- Email: [email protected]
Influence of Fat Content and Composition on Malonaldehyde Concentration in Chicken Meat and Skin
Influence of Fat Content and Composition on Malonaldehyde Concentration in Chicken Meat and Skin j . PIKUL Institute of Animal Products Technology, Agricultural University of Poznan, 60-624 Poznan, Wojska Polskiego 31, Poland D. E. LESZCZYNSKI1 and F. A. KUMMEROW2 Harlan E. Moore Heart Research Foundation, 503 South Sixth Street, Champaign, Illinois 61820
ABSTRACT Total fat extracted from breast meat, leg meat, and breast skin was analyzed for composition of phospholipids (PL), triglycerides (TG), and total cholesterol (C); and for fatty acid composition of PL and TG fractions. Whole fat was also analyzed for malonaldehyde (MA) by an improved thiobarbituric acid (TBA) assay with antioxidant protection and for certain secondary oxidation products by fluorescence excitation (360 nm) and emission (440 nm) spectra. Breast contained 1.1% fat composed of 58.4% PL and 35.5% TG; leg contained 2.4% fat composed of 32.1% PL and 62.9% TG, and skin contained 32.8% fat composed of 1.6% PL and 97.8% TG. Fatty acid compositions of TG fractions from all tissues were similar and contained almost no polyunsaturated fatty acids (PUFA) with 20 or 22 carbon atoms. Fatty acids from PL fractions of meat contained more than 20% arachidonic acid and substantial amounts of PUFA with 22 carbon atoms; skin PL contained approximately one-half the PUFA with 20 and 22 carbon atoms compared with meat. The MA concentration in breast fat was 1.9 times higher than in leg fat and 20.3 times higher than in skin fat. However, because of the different fat content of tissues, the TBA number of skin was higher than that of leg, which in turn was higher than that of breast. The relative levels of fluorescent products in fat from meat and skin tissues clearly paralleled the trend found for MA concentrations. It was concluded that the TBA number parameter is of little comparative value unless accompanied by fat content and composition data. (Key words: chicken, meat, skin, fat, malonaldehyde) 1985 Poultry Science 6 4 : 3 1 1 - 3 1 7
INTRODUCTION T h e t h i o b a r b i t u r i c acid (TBA) assay is t h e most p o p u l a r m e t h o d for measuring oxidative deterioration of lipids in muscle foods (Melton, 1 9 8 3 ) . T h e TBA assay measures t h e a m o u n t of m a l o n a l d e h y d e (MA), a secondary p r o d u c t obtained mainly from t h e oxidation of polyu n s a t u r a t e d fatty acids in food samples (Sinnhuber and Yu, 1 9 7 7 ; Pearson et al, 1983). T h e T B A n u m b e r is used as an indicator of food quality and is generally regarded t o be highly correlated with taste panel scores for oxidized and warmed-over flavors in muscle foods (Wilson et al, 1 9 7 6 ; Igene and Pearson, 1 9 7 9 ) .
1
To whom correspondence should be addressed. Burnsides Research Laboratory, Department of Food Science, University of Illinois, Urbana, IL 61801. J
Despite its popularity in t h e food industry, t h e TBA assay has been criticized for its reliability and usefulness as an i n d i c a t o r of food quality. A major technical criticism of t h e TBA assay is t h e e x t r e m e variability of assay results, which is generally d u e t o different ways t h a t t h e T B A assay is performed and due t o sample a u t o x i d a t i o n during preparation, e x t r a c t i o n or distillation, and t h e heating steps of these assays. These technical p r o b l e m s m a k e comparisons of TBA data a m o n g different studies virtually impossible ( R h e e , 1 9 7 8 ; M e l t o n , 1 9 8 3 ; Pikul et al, 1983). A second criticism of t h e T B A assay is t h a t t h e T B A n u m b e r b y itself is difficult t o interpret w i t h o u t additional inform a t i o n a b o u t t h e c o m p o s i t i o n of different kinds of food p r o d u c t s (Pearson et al, 1 9 8 3 ) . These criticisms are also i m p o r t a n t for analysis of p o u l t r y m e a t and o t h e r edible p r o d u c t s because of large differences in fat c o n t e n t and c o m p o s i t i o n a m o n g different anatomical parts
311
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
(Received for publication November 18, 1983)
312
PIKUL ET AL.
from the same bird, and also because of differences among birds due to age, sex, and breed. The purpose of our study was to determine the MA content and TBA numbers of chicken meat and skin using an improved TBA assay with antioxidant protection (Pikul et al., 1983) and to study the relative contributions of lipid quantity and composition in the generation of the TBA number in edible poultry products.
