Mutagenicity of pan residues and gravy from fried meat

Mutation Research, 187 (1987) 47-53

47

Elsevier MTR 01128

M u t a g e n i c i t y of p a n residues a n d gravy f r o m fried m e a t E. Overvik 1, L. Nilsson 1, L. Fredholm 3, 0. Levin 3, C.-E. Nord 2 and J.-A. Gustafsson 1 t Department of Medical Nutrition and 2 Department of Oral Microbiology, Karolinska Institute, Huddinge University Hospital, F 69, S-141 86 Huddinge (Sweden) and 3 Research Laboratories of Margarinbolaget, Stockholm (Sweden)

(Received 31 July 1986) (Accepted 13 August 1986)

Keywords: Fried meat; Pan residues; Gravy; (Ames Salmonella test).

Summary Lean pork meat was fried with or without the addition of frying-fat at 200 or 250 o C. The pan residues were collected by washing the hot pan with boiling water. When producing thickened gravy the water was substituted by a mixture of water and flour, milk and flour or cream and flour. The basic extracts were tested for mutagenicity in Ames' Salmonella test on strain TA98 with the addition of $9 mix. High amounts of mutagenicity were found in all samples. The amounts of mutagenicity in the pan residues were at a comparable level of the amounts found in the meat crusts. Thickening of the gravy caused only small changes in the mutagenicity.

The formation of mutagenic compounds in the crust of fried meat is well known (Spingarn et al., 1979; Pariza et al., 1979; 0vervik et al., 1984). Substantial mutagenicity has also been recovered in commercial beef extract as well as beef stock prepared by boiling water extracts from ground beef for prolonged periods (Commoner et al., 1978; Dolara et al., 1979, 1980; Taylor et al., 1984). More than 10 different compounds with varying mutagenlc potency in the Ames' Salmonella assay have been isolated from the crust of fried beef and fish (Kasai et al., 1980; Felton et al., 1984; Matsukura et al., 1981). The same type of compounds were shown to appear also in the muta-

Correspondence: Dr. Jan-,~kke Gustafsson, Department of Medical Nutrition, Karolinska Institute, F 69, Huddinge University Hospital, S-141 86 Huddinge (Sweden).

genic commercial beef extract and beef stock (Hargraves et al., 1983; Turesky et al., 1983; Taylor et al., 1985). Several of these food mutagens have been subjected to carcinogenicity tests on rats and mice and were shown to cause tumor formation in various sites on these animals (Matsukura et al., 1981; Ohgaki et al., 1984; Sugimura et al., 1985). The "Western-type" of diet often includes fried meat and fish several times a week. The meat is commonly served together with a gravy made from the pan residues. The aim of the present study was to evaluate the amount of mutagenicity formed in the pan residues during frying of meat and to compare this to the amount of mutagenicity formed in the crust. The effect on mutagenicity of thickening the gravy was also studied.

0165-1218/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

88 49 14

11 66 37

41 36 12

Sunflowerseed oil Butter Margarine A (solid)

Margarine B (solid) Margarine C (soft) Margarine D (liquid)

39 35 21

21 25 46

6 37 71

20 27 66

64 3 15

2 10 11

23 15

13

100 100

Lard

100

Olive oil

100

100

Sunflowerseed oil

Coconut oil

Polyenes

Saturated

Monoenes

Composition of fats % (w/w)

Fatty acids % ( w / w )

Coconut oil Lard Olive oil

Fat

100

Milk fat

31 10

8

Soybean oil

36 36

Hydrogenated soybean oil

23

Rapeseed oil

30

Hydrogenated rapeseed oil

39

Cottonseed oil

10

Palm oil

16

Hydrogenated Menhaden oil (fish oil)

10

Tallow

The oils and fats consist of 100% (w/w) fat. The margarines contain 80% fat, 7.5-10% skimmed milk and 1.5-1.8% salt. Butter contains 80% fat, 18% buttermilk and 1.3% salt.

