimentell überprüft und erweisen sich als unbegründet. Gegen den Einsatz von 0,3% des UV-Absorbers zur Stabilisierung von Polyolefinen für die Lebensmittelverpackung gibt es für die meisten Lebensmittel keine gesundheitlichen Bedenken. Im Falle fetthaltiger und stark alkoholischer Lebensmittel muß eine Zulassung jedoch von den Anwendungsbedingungen abhängig gemacht werden. Für die Stabilisierung von Polyolefinen gegen UV-Licht wird empfohlen, bei Polypropylen 0,3 bis 0,6% und bei Polyäthylen 0,2 bis 0,4% 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)-5-chlorbenztriazol 1 einzusetzen [1]. Infolge seiner geringen Flüchtigkeit bei hohen Temperaturen und seiner guten Beständigkeit gegen thermische Zersetzung kann es ohne nennenswerte Verluste und Zersetzung bei der Verarbeitung eingesetzt werden. Neben der eigentlichen Erhöhung der Lichtschutzstabilisierung des Plastmaterials selbst kann die Anwesenheit des UV-Absorbers auch auf das verpackte Lebensmittel hinsichtlich der Haltbarkeit einen günstigen Einfluß ausüben. Die LD 5 0 per os für Mäuse und Ratten ist mit > 5000 mg/kg Körpergewicht der Versuchstiere sehr hoch. Die einmal täglich über 4 Wochen vorgenommene Applikation einer 5%igen Emulsion des Präparates in wäßrigem Gummiarabikum ergab keine lokale Irritation und keine resorptive Giftwirkung. Auch Untersuchungen zur subchronischen Toxizität (90-Tage-Versuch) verliefen befriedigend [12]. Auf eine mögliche photosensibilisierende Wirkung ist von JANECKOVA u. a. [14] hingewiesen worden. Auf Grund der ausgedehnten toxikologischen Prüfungen kann der UV-Absorber in den geprüften Konzentrationen als nicht toxische Substanz angesehen werden. Als tolerierbarer Migrationswert werden i o p p m vorgeschlagen [13].

Im Rahmen der hier vorliegenden Arbeit soll nachgewiesen werden, ob dieser in Polyolefinen vorhandene UV-Absorber vom Benztriazol-Typ von LebensmittelModellflüssigkeiten herausgelöst wird. Zu diesem Zweck mußten zunächst geeignete 1

Tinuvin 326, Handelsname der C I B A - G E I G Y AG, Basel (Schweiz)

706

UHDE/HORÄCEK

analytische Methoden erarbeitet werden. Insbesondere galt es, zuverlässige Isolierungsverfahren zu entwickeln. Auch der bestehende Verdacht hinsichtlich einer pliotosensibilisierenden Wirkung des o. g. UV-Absorbers sollte nachgeprüft werden. Versuchsdurchführung

und Methodik der

Migrationsuntersuchungen

Als Probenmaterial standen der technisch hergestellte UV-Absorber und damit stabilisierte Platten aus Niederdruck-Polyäthylen, Hochdruck-Polyäthylen und Polypropylen zur Verfügung. Die Abmessungen der Platten betrugen 10 x 10 cm, die Dicke lag bei etwa i mm. Der Anteil an UV-Absorber war abgestuft und betrug max. 0,6%. Bei der Versuchsdurchführung wurden sowohl die Bedingungen des DDR-Gebrauchstestes für Plaste im Lebensmittelverkehr [2] als auch die in der CSSR gültigen Untersuchungsvorschriften [3] zugrunde gelegt. Über Trennung und Nachweis von UV-Absorbern auf Basis von Hydroxyphenylbenztriazol ist nur wenig bekannt. VAN DER HEIDE [4] beschreibt ein spektrophotometrisches Verfahren, das eine direkte Bestimmung einiger Hydroxyphenylbenztriazole in verschiedenen lebensmittelersetzenden Flüssigkeiten ermöglichen soll. SOUCEK u. a. [5] identifizierten und bestimmten UV-Absorber in Polypropylen durch differentiale UV-Spektrophotometrie. Nach früher durchgeführten Untersuchungen lassen sich diese UV-Absorber semiquantitativ mittels Dünnschichtchromatographie und quantitativ mittels Polarographie gut bestimmen [6]. Auch die Gaschromatographie wurde von verschiedenen Autoren vorgeschlagen [4, 7, 8, 15]. ROBERT U. a. [8] haben dabei das in Methylenchlorid gelöste Polymere direkt in den Gaschromatographen eingespritzt. Trotz der skeptischen Meinung von CROMPTON [9] haben wir mit gutem Erfolg die Gaschromatographie im Rahmen unserer Migrationsuntersuchungen eingesetzt. Bei dieser Methode stören die üblicherweise verwendeten Plastadditive nicht [10].