MATERIALS AND METHODS
Measurement of Fluorescent Products. For fluorescence analysis, tissue samples were extracted with chloroform-methanol according to the methods of Folch et al. (1957). After phase separation, samples from the organic and aqueous layers of Folch extractions were appropriately diluted and measured in a Perkin-Elmer Model 650-lOs fluorescence spectrophotometer with 10" 8 M quinine sulfate (DAB standard, Fluka, Switzerland) set equal to 50 fluorescence units as described by Golstein et al. (1979). Fluorescence in the aqueous layer was
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
Materials. Meat and skin samples were prepared from 4-month-old New Hampshire X Columbian pullets fed a high protein-low fat corn-soybean meal based starter ration. Chickens were sacrificed by decapitation and hung until bleeding was complete. Samples of breast skin were removed after manual defeathering; then whole, skinless, manually deboned breast and leg meat were collected. Samples were individually wrapped in aluminum foil, kept at 4 C for 2 hr, and then stored at —18 C until 1 day prior to extraction. Extraction and Chemical Analysis of Total Fat. Breast meat, leg meat, and breast skin were extracted for total lipid according to the basic methods of Kates (1972), with several modifications, including antioxidant protection during extraction (Pikul et al., 1983). Tissues were homogenized and extracted three times with chloroform-methanol; supernatants were mixed with .2 parts of water and allowed to separate overnight at 4 C. The organic layer was collected, dried with anhydrous sodium sulfate, filtered clear, evaporated with nitrogen gas in preparation for gravimetric weighing (PerkinElmer, Model AD-2 Autobalance), and diluted with chloroform for lipid analysis. Total fat extracted from tissues was used for the determination of triglycerides (TG) by the methods of Foster and Dunn (1973), total cholesterol (C) by modified methods of Glick et al. (1964), and phosphorus by the methods of Eng and Noble (1968), which was multipled by the factor 25.5 to estimate total phospholipids (PL). Malonaldehyde concentrations in total fat extracts from tissues were determined by improved TBA assay with antioxidant protection (Pikul et al, 1983). The TBA numbers, expressed as milligrams MA per kilogram tissues, were calculated from the percentage of total fat in tissue, and the concentration of MA in total fat. Separation and Fatty Acid Analysis of Phos-
pholipids and Triglycerides. Total fat, dissolved in chloroform, was applied in a nitrogen atmosphere on 20 X 20 cm precoated silica gel G glass plates (500 /Urn thick, Alltech Associates, Inc.); a standard mixture containing phosphatidyl choline, trioleoylglycerol, oleic acid, cholesterol, and cholesteryl oleate was simultaneously spotted on both sides of the sample. Total fat was separated into PL, TG, free fatty acid, cholesterol, and cholesterol esters by thin layer chromatography (TLC) with hexane-diethyl ether-glacial acetic acid (85:15:2, v/v). After development, the solvent was evaporated from plates with nitrogen, and the locations of separated bands were identified under ultraviolet light after spraying standards with rhodamine 6G solution. Bands corresponding to PL and TG were scraped from plates into centrifuge tubes and extracted three times with chloroform-methanol (1:2) for PL and (2:1) for TG. These extracts were evaporated to dryness under nitrogen and then dissolved in benzene. Fatty acids present in benzene were hydrolyzed and converted to methyl esters by adding an equal volume of 5% sulfuric acid in anhydrous methanol (v/v) and heating in a closed container at 90 C for 2 hr. Fatty acid methyl esters were collected by adding one part water and extracting three times with petroleum ether. Combined ether extracts were evaporated under nitrogen and resuspended in proper concentrations with carbon dioxide. Analysis of fatty acid methyl esters was accomplished with a Hewlett-Packard Model 5890A gas chromatography machine equipped with a 30 m X .24 mm fused silica capillary column packed with 20% SP 2340 (Supelco, Inc.). Peaks were identified by comparison with retention times of authentic fatty acid methyl esters mixtures (Nu Chek Prep, Inc.; Supelco, Inc.), and results were expressed as percentage of total fatty acids, as determined by a Hewlett-Packard 3390A integrator.
FAT AND MALONALDEHYDE expressed per micrograms p r o t e i n (Lowry et al., 1 9 5 1 ) ; and t h e fluorescence in t h e organic layer was expressed per milligram total lipid as determined gravimetrically.
RESULTS
and leg m e a t tissues, total fat was c o m p o s e d mostly of PL and T G ; total fat from skin was c o m p o s e d almost entirely of T G . Leg m e a t contained m o r e visible yellow intramuscular fat, which was similar in appearance t o adipose tissue t h a n breast muscle; consequently leg m e a t contained m o r e fat, which had a higher T G c o n t e n t . Large a m o u n t s of attached visible fat, which was rich in T G , were present in skin tissue. T h e fatty acid c o m p o s i t i o n of t h e isolated T G fractions from m e a t and skin fat are presented in Table 2. T h e general c o m p o s i t i o n of TG fatty acids was similar for all three tissues. Oleic acid ( 1 8 : 1 ) and palmitic acid ( 1 6 : 0 ) were t h e m o s t a b u n d a n t fatty acids; linoleic ( 1 8 : 2 ) acid averaged 2 1 % of t h e total fatty acids and
TABLE 1. Content and composition of the lipid extracted from breast, leg, and skin tissues Type of fat
Breast meat 1
Total lipids Phospholipids Triglycerides Cholesterol
1,098.4 641.5 389.9 61.5
± ± ± ±
77.61 29.82 14.68 5.01
Skin1
Leg meat 1 2,348.7 753.9 1,477.3 108.0
+ 157.16 ± 36.41 ± 49.82 ± 7.04
32,808.5 524.9 32,086.7 118.1
± 3,480.12 ± 78.56 + 1,384.28 ± 12.45
1 Data expressed as milligrams lipid class per 100 gram wet tissue and presented as means ± standard deviations of 5 samples.
TABLE 2. Fatty acid composition of isolated triglyceride fraction from breast, leg, and skin tissues
1 2
Fatty acid 1
Breast meat 2
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:2 20:4 22:0
.88 .28 25.86 5.33 6.38 37.27 21.30 1.25 .28 .24 .70 .28
± .01 ± .10 ± .35 ± .07 ± .08 + .16 ± .02 ± .01 + .01 + .09 ± .01 ±.01
Leg meat 2 .75 ± .14 .17 + .04 24.15 ± .97 5.45 ± .37 5.87 ± .01 37.65 ± 1.46 2 2 . 5 0 ± .01 1.27 + .03 .28 + .02 . 3 5 + .02 1.14 ± .06 .40 ± . 0 2
Skin 2 .66 .17 26.06 4.85 6.18 41.19 19.27 .66 .48 .18 .40 .23
± ± + ± ± ± ± + ± ± ± ±
.06 .08 .37 .55 .16 .46 .28 .02 .01 .01 .01 .01
Number of carbon atoms:number of double bonds.