COMPOSITION OF FATS USED FOR F R Y I N G

TABLE 1

49

Materials and methods Lean ( - 5% fat) pork meat was acquired from pigs reared for progeny testing and thus fed in a standardized manner. The temperature was measured with Cr-Al-thermocouples in the bottom of the frying-pan, in the centre of the meat and at the surface of contact between the meat and the pan. The starting temperature in the bottom of the frying pan was 200 or 250°C. The meat was fried for 10.5 or 10 min, respectively, and turned once during frying. Two slices, each weighing - 1 0 0 g, were fried at the same time. In the cases where the meat was fried with fat, 16 g fat forming a fat layer with an area of 254 cm 2 and a depth of 0.7 m m was added to the pan for each frying of two meat slices. 9 different frying fats were used, their composition is shown in Table 1. Fat was added when the pan had reached the desired temperature, 200 or 250°C. The addition of fat caused a lowering of the pan temperature of around 25°C. When the original temperature was reached again, the meat was added. After removing the fried meat slices, 50 ml tap water was added to the hot pan. The water was boiled for a few minutes and stirred so that the residues in the b o t t o m of the frying-pan were transferred into the water. The sample collected in this manner is called the pan residue. For preparation of gravy, 2 g wheat flour of c o m m o n household quality was mixed with either 50 ml tap-water, 50 ml milk (3% fat) or 50 ml cream (40% fat). The mixture was added to the hot pan in the same manner as when preparing the pan residues. Thickened gravy was prepared only at 200 ° C, and only for samples fried without fat or with margarine B.

Treatment of samples The crust, - 2 m m thick, was separated from the inner parts of the meat and lyophilized. Pan residues or gravy were collected separately for each frying. All samples were stored in a freezer ( - 18°C) for a few weeks prior to extraction.

Extraction procedures The extraction of the crust was performed according to Fig. 1A, twice with C H C 1 3 / C H 3 O H

(1 : 1, v / v ) and twice with CH3OH. In each of these steps 5 ml of solvent was added per g dry substance (DS) and the mixture was stirred for 5 h. The mixture was filtered through a sintered glass funnel, and the extraction repeated with the same amount of solvent. The filtrates were combined, placed in a freezer ( - 1 8 ° C ) overnight and refiltered to remove precipitated protein. The clear extracts were concentrated to dryness on a rotary evaporator. The dry extracts were dissolved in a solution of 70% ethanol in water, p H 2, and washed 3 times with equivalent amounts of petroleum ether, b.p. 35-40 o C. The ethanol-water phases were adjusted to p H 10-12 and extracted 3 times with dichloromethane. The organic fractions containing the basic components were evaporated to dryness and dissolved in dimethyl sulfoxide. The pan residues and the gravy samples were centrifuged at 2000 × g for 10 rain (Fig. 1B). The solids were treated in the same manner as the crust (cf. above). The supernatants were passed through an Amberlite XAD-2 resin, 12.5 x 1.5 cm, prewashed with ethanol, acetone and distilled H20. The samples were passed through the column at a speed of 1 m l / m i n , the resin was washed with 100 ml redistilled H 2 0 and eluted with 150 ml C H C l J C H 3 O H 1 : 1. The solvent was evaporated, the dry samples were dissolved in 70% ethanol in water, p H 2, and acids and bases were separated as described above. Finally, the basic components were combined with the corresponding extracts from the solid phase of the pan residue and gravy, respectively.