A rbeitsvor schrift Apparate und Reagenzien Kathodenstrahlpolarograph K 1000 der Southern Analytical Ltd., polarographische Zelle nach NOVAK mit seitlich angesetztem Entlüftungsrohr und Bodenquecksilber Gaschromatograph, Packard-Instr., Modell Becker 419 mit Zubehör Standard-Ausrüstung für die Dünnschicht-Chromatographie Kieselgel G der Fa. Merck, Darmstadt (BRD) Analysenquarzlampe, UV-Licht 365 nm Bombenstickstoff mit Pyrogallollösung und Wasser gewaschen Leitelektrolyt: 160 ml Benzol, 120 ml Perchlorsäure (5 N), 40 ml Wasser und Äthanol ad 1000 ml Sprühreagenz I: Lösung von 200 mg Echtrotsalz A L und 100 mg Natriumacetat in 10 ml Wasser Sprühreagenz I I : Nacheinanderfolgendes Besprühen mit 5%iger Eisen(III)-chlorid-Lösung, 5%iger Kaliumhexacyanoferrat(III)-Lösung und 1 N Salzsäure Fließmittel: n-Heptan + Chloroform (4 + 1).

Halbquantitative

dünnschicht-chromatographische

Bestimmung

(Screening-Methode)

Die halbquantitative Bestimmung der Hydroxyphenylbenztriazole erfolgt nach dem visuellen Fleckenvergleich auf den mit Kieselgel G beschichteten Glasplatten der Größe 20 x 20 cm. Auf die Startpunkte 1, 3, 5 und 7 werden in einem Abstand von 2 cm zunehmende Mengen der UV-AbsorberVergleichslösung (o,i%ige Lösung in Chloroform) und auf die Startpunkte 8, 6, 4 und 2 ebenfalls zunehmende Mengen der eingeengten Extraktlösung 2 aufgetragen. Zum Sichtbarmachen der Flecken wird die Platte unter der Analysenquarzlampe bei 365 nm betrachtet. Die Flecken können auch durch 2 Bei Verwendung von Sonnenblumenöl als Testflüssigkeit werden 20 ml mit 50 ml n-Pentan verdünnt und dreimal mit je xo ml wäßrigem Acetonitril (5% Wasser) 5 min kräftig im Scheidetrichter geschüttelt. Die vereinigten Acetonitrilextrakte werden nach Zugabe von 50 ml n-Pentan ausgeschüttelt, vorsichtig im Vakuum in einem Spitzlcölbchen auf 1 ml eingeengt und chromatographiert.

Migration und Toxikologie von H y d r o x y b e n z t r i a z o l

707

Besprühen m i t E c h t r o t s a l z - A L - L ö s u n g (Sprühreagenz I) oder mit Berliner-Blau-Reagenz (Sprühreagenz II) nach kurzzeitigem E r w ä r m e n auf 100 °C sichtbar gemacht werden. Die für den Fleckenvergleich zweckmäßige Menge sollte durch ein Vorchromatogramm ermittelt werden. Dasjenige Fleckenpaar (Analysen- und Vergleichslösung), das hinsichtlich Fleckengröße und Intensität miteinander gleich ist, dient entsprechend der aufgetragenen Vergleichsmenge als Berechnungsgrundlage. Nachweisgrenze: 0,01 p p m .

Gaschromatographische Bestimmung Säule: Edelstahl, 3 m m Außendurchmesser, 1 m Länge, gefüllt mit Chromosorb W (80 — 100 mesh), belegt m i t 1 0 % SE-30 Gase: Stickstoff, 20 ml/min; Wasserstoff, 30 ml/min; L u f t , 300 ml/min I n j e k t o r t e m p e r a t u r : 260 °C F I D - T e m p e r a t u r : 280 °C Temperaturprogrammierung: Start 180 °C, nach 2 min Temperaturgradient 5 °C/min bis 270 "C. Migrat wird bis zur Trockne eingedampft und mit Chloroform aufgenommen; d a v o n werden wie üblich 2 — 5 fil injiziert. Auswertung der Gaschromatogramme erfolgte anhand einer unter den gleichen Bedingungen aufgestellten Eichkurve. Nachweisgrenze: 0,06 ppm.