Fatty acid values are expressed as percentages of total integrated mass of fatty acid methyl esters detected during chromatography. Data are presented as means ± standard deviations of two separate determinations from pooled extract.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
Total fat contained in chicken m e a t and skin was o b t a i n e d b y e x t r a c t i o n three times with c h l o r o f o r m - m e t h a n o l . Breast m e a t , leg m e a t , and breast skin were found t o contain 1.10, 2 . 3 5 , and 3 2 . 8 1 % total fat, respectively (Table 1). Fat from breast meat was c o m p o s e d of 58.4% PL, 3 5 . 5 % T G , and 5.6% C; t h e composition of leg fat was 3 2 . 1 % PL, 6 2 . 9 % T G , and 4.6% C; t h e c o m p o s i t i o n of skin fat was 1.6% PL, 9 7 . 8 % T G , and .4% C (Table 1). In breast
313
314
PIKULETAL. breast or leg meat. These results demonstrate that TBA numbers alone are of little value for judging tissue quality unless data concerning the fat content and composition of tissue are also known. The relative levels of fluorescent products in the organic layers obtained from Folchextracted breast meat, leg meat, and breast skin are presented in Table 5. Fluorescent products were almost two times more concentrated in fat from breast meat compared with leg meat and more than twenty-times more concentrated in breast fat compared with skin fat. These relative levels of fluorescent products in fat from breast, leg, and skin tissues were closely parallel to the relative levels of MA in fat as reported in Table 4. Fluorescent products in the aqueous layers obtained from Folch-extracted tissue were most concentrated in skin and least concentrated in breast meat when expressed per unit of protein (Table 5); however, in all three tissues, only .2% of the total protein of the tissues was extracted into the aqueous layer. DISCUSSION Reports of TBA numbers for chicken meat
TABLE 3. Fatty acid composition of isolated phospholipid fraction from breast, leg, and skin tissues Fatty
1
acid1
Breast m e a t 2
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:2 20:4 22:0 22:4 22:5 22:6 24:0 24:1
.08 .04 22.47 .26 16.71 15.69 7.66 .12 .14 .36 22.89 1.06 2.74 1.53 3.62 .19 2.26
± ± + ± ± ± ± ± ± ± ± ± ± ± + ± ±
.01 .01 .64 .03 .09 .07 .09 .14 .01 .01 .31 .02 .01 .01 .02 .01 .01
Leg m e a t 2 .09 ± .04 ± 15.36 ± .44 ± 20.89 ± 13.10± 14.71 ± .11 ± .31 ± .31 ± 20.98 ± .79 ± 2.99 ± 1.06 ± 3.24 ± .29 ± 2.24 +
.01 .01 .29 .01 .02 .05 .06 .01 .01 .01 .31 .01 .08 .02 .08 .01 .05
Skin 2 .47 .11 27.81 1.16 20.19 19.52 13.82 .42 1.22 .31 10.45 .42 1.63 .79 1.06 .22 .21
± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± +
.03 .01 .52 .05 .29 .23 .58 .01 .14 .02 .22 .01 .01 .10 .02 .03 .04
Number of carbon atoms: number of double bonds. Fatty acid values are expressed as percentages of total integrated mass of fatty acid methyl esters detected during chromatography. Data are presented as means ± standard deviations of two separate determinations from pooled extract. 2
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
was the only polyunsaturated fatty acid (PUFA) present in a large amount; PUFA with 22 carbons were not detectable. These TG fatty acid composition patterns were in sharp contrast to the patterns found in the isolated PL fractions presented in Table 3. Fatty acids from the isolated PL fraction of breast and leg meat contained more than 20% arachidonic acid (20:4) and substantial amounts of PUFA with 22 carbon atoms (22:4, 22:5, 22:6). The fatty acids from skin PL contained approximately one-half the amount of PUFA with 20 and 22 carbon atoms compared with fatty acids from meat tissues (Table 3). Fat from breast meat contained a higher proportion of PL, which was rich in PUFA, than leg meat; breast fat also contained a higher concentration of malonaldehyde (MA) compared with leg as indicated in Table 4. However, leg meat contained two times more total fat than breast meat, and consequently, leg meat had a slightly higher TBA number than breast meat. Fat from skin had a very low proportion of PL and, consequently, a low concentration of MA; but because of high total fat content, the calculated TBA number of skin was significantly higher (P<.05) than values obtained from either
FAT AND MALONALDEHYDE
Even though the autoxidation problem of the TBA assay can be eliminated, we have found that TBA analysis, by itself, is not a useful parameter for evaluating food quality. For instance, in our study, chicken skin was found to have a higher TBA number than chicken meat, which is a result compatible with the findings of others (Marion and Woodroof, 1966; Marion, 1969). These TBA results taken alone indicate that skin tissue might be more unstable for oxidation than meat. However, when more detailed lipid analysis is provided, it becomes obvious that the TBA number of skin is higher than meat because skin contains 15 times more total fat than leg and 30 times more than breast. In addition, it is apparent that fat from skin is actually more stable than fat from meat because of a far lower concentration of MA due to a small PL fraction and an overall low concentration of PUFA. This conclusion is supported by results of others who reported
TABLE 4. Malonaldehyde (MA) concentration and thiobarbituric acid (TBA) number of fresh breast, leg, and skin tissues Type of tissues
Mg MA/g Fat 1
Breast meat Leg meat Skin
28.4 ± 1.84c 15.3 ± . 8 1 b 1.4 ± .08 a
TBA number 1
% Fat 1 1.10 ± .08 a 2.35 ± .16 b 32.81 + 3.48 c
.31 ± .02* .36 ± .02 a .46 + . 0 3 b
a,b,c Mean values within each column with different superscripts are significantly different (P<.05, two-tailed t test). 1
Data are presented as means ± standard deviations of 5 samples.
TABLE 5. Relative levels of fluorescent products presented in the organic and aqueous layers from Folch-extracted meat and skin tissues Type of tissues
Breast meat Leg meat Skin
Organic1
Aqueous 1
(FU'/mg fat)
(FU//zg protein)
98.8 ± 4.02 c 53.7 ± 2 . 7 1 b 4.4 ± .22 a
1.16 ± .04 a 1.51 + . 0 5 b 1.81 ± .07 c
' ' Mean values within each column with different superscripts are significantly different (P<.05, two-tailed t test). 1
Data are presented as means ± standard deviations of 5 samples.
2
FU = Fluorescent units.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
and skin vary widely. Some of this variability may be due to differences in age, sex, diet, or breed of chickens studied. But some reported TBA values are so high that artifacts due to sample autoxidation can be suspected. Several investigators have tried to avoid the problem of autoxidation by adding antioxidants to meat samples during preparation, extraction, or distillation, and during the critical heating steps of the TBA assay (Moerck and Ball, 1974; Rhee, 1978; Rhee and Ziprin, 1981; Yamauchi et al, 1982). We have recently demonstrated that the addition of butylated hydroxy toluene to fat from chicken tissues can eliminate the problem of sample autoxidation in the TBA assay (Pikul et al, 1983), and this improved method was used in our present study. The TBA numbers reported in our study are similar to values reported elsewhere for similar material (Jantawat and Dawson, 1977; Igene et al, 1979; Yamauchi etal, 1982).
315
316
PIKUL ET AL.