Mutagenicity assay The mutagenicity of the extracts was assayed in Ames' Salmonella/mammalian microsome test on Salmonella typhimurium TA98 which was kindly supplied by Dr. Bruce N. Ames, University of Berkeley, CA. The test was conducted according to the original method (Ames et al., 1975). All measurements were made on triplicate plates and repeated on 4 different occasions. The spontaneous reversion rate was 27 d- 3 revertants/plate; this value was not subtracted from any of the results shown. All measurements were made with the addition of $9 mix prepared from livers of

50

B A meat c r u s t

measured by the method of Lowry et al. (1951). All samples were tested at 7 dose levels. The colonies were counted in an automatic colony counter.

pan - residue or gravy

p/solids ~'-

CHCl3:CH3OH ~ K . 1:1 "2 I~ ~ :~ solids

1 centrifugalion liquid/ phase

or anic

|

p~se

CH3OH'2 1 ~ so,as ~-~'1/" discarded;

Statistical procedures

XAD2 * I c.c.,/

From the linear part of the dose-response curves the slope, intercept and correlation coefficient of each line were calculated. Significance of the dose-responses and comparison between the mean values of the samples were determined by Student's t-test.

~ CH30H 1:1

organic phase

organic phase

evaporatedtodryrtess dissolvedinTO';C21t,3OHin water pH2 \ \

evapolatadto dryness dissolved in 70"/.C2H~:OHin / / waterpH2

/ ~tr~um

ether- 3

~ p ether phase discarded

70% C2HsOH phase pH 10-12 C2H5OH-phase discarded CH2CI2 x3

Results

basic extract

Fig. 1. Scheme for extraction of mutagenic compounds in crust (A) or pan residue (B) of fried meat.

male Sprague-Dawley rats induced with Aroclor 1254. The amount of $9 was 70 # l / p l a t e corresponding to 1.89 mg microsomal protein/plate as

500: W i t h o u t

fat

All samples - - meat, pan residues and gravy were mutagenic with highly significant dose-responses, p < 0.005, in all cases. The temperature dependence of the mutagenforming reactions has earlier been shown for meat crust (Spingarn et al., 1979; Nagao et al., 1977) and was also obvious for the pan residues (Fig. 2). Most of the samples fried at 250°C had a signifi--

Coconut o i l , / Lard

Sunflowerseed oil

Oliveoil

400. 300 200 ffJ

"~ lO0, +

~5oo,

Butter

Margarine/, Margarine

B

Margarine

C

400 / ~ , 300. 200 1 0 0 ~ 0~5 1.'0 1:5

O.'5

1.~

1;5

0;5 1'.0 1~'

0~5 1.'0 1:5

7

/

Margarine

// O.'5

1"0

g meat

D

1:5

Fig. 2. Mutagenicity of pan residues from meat fried without fat or with the addition of different frying-fats. 0 , frying temperature 200°C; (~, frying temperature 250°C.

51 TABLE 2 MUTAGENIC ACTIVITY FORMED DURING FRYING OF 100 g LEAN PORK. DISTRIBUTION BETWEEN CRUST AND PAN RESIDUES Sample

Amount of his-revertants per 100 g meat Sum

% in pan residue

• in crust

Meat fried at 200°C Without fat 1.7x104 _+0.4×104 of all 2.5×104_+0.9×104 fryings with fat

49 58

42

Meat fried at 250°C Without fat 2.6×104 +0.6×104 of all 5.2×104_+0.9×104 fryings with fat

51 43

51

49

57

cantly higher mutagenic activity than the samples fried at 200°C. The total mutagenicity formed from 100 g meat was calculated as the sum of the mutagenic activity in the crust and in the pan residue.