Polaro graphische

Bestimmung

Z u r B e s t i m m u n g des U V - A b s o r b e r s wird der R ü c k s t a n d der zur Trockne g e d a m p f t e n Testlösung sorgfältig in 3 ml der o. g. Leitelektrolytlösung aufgenommen. Z u r E n t f e r n u n g des Luftsauerstoffs wird 5 min Stickstoff durchgeleitet und anschließend i m Bereich v o n — 1 , 3 bis — 1 , 8 V o l t polarographiert. D a s Halbstufenpotential liegt bei — 1 , 5 Volt, gemessen gegen Bodenquecksilber. Die A u s w e r t u n g der P e a k h ö h e erfolgt nach einer Eichkurve. Zur Aufstellung der E i c h k u r v e werden aliquote Teile einer chloroformischen Stammlösung im Trockenschrank bei 100 °C eingedampft und der R ü c k s t a n d mit einer entsprechenden Menge Leitelektrolytlösung aufgenommen. Nachweisgrenze: 0,1 ppm. D a beim A b d a m p f e n der wäßrigen, sauren und alkoholischen Testflüssigkeiten größere Verluste an U V - A b s o r b e r auftreten, ist dieser durch E x t r a k t i o n mit n - H e p t a n zu isolieren. Z u m Ausschütteln v o n 250 ml Testflüssigkeit werden zweimal je 20 ml und einmal 10 ml n - H e p t a n verwendet. B e i den 5 0 % i g e n alkoholischen Testflüssigkeiten ist v o r der E x t r a k t i o n zur besseren Trennung der Phasen etwas Wasser hinzuzufügen. Die H e p t a n e x t r a k t e werden im V a k u u m zur Trockne gedampft, der R ü c k s t a n d wird sofort sorgfältig im Leitelektrolyt gelöst und anschließend polarographiert. 1 Bei V e r w e n d u n g v o n Sonnenblumenöl wird der U V - A b s o r b e r vorher säulenchromatographisch v o m Öl abgetrennt [11]. Zur Bestimmung wird dann das als Elutionsmittel verwendete Cyclohexan a b g e d a m p f t , der R ü c k s t a n d in Leitelektrolyt-Lösung aufgenommen und wie oben beschrieben verfahren.

Migrationsergebnisse Ausgewählte Ergebnisse der Migrationsuntersuchungen sind in Tab. 1 — 3 zusammengefaßt. Bei der Berechnung der Werte wurde von der Annahme ausgegangen, daß 2000 cm 2 Verpackungsoberfläche mit 1 kg bzw. 1 1 Lebensmittel in Berührung kommen. Diesem Verhältniswert liegen internationale Erfahrungen zugrunde und schließen schon einen Sicherheitsfaktor ein. Die in Wasser, verdünnte Essigsäure und verdünnten Alkohol migrierte UVAbsorber-Menge liegt bei allen drei geprüften Plasten (Hochdruck-Polyäthylen, Niederdruck-Polyäthylen, Polypropylen) unter 1 ppm. Bei 5o%igem Alkohol, Sonnenblumenöl und bei dem in der CSSR als fettsimulierende Testlösung üblichen n-Heptan sind wesentlich höhere Migrationswerte zu verzeichnen. Toxikologische Untersuchungen zur

Photosensibilisierung

Zur Prüfung, ob mit der o. a. möglichen photosensibilisierenden Wirkung [14] tatsächlich gerechnet werden muß, wurde Meerschweinchen an 20 aufeinanderfolgenden

Uhde/Horä£ek

708

Tabelle i Migration von 2-(2'-Hydroxy-3'-tert.butyl-5'-methylphenyl)-5-chlorbenztriazol aus Polypropylen in Lebensmittel-Modellflüssigkeiten [ppm] Testflüssigkeit

UV-Absorber [%]

10 d bei 20 °C

o-3 o.5