ACKNOWLEDGMENTS This work was s u p p o r t e d b y a FulbrightHays Exchange Scholarship, a grant from t h e American Heart Association, Illinois Affiliate, and a grant from t h e Wallace Genetic F o u n d a tion. T h e a u t h o r s recognize t h e technical assistance of Regina Galer-Unti and J o h n Catlow. REFERENCES Chio, K. S., and A. L. Tappel, 1969. Synthesis and characterization of the fluorescent products derived from malonaldehyde and amino acids. Biochemistry 8:2821-2827. Dawson, L. E., and K. Schierholz, 1976. Influence of grinding, cooking, and refrigerated storage on lipid stability in turkey. Poultry Sci. 55:618— 622. Dillard, C. J., and A. L. Tappel, 1973. Fluorescent products from reaction of peroxidizing polyun-
saturated fatty acids with phosphatidyl ethanolamine and phenylalanine. Lipids 8:183—189. Eng, L. F., and E. P. Noble, 1968. The maturation of rat brain myelin. Lipids 3:157—162. Folch, J., M. Lees, and G. M. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Foster, L. B., and R. T. Dunn, 1973. Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin. Chem. 19:338-340. Glick, D., B. F. Fell, and K. E. Sjolin, 1964. Spectrophotometry determination of nanogram amounts of total cholesterol in microgram quantities of tissue or microliter volumes of serum. Anal. Chem. 36:1119-1121. Goldstein, B. D., M. G. Rozen, and M. A. Amoruso, 1979. Relation of fluorescence in lipid-containing red cell membrane extracts to in vivo lipid peroxidation. J. Lab. Clin. Med. 93:687-694. Igene, J. O., and A. M. Pearson, 1979. Role of phospholipids and triglycerides in warmed-over flavor development in meat model systems. J. Food Sci. 44:1285-1290. Jantawat, P. P., and L. E. Dawson, 1977. Stability of broiler pieces during frozen storage. Poultry Sci. 56:2026-2030. Kates, M., 1972. Techniques of lipidology. Pages 347— 353 in Laboratory Techniques in Biochemistry and Molecular Biology. T. S. Work and E. Work, ed. North Holland Publ. Co., Amsterdam. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. MacDonald, B., J. I. Gray, Y. Kakuda, and M. L. Lee, 1980. Role of nitrite in cured meat flavor chemical analysis. J. Food Sci. 45:889-892. Marion, J. E., 1969. Oxidation of chicken tissue lipids as influenced by age and sex. Poultry Sci. 48: 301-304. Marion, J. E., and J. G. Woodroof, 1966. Composition and stability of broiler carcasses as affected by dietary protein and fat. Poultry Sci. 45:241 — 247. Melton, S. L., 1983. Methodology for following lipid oxidation in muscle foods. Food Technol. 37: 1 0 5 - 1 1 1 , 116. Moerck, K. E., and H. R. Ball, 1974. Lipid autoxidation in mechanically deboned chicken meat. J. Food Sci. 39:876-879. Pearson, A. M., J. I. Gray, A. M. Wolzak, and N. A. Horenstein, 1983. Safety implications of oxidized lipids in muscle foods. Food Technol. 37: 121-129. Pikul, J., D. E. Leszczynski, and F. A. Kummerow, 1983. Elimination of sample autoxidation by butylated hydroxytoluene additions before thiobarbituric acid assay for malonaldehyde in fat from chicken meat. J. Agric. Food Chem. 3 1 : 1338-1342. Rhee, K. S., 1978. Minimization of further lipid peroxidation in the distillation 2-thiobarbituric acid test of fish and meat. J. Food Sci. 43:1776— 1778, 1781. Rhee, K. S., and Y. A. Ziprin, 1981. Oilseed protein
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
TBA n u m b e r s for raw p o u l t r y skin t h a t were higher t h a n raw p o u l t r y m e a t , b u t t h a t after cooking and refrigerated storage, T B A n u m b e r s of skin were equal t o or lower t h a n values for m e a t (Marion, 1 9 6 9 ; Dawson and Schierholz, 1976). In addition t o MA and TBA n u m b e r determ i n a t i o n s , fluorescence excitation and emission spectra were used t o measure certain secondary p r o d u c t s of lipid o x i d a t i o n , which are believed t o represent covalent crosslinkage of MA with amino acids, peptides, lipids, and itself (Chio a n d Tappel, 1 9 6 9 ; Pearson et al, 1 9 8 3 ) . Meas u r e m e n t of fluorescent p r o d u c t s from b o t h t h e organic and a q u e o u s layers of Folch-extracted tissues has been used as an analytical m e t h o d for q u a n t i t a t i o n of p e r o x i d a t i o n damage (Dillard and Tappel, 1 9 7 3 ; MacDonald et al, 1 9 8 0 ; Pearson et al, 1 9 8 3 ) . F r o m Folche x t r a c t e d m e a t and skin, t h e relative concentrations of fluorescent p r o d u c t s in t h e organic layers, which contain t h e whole e x t r a c t e d fat, showed a trend t h a t was very similar t o t h e relative c o n c e n t r a t i o n s of MA in those tissues. Because MA is a highly reactive c o m p o u n d , samples that contain high c o n c e n t r a t i o n s of free M A can b e e x p e c t e d t o contain high concentrations of crosslinked MA. T h e relative c o n c e n t r a t i o n s of fluorescent p r o d u c t s in t h e aqueous layers of Folch-extracted m e a t and skin did n o t follow t h e p a t t e r n of MA o r fluorescent p r o d u c t c o n c e n t r a t i o n s in total fat e x t r a c t . These results indicate t h a t free MA m a y selectively react with lipid molecules, especially PL, r a t h e r t h a n amino acids or peptides in fresh tissue samples.