At 250°C frying with fat caused a higher mutagenie activity than frying without fat. At 200°C the result was not as clear-cut. Most samples fried in fat were more mutagenic than the samples without fat, with the exception of samples fried in lard, olive oil or butter where the mutagenicity was in fact slightly lower than in the samples fried without fat. In an effort to simplify comparison a mean value was calculated from all samples fried with fat and this mean mutagenicity value was compared to a mean value calculated from two separate fryings of meat without added fat (Table 2). The difference in mutagenicity between meat samples fried with or without fat was significant at both 200 and 250°C. The mutagenic activity of the pan residues was high and at a comparable level to the activity in the meat crusts (Table 2 and Fig. 3). This was true whether the samples were fried with or without fat, and for both pan temperatures tested. Thickening of the gravy did not alter the mutagenicity of pan residues from meat flied without fat. For pan residues from meat fried with

_7 -pan residue [ ~ - Crust

6

t

x u~

/ / / i

¢1-

F/ /I / / / / / / / /

i / / / / / / / i / / /

///.,,

, / / / ~.///

200 ° 250 ° Without

fat

Coconut

oil

Butter

Lard

Olive oil

Margarine

A Margarine

B Margarine

C Sunflower

oli Margarine

D

Fig. 3. Mutagen/eity in crust and pan residues of 100 g lean pork fried at 200 ° and 250°C with or without the addition of frying fat.

52

margarine B, thickening caused a small raise in mutagenicity. The differences in mutagenicity between the various types of thickening were also small (Figs. 4 and 5).

Z1- H 2 0 + flour

& - 1-12o m . Milk +flour

0 " Cream +flour

Discussion

ilk+ flour 'earn+flour

'

'

o~5

f:o

0 m°.t"

Fig. 4. Mean mutagenicity of thickened gravy prepared .with water and flour, milk and flour or cream and flour, and pan residue from meat fried without the addition of fat.

- H20 + flour

, , - H~O I I - Milk + flour N-Cream

Cream + flour

+flour

//~/I.t,O + flour

~lilk + flour

H20

o~e

t:o

ome*t

Fig. 5. Mean mutagenicity of thickened gravy prepared with water and flour, milk and flour or cream and flour and pan residue from meat fried with the addition of margarine B.

When preparing a meal containing fried meat the pan residues are commonly served and consumed along with the meat. Pan residues from fried meat were in this study shown to contain similar amounts of mutagenic activity to the meat itself and can thus be considered to be of equal importance as a source of mutagenic compounds in the diet. For all samples fried at 250°C and for all except 3 samples fried at 200°C addition of frying fat to the pan caused a higher formation of mutagens in the pan residues as well as in the meat crust compared to frying without fat. A probable explanation is that fat is an effective heat-transferring agent, causing a higher temperature at the surface of the meat (Nilsson et al., 1986; Holtz et al., 1985). The strong temperature dependence of the mutagen-forming reactions was previously shown (0vervik et al., 1984) and it is thus not surprising that frying of meat in the presence of fat leads to a higher amount of mutagens in the pan residues. For the meat samples fried at 200 o C in lard, olive oil and butter the above is obviously not applicable, since the mutagenicity of these samples did not exceed that of the sample fried without fat. It is difficult to find a valid explanation for this result. All 3 fats have a low content of polyunsaturated fatty acids but so has coconut oil which resulted in samples with a high mutagenic content at both 200 and 250°C. Neither is there an association with the content of monounsaturated fatty acids which is high in olive oil, but not in lard or butter. During frying, meat juice leaks out from the meat slices into the frying-pan. Depending on several factors such as the muscle tension and direction of the muscle fibers, the amount of meat juice leaking out of the meat slices during frying varies considerably (Hamm, 1977). This might partly explain the varying mutagenic content of the pan residues from different samples (cf. Fig. 2).

53 The m u t a g e n i c i t y of the p a n residues persisted i n the thickened gravy p r e p a r e d from meat fried with as well as w i t h o u t fat. T h e small differences that occurred b e t w e e n the samples c a n p r o b a b l y b e explained b y the factors m e n t i o n e d above, i.e. v a r y i n g a m o u n t s of meat j u i c e leaking out from the samples. E s t i m a t i o n of the total a m o u n t of food m u t a gens ingested b y m a n is a difficult task. More data o n m u t a g e n i c i t y of different food items i n c l u d e d i n a meal as well as data o n cooking practices m u s t be collected before a n y risk assessment c a n possibly be carried out. This work shows that the p a n residues formed d u r i n g frying of meat should be i n c l u d e d in such a risk assessment.