FAT AND MALONALDEHYDE ingredients as antioxidant for meat in food service. J. Food Prot. 4 4 : 2 5 4 - 2 5 6 . Sinnhuber, R. O., and T. C. Yu, 1977. The 2-thiobarbituric acid reaction, an objective measure of the oxidative deterioration occurring in fats and oils. Yukagaku 26:259-267. Wilson, B. R., A. M. Pearson, and F. B. Shorland, 1976. Effect of total lipids and phospholipids on warmed-over flavor in red and white muscle from
317
several species as measured by thiobarbituric acid analysis. J. Agric. Food Chem. 24:7— 11. Yamauchi, K., Y. Nagai, and T. Ohashi, 1982. Quantitative relationship between alpha-tocopherol and polyunsaturated fatty acids and its connection to development of oxidative rancidity in chicken skeletal muscle. Agric. Biol. Chem. 46:2719— 2724.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
ABSTRACT Total fat extracted from breast meat, leg meat, and breast skin was analyzed for composition of phospholipids (PL), triglycerides (TG), and total cholesterol (C); and for fatty acid composition of PL and TG fractions. Whole fat was also analyzed for malonaldehyde (MA) by an improved thiobarbituric acid (TBA) assay with antioxidant protection and for certain secondary oxidation products by fluorescence excitation (360 nm) and emission (440 nm) spectra. Breast contained 1.1% fat composed of 58.4% PL and 35.5% TG; leg contained 2.4% fat composed of 32.1% PL and 62.9% TG, and skin contained 32.8% fat composed of 1.6% PL and 97.8% TG. Fatty acid compositions of TG fractions from all tissues were similar and contained almost no polyunsaturated fatty acids (PUFA) with 20 or 22 carbon atoms. Fatty acids from PL fractions of meat contained more than 20% arachidonic acid and substantial amounts of PUFA with 22 carbon atoms; skin PL contained approximately one-half the PUFA with 20 and 22 carbon atoms compared with meat. The MA concentration in breast fat was 1.9 times higher than in leg fat and 20.3 times higher than in skin fat. However, because of the different fat content of tissues, the TBA number of skin was higher than that of leg, which in turn was higher than that of breast. The relative levels of fluorescent products in fat from meat and skin tissues clearly paralleled the trend found for MA concentrations. It was concluded that the TBA number parameter is of little comparative value unless accompanied by fat content and composition data. (Key words: chicken, meat, skin, fat, malonaldehyde) 1985 Poultry Science 6 4 : 3 1 1 - 3 1 7
INTRODUCTION T h e t h i o b a r b i t u r i c acid (TBA) assay is t h e most p o p u l a r m e t h o d for measuring oxidative deterioration of lipids in muscle foods (Melton, 1 9 8 3 ) . T h e TBA assay measures t h e a m o u n t of m a l o n a l d e h y d e (MA), a secondary p r o d u c t obtained mainly from t h e oxidation of polyu n s a t u r a t e d fatty acids in food samples (Sinnhuber and Yu, 1 9 7 7 ; Pearson et al, 1983). T h e T B A n u m b e r is used as an indicator of food quality and is generally regarded t o be highly correlated with taste panel scores for oxidized and warmed-over flavors in muscle foods (Wilson et al, 1 9 7 6 ; Igene and Pearson, 1 9 7 9 ) .
1
To whom correspondence should be addressed. Burnsides Research Laboratory, Department of Food Science, University of Illinois, Urbana, IL 61801. J
Despite its popularity in t h e food industry, t h e TBA assay has been criticized for its reliability and usefulness as an i n d i c a t o r of food quality. A major technical criticism of t h e TBA assay is t h e e x t r e m e variability of assay results, which is generally d u e t o different ways t h a t t h e T B A assay is performed and due t o sample a u t o x i d a t i o n during preparation, e x t r a c t i o n or distillation, and t h e heating steps of these assays. These technical p r o b l e m s m a k e comparisons of TBA data a m o n g different studies virtually impossible ( R h e e , 1 9 7 8 ; M e l t o n , 1 9 8 3 ; Pikul et al, 1983). A second criticism of t h e T B A assay is t h a t t h e T B A n u m b e r b y itself is difficult t o interpret w i t h o u t additional inform a t i o n a b o u t t h e c o m p o s i t i o n of different kinds of food p r o d u c t s (Pearson et al, 1 9 8 3 ) . These criticisms are also i m p o r t a n t for analysis of p o u l t r y m e a t and o t h e r edible p r o d u c t s because of large differences in fat c o n t e n t and c o m p o s i t i o n a m o n g different anatomical parts
311
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
(Received for publication November 18, 1983)
312
PIKUL ET AL.
from the same bird, and also because of differences among birds due to age, sex, and breed. The purpose of our study was to determine the MA content and TBA numbers of chicken meat and skin using an improved TBA assay with antioxidant protection (Pikul et al., 1983) and to study the relative contributions of lipid quantity and composition in the generation of the TBA number in edible poultry products.
MATERIALS AND METHODS
Measurement of Fluorescent Products. For fluorescence analysis, tissue samples were extracted with chloroform-methanol according to the methods of Folch et al. (1957). After phase separation, samples from the organic and aqueous layers of Folch extractions were appropriately diluted and measured in a Perkin-Elmer Model 650-lOs fluorescence spectrophotometer with 10" 8 M quinine sulfate (DAB standard, Fluka, Switzerland) set equal to 50 fluorescence units as described by Golstein et al. (1979). Fluorescence in the aqueous layer was
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
Materials. Meat and skin samples were prepared from 4-month-old New Hampshire X Columbian pullets fed a high protein-low fat corn-soybean meal based starter ration. Chickens were sacrificed by decapitation and hung until bleeding was complete. Samples of breast skin were removed after manual defeathering; then whole, skinless, manually deboned breast and leg meat were collected. Samples were individually wrapped in aluminum foil, kept at 4 C for 2 hr, and then stored at —18 C until 1 day prior to extraction. Extraction and Chemical Analysis of Total Fat. Breast meat, leg meat, and breast skin were extracted for total lipid according to the basic methods of Kates (1972), with several modifications, including antioxidant protection during extraction (Pikul et al., 1983). Tissues were homogenized and extracted three times with chloroform-methanol; supernatants were mixed with .2 parts of water and allowed to separate overnight at 4 C. The organic layer was collected, dried with anhydrous sodium sulfate, filtered clear, evaporated with nitrogen gas in preparation for gravimetric weighing (PerkinElmer, Model AD-2 Autobalance), and diluted with chloroform for lipid analysis. Total fat extracted from tissues was used for the determination of triglycerides (TG) by the methods of Foster and Dunn (1973), total cholesterol (C) by modified methods of Glick et al. (1964), and phosphorus by the methods of Eng and Noble (1968), which was multipled by the factor 25.5 to estimate total phospholipids (PL). Malonaldehyde concentrations in total fat extracts from tissues were determined by improved TBA assay with antioxidant protection (Pikul et al, 1983). The TBA numbers, expressed as milligrams MA per kilogram tissues, were calculated from the percentage of total fat in tissue, and the concentration of MA in total fat. Separation and Fatty Acid Analysis of Phos-
pholipids and Triglycerides. Total fat, dissolved in chloroform, was applied in a nitrogen atmosphere on 20 X 20 cm precoated silica gel G glass plates (500 /Urn thick, Alltech Associates, Inc.); a standard mixture containing phosphatidyl choline, trioleoylglycerol, oleic acid, cholesterol, and cholesteryl oleate was simultaneously spotted on both sides of the sample. Total fat was separated into PL, TG, free fatty acid, cholesterol, and cholesterol esters by thin layer chromatography (TLC) with hexane-diethyl ether-glacial acetic acid (85:15:2, v/v). After development, the solvent was evaporated from plates with nitrogen, and the locations of separated bands were identified under ultraviolet light after spraying standards with rhodamine 6G solution. Bands corresponding to PL and TG were scraped from plates into centrifuge tubes and extracted three times with chloroform-methanol (1:2) for PL and (2:1) for TG. These extracts were evaporated to dryness under nitrogen and then dissolved in benzene. Fatty acids present in benzene were hydrolyzed and converted to methyl esters by adding an equal volume of 5% sulfuric acid in anhydrous methanol (v/v) and heating in a closed container at 90 C for 2 hr. Fatty acid methyl esters were collected by adding one part water and extracting three times with petroleum ether. Combined ether extracts were evaporated under nitrogen and resuspended in proper concentrations with carbon dioxide. Analysis of fatty acid methyl esters was accomplished with a Hewlett-Packard Model 5890A gas chromatography machine equipped with a 30 m X .24 mm fused silica capillary column packed with 20% SP 2340 (Supelco, Inc.). Peaks were identified by comparison with retention times of authentic fatty acid methyl esters mixtures (Nu Chek Prep, Inc.; Supelco, Inc.), and results were expressed as percentage of total fatty acids, as determined by a Hewlett-Packard 3390A integrator.
FAT AND MALONALDEHYDE expressed per micrograms p r o t e i n (Lowry et al., 1 9 5 1 ) ; and t h e fluorescence in t h e organic layer was expressed per milligram total lipid as determined gravimetrically.
RESULTS
and leg m e a t tissues, total fat was c o m p o s e d mostly of PL and T G ; total fat from skin was c o m p o s e d almost entirely of T G . Leg m e a t contained m o r e visible yellow intramuscular fat, which was similar in appearance t o adipose tissue t h a n breast muscle; consequently leg m e a t contained m o r e fat, which had a higher T G c o n t e n t . Large a m o u n t s of attached visible fat, which was rich in T G , were present in skin tissue. T h e fatty acid c o m p o s i t i o n of t h e isolated T G fractions from m e a t and skin fat are presented in Table 2. T h e general c o m p o s i t i o n of TG fatty acids was similar for all three tissues. Oleic acid ( 1 8 : 1 ) and palmitic acid ( 1 6 : 0 ) were t h e m o s t a b u n d a n t fatty acids; linoleic ( 1 8 : 2 ) acid averaged 2 1 % of t h e total fatty acids and
TABLE 1. Content and composition of the lipid extracted from breast, leg, and skin tissues Type of fat
Breast meat 1
Total lipids Phospholipids Triglycerides Cholesterol
1,098.4 641.5 389.9 61.5
± ± ± ±
77.61 29.82 14.68 5.01
Skin1
Leg meat 1 2,348.7 753.9 1,477.3 108.0
+ 157.16 ± 36.41 ± 49.82 ± 7.04
32,808.5 524.9 32,086.7 118.1
± 3,480.12 ± 78.56 + 1,384.28 ± 12.45
1 Data expressed as milligrams lipid class per 100 gram wet tissue and presented as means ± standard deviations of 5 samples.
TABLE 2. Fatty acid composition of isolated triglyceride fraction from breast, leg, and skin tissues
1 2
Fatty acid 1
Breast meat 2
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:2 20:4 22:0
.88 .28 25.86 5.33 6.38 37.27 21.30 1.25 .28 .24 .70 .28
± .01 ± .10 ± .35 ± .07 ± .08 + .16 ± .02 ± .01 + .01 + .09 ± .01 ±.01
Leg meat 2 .75 ± .14 .17 + .04 24.15 ± .97 5.45 ± .37 5.87 ± .01 37.65 ± 1.46 2 2 . 5 0 ± .01 1.27 + .03 .28 + .02 . 3 5 + .02 1.14 ± .06 .40 ± . 0 2
Skin 2 .66 .17 26.06 4.85 6.18 41.19 19.27 .66 .48 .18 .40 .23
± ± + ± ± ± ± + ± ± ± ±
.06 .08 .37 .55 .16 .46 .28 .02 .01 .01 .01 .01
Number of carbon atoms:number of double bonds.