Acknowledgements Ms. L u d m i l a O t t o w a a n d Ms. Christina T h u l i n are gratefully acknowledged for their technical assistance, a n d Ms. A n n a Bertling for typing the m a n u s c r i p t . Two of us (Lena N i l s s o n a n d Eva t)vervik) are grateful to the Ekhaga F o u n d a t i o n for fellowships. The work was supported b y a g r a n t from the Bank of Sweden T e r c e n t e n a r y Foundation.

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Hargraves, W.A., and M.W. Pariza (1983) Purification and mass spectral characterization of bacterial mutagens from commercial beef extract, Cancer Res., 43, 1467-1472. Holtz E., C. Skjt~ldebrand,M. Jiigerstad, A. Laser-Reuterswiird and P.-E. Isberg (1985) The effect of recipes on crust formation and mutagenicity in meat products during baking, J. Food Technol., 20, 57-66. Kasai, H., Z. Yamaizumi, K. Wakabayashi, M. Nagao, T. Sugimura, S. Yokoyama, T. Miyazawa, N.E. Spingarn, J.H. Weisburger and S. Nishimura (1980) Potent novel mutagens produced by broiling fish under normal conditions, Proc. Jpn. Acad., 56, Ser. B, 278-283. Matsukura, N., T. Kawachi, K. Morino, H. Ohgaki, T. Sugimura and S. Takayama (1981) Carcinogenicity in mice of mutagenic compounds from a tryptophan pyrolysate, Science, 213, 346-347. Nagao, M., M. Honda, Y. Seino, T. Yahagi and T. Sugimura (1977) Mutagenicity of smoke condensates and the charred surface of fish and meat, Cancer Lett., 2, 221-226. Nilsson, L., E. 0vervik, L. Fredholm, 0, Levin, C.-E. Nord and J.-.~. Gustafsson (1986) Influence of frying fat on mutagenic activity in lean pork meat, Mutation Res., 171, 115-121. t)vervik, E., L. Nilsson, L. Fredholm, t). Levin, C.-E. Nord and J.-.A. Gustafsson (1984) High mutagenic activity formed in pan-broiled pork, Mutation Res., 135, 149-157. Ohgaki, H., K. Kusama, N. Matsukura, K. Morino, H. Hasegawa, S. Sato, S. Takayama and T. Sugimura (1984) Carcinogenicity in mice of a mutagenic compound, 2amino-3-methylimidazo[4,5-f]quinoline, from broiled sardine, cooked beef and beef extract, Carcinogenesis, 5, 921-924. Pariza, M.W., S.H. Ashoor and F.S. Chu (1979) Mutagens in heat-processed meat, bakery and cereal products, Food Cosmet. Toxicol., 17, 429-430. Spingarn, N.E., and J.H. Weisburger (1979) Formation of mutagens in cooked foods, I. Beef, Cancer Lett., 7, 259-264. Sugimura, T. (1985) Carcinogenicityof mutagenic, heterocyclic amines formed during the cooking process, Mutation Res., 150, 33-41. Taylor, R.T., E. Fultz and V. Shore (1984) Mutagen formation in a model beef boiling system, I. Conditions with a soluble beef derived fraction, J. Environ. Sci. Health, A 19 (7), 791-81-7. Taylor, R.T., E. Fultz and M. Knize (1985) Mutagen formation in a model beef boiling system, III. Purification and identification of three heterocyclic amine mutagens-carcinogens, J. Environ. Sci. Health, A 20 (2), 135-148. Turesky, R.J., J.S. Wishnok, S.R. Tannenbaum, R.A. Pfund and G.H. Buchi (1983) Qualitative and quantitative characterization of mutagens in commercial beef extract, Carcinogenesis, 7, 863-866.