Fatty acid values are expressed as percentages of total integrated mass of fatty acid methyl esters detected during chromatography. Data are presented as means ± standard deviations of two separate determinations from pooled extract.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
Total fat contained in chicken m e a t and skin was o b t a i n e d b y e x t r a c t i o n three times with c h l o r o f o r m - m e t h a n o l . Breast m e a t , leg m e a t , and breast skin were found t o contain 1.10, 2 . 3 5 , and 3 2 . 8 1 % total fat, respectively (Table 1). Fat from breast meat was c o m p o s e d of 58.4% PL, 3 5 . 5 % T G , and 5.6% C; t h e composition of leg fat was 3 2 . 1 % PL, 6 2 . 9 % T G , and 4.6% C; t h e c o m p o s i t i o n of skin fat was 1.6% PL, 9 7 . 8 % T G , and .4% C (Table 1). In breast
313
314
PIKULETAL. breast or leg meat. These results demonstrate that TBA numbers alone are of little value for judging tissue quality unless data concerning the fat content and composition of tissue are also known. The relative levels of fluorescent products in the organic layers obtained from Folchextracted breast meat, leg meat, and breast skin are presented in Table 5. Fluorescent products were almost two times more concentrated in fat from breast meat compared with leg meat and more than twenty-times more concentrated in breast fat compared with skin fat. These relative levels of fluorescent products in fat from breast, leg, and skin tissues were closely parallel to the relative levels of MA in fat as reported in Table 4. Fluorescent products in the aqueous layers obtained from Folch-extracted tissue were most concentrated in skin and least concentrated in breast meat when expressed per unit of protein (Table 5); however, in all three tissues, only .2% of the total protein of the tissues was extracted into the aqueous layer. DISCUSSION Reports of TBA numbers for chicken meat
TABLE 3. Fatty acid composition of isolated phospholipid fraction from breast, leg, and skin tissues Fatty
1
acid1
Breast m e a t 2
14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:2 20:4 22:0 22:4 22:5 22:6 24:0 24:1
.08 .04 22.47 .26 16.71 15.69 7.66 .12 .14 .36 22.89 1.06 2.74 1.53 3.62 .19 2.26
± ± + ± ± ± ± ± ± ± ± ± ± ± + ± ±
.01 .01 .64 .03 .09 .07 .09 .14 .01 .01 .31 .02 .01 .01 .02 .01 .01
Leg m e a t 2 .09 ± .04 ± 15.36 ± .44 ± 20.89 ± 13.10± 14.71 ± .11 ± .31 ± .31 ± 20.98 ± .79 ± 2.99 ± 1.06 ± 3.24 ± .29 ± 2.24 +
.01 .01 .29 .01 .02 .05 .06 .01 .01 .01 .31 .01 .08 .02 .08 .01 .05
Skin 2 .47 .11 27.81 1.16 20.19 19.52 13.82 .42 1.22 .31 10.45 .42 1.63 .79 1.06 .22 .21
± ± ± ± ± + ± ± ± ± ± ± ± ± ± ± +
.03 .01 .52 .05 .29 .23 .58 .01 .14 .02 .22 .01 .01 .10 .02 .03 .04
Number of carbon atoms: number of double bonds. Fatty acid values are expressed as percentages of total integrated mass of fatty acid methyl esters detected during chromatography. Data are presented as means ± standard deviations of two separate determinations from pooled extract. 2
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
was the only polyunsaturated fatty acid (PUFA) present in a large amount; PUFA with 22 carbons were not detectable. These TG fatty acid composition patterns were in sharp contrast to the patterns found in the isolated PL fractions presented in Table 3. Fatty acids from the isolated PL fraction of breast and leg meat contained more than 20% arachidonic acid (20:4) and substantial amounts of PUFA with 22 carbon atoms (22:4, 22:5, 22:6). The fatty acids from skin PL contained approximately one-half the amount of PUFA with 20 and 22 carbon atoms compared with fatty acids from meat tissues (Table 3). Fat from breast meat contained a higher proportion of PL, which was rich in PUFA, than leg meat; breast fat also contained a higher concentration of malonaldehyde (MA) compared with leg as indicated in Table 4. However, leg meat contained two times more total fat than breast meat, and consequently, leg meat had a slightly higher TBA number than breast meat. Fat from skin had a very low proportion of PL and, consequently, a low concentration of MA; but because of high total fat content, the calculated TBA number of skin was significantly higher (P<.05) than values obtained from either
FAT AND MALONALDEHYDE
Even though the autoxidation problem of the TBA assay can be eliminated, we have found that TBA analysis, by itself, is not a useful parameter for evaluating food quality. For instance, in our study, chicken skin was found to have a higher TBA number than chicken meat, which is a result compatible with the findings of others (Marion and Woodroof, 1966; Marion, 1969). These TBA results taken alone indicate that skin tissue might be more unstable for oxidation than meat. However, when more detailed lipid analysis is provided, it becomes obvious that the TBA number of skin is higher than meat because skin contains 15 times more total fat than leg and 30 times more than breast. In addition, it is apparent that fat from skin is actually more stable than fat from meat because of a far lower concentration of MA due to a small PL fraction and an overall low concentration of PUFA. This conclusion is supported by results of others who reported
TABLE 4. Malonaldehyde (MA) concentration and thiobarbituric acid (TBA) number of fresh breast, leg, and skin tissues Type of tissues
Mg MA/g Fat 1
Breast meat Leg meat Skin
28.4 ± 1.84c 15.3 ± . 8 1 b 1.4 ± .08 a
TBA number 1
% Fat 1 1.10 ± .08 a 2.35 ± .16 b 32.81 + 3.48 c
.31 ± .02* .36 ± .02 a .46 + . 0 3 b
a,b,c Mean values within each column with different superscripts are significantly different (P<.05, two-tailed t test). 1
Data are presented as means ± standard deviations of 5 samples.
TABLE 5. Relative levels of fluorescent products presented in the organic and aqueous layers from Folch-extracted meat and skin tissues Type of tissues
Breast meat Leg meat Skin
Organic1
Aqueous 1
(FU'/mg fat)
(FU//zg protein)
98.8 ± 4.02 c 53.7 ± 2 . 7 1 b 4.4 ± .22 a
1.16 ± .04 a 1.51 + . 0 5 b 1.81 ± .07 c
' ' Mean values within each column with different superscripts are significantly different (P<.05, two-tailed t test). 1
Data are presented as means ± standard deviations of 5 samples.
2
FU = Fluorescent units.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
and skin vary widely. Some of this variability may be due to differences in age, sex, diet, or breed of chickens studied. But some reported TBA values are so high that artifacts due to sample autoxidation can be suspected. Several investigators have tried to avoid the problem of autoxidation by adding antioxidants to meat samples during preparation, extraction, or distillation, and during the critical heating steps of the TBA assay (Moerck and Ball, 1974; Rhee, 1978; Rhee and Ziprin, 1981; Yamauchi et al, 1982). We have recently demonstrated that the addition of butylated hydroxy toluene to fat from chicken tissues can eliminate the problem of sample autoxidation in the TBA assay (Pikul et al, 1983), and this improved method was used in our present study. The TBA numbers reported in our study are similar to values reported elsewhere for similar material (Jantawat and Dawson, 1977; Igene et al, 1979; Yamauchi etal, 1982).
315
316
PIKUL ET AL.
ACKNOWLEDGMENTS This work was s u p p o r t e d b y a FulbrightHays Exchange Scholarship, a grant from t h e American Heart Association, Illinois Affiliate, and a grant from t h e Wallace Genetic F o u n d a tion. T h e a u t h o r s recognize t h e technical assistance of Regina Galer-Unti and J o h n Catlow. REFERENCES Chio, K. S., and A. L. Tappel, 1969. Synthesis and characterization of the fluorescent products derived from malonaldehyde and amino acids. Biochemistry 8:2821-2827. Dawson, L. E., and K. Schierholz, 1976. Influence of grinding, cooking, and refrigerated storage on lipid stability in turkey. Poultry Sci. 55:618— 622. Dillard, C. J., and A. L. Tappel, 1973. Fluorescent products from reaction of peroxidizing polyun-
saturated fatty acids with phosphatidyl ethanolamine and phenylalanine. Lipids 8:183—189. Eng, L. F., and E. P. Noble, 1968. The maturation of rat brain myelin. Lipids 3:157—162. Folch, J., M. Lees, and G. M. Sloane-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Foster, L. B., and R. T. Dunn, 1973. Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method. Clin. Chem. 19:338-340. Glick, D., B. F. Fell, and K. E. Sjolin, 1964. Spectrophotometry determination of nanogram amounts of total cholesterol in microgram quantities of tissue or microliter volumes of serum. Anal. Chem. 36:1119-1121. Goldstein, B. D., M. G. Rozen, and M. A. Amoruso, 1979. Relation of fluorescence in lipid-containing red cell membrane extracts to in vivo lipid peroxidation. J. Lab. Clin. Med. 93:687-694. Igene, J. O., and A. M. Pearson, 1979. Role of phospholipids and triglycerides in warmed-over flavor development in meat model systems. J. Food Sci. 44:1285-1290. Jantawat, P. P., and L. E. Dawson, 1977. Stability of broiler pieces during frozen storage. Poultry Sci. 56:2026-2030. Kates, M., 1972. Techniques of lipidology. Pages 347— 353 in Laboratory Techniques in Biochemistry and Molecular Biology. T. S. Work and E. Work, ed. North Holland Publ. Co., Amsterdam. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. MacDonald, B., J. I. Gray, Y. Kakuda, and M. L. Lee, 1980. Role of nitrite in cured meat flavor chemical analysis. J. Food Sci. 45:889-892. Marion, J. E., 1969. Oxidation of chicken tissue lipids as influenced by age and sex. Poultry Sci. 48: 301-304. Marion, J. E., and J. G. Woodroof, 1966. Composition and stability of broiler carcasses as affected by dietary protein and fat. Poultry Sci. 45:241 — 247. Melton, S. L., 1983. Methodology for following lipid oxidation in muscle foods. Food Technol. 37: 1 0 5 - 1 1 1 , 116. Moerck, K. E., and H. R. Ball, 1974. Lipid autoxidation in mechanically deboned chicken meat. J. Food Sci. 39:876-879. Pearson, A. M., J. I. Gray, A. M. Wolzak, and N. A. Horenstein, 1983. Safety implications of oxidized lipids in muscle foods. Food Technol. 37: 121-129. Pikul, J., D. E. Leszczynski, and F. A. Kummerow, 1983. Elimination of sample autoxidation by butylated hydroxytoluene additions before thiobarbituric acid assay for malonaldehyde in fat from chicken meat. J. Agric. Food Chem. 3 1 : 1338-1342. Rhee, K. S., 1978. Minimization of further lipid peroxidation in the distillation 2-thiobarbituric acid test of fish and meat. J. Food Sci. 43:1776— 1778, 1781. Rhee, K. S., and Y. A. Ziprin, 1981. Oilseed protein
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015
TBA n u m b e r s for raw p o u l t r y skin t h a t were higher t h a n raw p o u l t r y m e a t , b u t t h a t after cooking and refrigerated storage, T B A n u m b e r s of skin were equal t o or lower t h a n values for m e a t (Marion, 1 9 6 9 ; Dawson and Schierholz, 1976). In addition t o MA and TBA n u m b e r determ i n a t i o n s , fluorescence excitation and emission spectra were used t o measure certain secondary p r o d u c t s of lipid o x i d a t i o n , which are believed t o represent covalent crosslinkage of MA with amino acids, peptides, lipids, and itself (Chio a n d Tappel, 1 9 6 9 ; Pearson et al, 1 9 8 3 ) . Meas u r e m e n t of fluorescent p r o d u c t s from b o t h t h e organic and a q u e o u s layers of Folch-extracted tissues has been used as an analytical m e t h o d for q u a n t i t a t i o n of p e r o x i d a t i o n damage (Dillard and Tappel, 1 9 7 3 ; MacDonald et al, 1 9 8 0 ; Pearson et al, 1 9 8 3 ) . F r o m Folche x t r a c t e d m e a t and skin, t h e relative concentrations of fluorescent p r o d u c t s in t h e organic layers, which contain t h e whole e x t r a c t e d fat, showed a trend t h a t was very similar t o t h e relative c o n c e n t r a t i o n s of MA in those tissues. Because MA is a highly reactive c o m p o u n d , samples that contain high c o n c e n t r a t i o n s of free M A can b e e x p e c t e d t o contain high concentrations of crosslinked MA. T h e relative c o n c e n t r a t i o n s of fluorescent p r o d u c t s in t h e aqueous layers of Folch-extracted m e a t and skin did n o t follow t h e p a t t e r n of MA o r fluorescent p r o d u c t c o n c e n t r a t i o n s in total fat e x t r a c t . These results indicate t h a t free MA m a y selectively react with lipid molecules, especially PL, r a t h e r t h a n amino acids or peptides in fresh tissue samples.
FAT AND MALONALDEHYDE ingredients as antioxidant for meat in food service. J. Food Prot. 4 4 : 2 5 4 - 2 5 6 . Sinnhuber, R. O., and T. C. Yu, 1977. The 2-thiobarbituric acid reaction, an objective measure of the oxidative deterioration occurring in fats and oils. Yukagaku 26:259-267. Wilson, B. R., A. M. Pearson, and F. B. Shorland, 1976. Effect of total lipids and phospholipids on warmed-over flavor in red and white muscle from
317
several species as measured by thiobarbituric acid analysis. J. Agric. Food Chem. 24:7— 11. Yamauchi, K., Y. Nagai, and T. Ohashi, 1982. Quantitative relationship between alpha-tocopherol and polyunsaturated fatty acids and its connection to development of oxidative rancidity in chicken skeletal muscle. Agric. Biol. Chem. 46:2719— 2724.
Downloaded from http://ps.oxfordjournals.org/ at Universitat Autonoma Barcelona on June 2, 2015