E ect of irradiation and packaging conditions after cooking on the formation of cholesterol and lipid oxidation products in meats during storage $

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1 Meat Science 57 (2001) 413±418 E ect of irradiation and packaging conditions after cooking on the formation of cholesterol and lipid oxidation products in meats during storage $ D.U. Ahn *, K.C. Nam, M. Du, C. Jo Department of Animal Science, Iowa State University, Ames, IA , USA Received 25 July 2000; received in revised form 18 September 2000; accepted 18 September 2000 Abstract The e ect of irradiation and packaging conditions on the content of cholesterol oxidation products (COPs) and lipid oxidation in cooked turkey, beef, and pork during storage was studied. Ground turkey leg, beef, and pork were cooked, packaged either in oxygen-permeable or oxygen-impermeable bags, and irradiated at 0 or 4.5 kgy. Lipid oxidation and COPs were determined after 0 and 7 days of storage at 4 C. Packaging of cooked meat was more important than irradiation in developing COPs and lipid oxidation in cooked meats during storage. 7a-Hydroxycholesterol, 7b-hydroxycholesterol, b-epoxide, and 7-ketocholesterol were among the major COPs formed in cooked turkey, beef, and pork after storage, and their amounts increased dramatically during the 7-day storage in aerobic conditions. Irradiation had no signi cant e ect on the amounts of any of the COPs found in cooked turkey and beef, but increased (P<0.05) the amounts of a- plus 7b-hydroxycholesterol, b-epoxide, 7-ketocholesterol, and total COPs in aerobically packaged cooked pork. The amounts of COPs and lipid oxidation products (TBARS) closely related to the proportion of polyunsaturated fatty acids in meat. The results indicated that the composition of fats in meat is important on the oxidation rates of lipids and cholesterol, and packaging is far more important than irradiation in the formation of COPs and lipid oxidation in cooked meat. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Irradiation; Packaging; Cholesterol oxidation products; Lipid oxidation; Cooked meat 1. Introduction Cholesterol oxidation products (COPs) have received considerable attention in recent years because of their biological activities associated with human diseases. Animal studies suggested that COPs in the diet could be associated with heart and vascular diseases. Human studies also demonstrated that the quantity of oxidized lipids in the diet was directly related to the level of oxidized lipids in serum postprandial chylomicrons (Staprans, Rapp, & Pan, 1994), which provides a mechanism by which dietary oxidized lipids can a ect the oxidative states of endogenous lipoproteins. $ Journal paper No. J of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 3322, and supported by the S-292 Regional Project and State of Iowa funds. * Corresponding author. Tel.: ; fax: address: duahn@iastate.edu (D.U. Ahn). Staprans, Pan, and Rapp (1998) showed that oxidized cholesterol in the diet could be directly absorbed into circulation, and that COPs accelerated the development of atherosclerosis in rabbits. Despite the wide existence of COPs in foods and its adverse e ect on health, little work has been done on the combined e ect of processing, including irradiation, cooking, packaging, and storage methods, on the formation of COPs in meat. During food processing and storage, polyunsaturated fatty acids tend to be oxidized. Cholesterol can be oxidized by the same mechanism as fatty acids. Therefore, lipid radicals formed during processing and storage of foods can accelerate the formation of COPs (Chan, Gray, Gomaa, Harte, Kelly, & Buckley 1993; Paniangvait, King, Jones, & German 1995). The exposure of foods containing cholesterol to heat, air, or irradiation increases the production of COPs (Pie, Spahis, & Seillan, 1990; Yan & White, 1990; Lebovics & Gaal, 1994). A variety of COPs were found in foods of animal origin (Paniangvait et al., Li, Ohishima, Shozen, Ushio, /01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S (00)

2 414 D.U. Ahn et al. / Meat Science 57 (2001) 413±418 and Koizumi (1994) showed that the formation of COPs was accelerated by polyunsaturated fatty acids present in lipids. Because meats from di erent animal species have di erent fatty acid composition, the rates of COPs formation can also be di erent. However, little information is available about COPs formation in meats from di erent animal species. Ionizing radiation has been used in food processing to control microbial growth (Farkas, 1998). Ionizing radiation induces oxidation, and the quantity of oxidation products formed by irradiation increased in a dosedependent manner (Lebovics, Gaal, Somagyi, & Farkas 1992). Hwang and Maerker (1993a) reported that irradiation of raw beef, pork and veal at 10 kgy increased the contents of COPs, and the increases of COPs in those meats during storage were greater in the irradiated than in the nonirradiated. Du and Ahn (2000) reported that radiation increased the formation of COPs in egg yolk powder, and the presence of oxygen had a signi cant e ect on the rate of formation. Cooked meat is very susceptible to oxidative change because of the destruction of phospholipid membrane structure by heat denaturation (Ahn, Olson, Lee, Jo, Chen, & Wu 1998). Lipid oxidation in cooked meat was accelerated under aerobic conditions during storage. Therefore, signi cant amounts of COPs can be formed if irradiation, cooking, and storage in aerobic packaging are combined. The objective of this study was to determine the e ect of irradiation and packaging on the content of COPs in cooked turkey, beef, and pork during storage. 2. Materials and methods 2.1. Sample preparation Turkey leg, beef loin, and pork loin muscles, purchased from four local supermarkets, were trimmed of all fat from the surface, the lean muscles were ground separately through a 3-mm plate, and patties (approximately 100 g each) were prepared. Meats from each supermarket were used as a replication. Patties were individually packaged in oxygen-permeable zipper bags (46, 2 MIL, Associated Bag Company, Milwaukee, WI), stored overnight at 4 C and cooked in bags in an 85 C water bath for 25 min. After cooling for 30 min at room temperature and draining meat juice o from the bag, the meat patties were repackaged either in polyethylene oxygen-permeable bags or nylon/polyethylene vacuum bags (O 2 permeability, 9.3 ml O 2 /m 2 /24 h at 0 C; Koch, Kansas City, MO). After packaging, patties were stored overnight at 4 C and then irradiated at ambient temperature at 0 or 4.5 kgy using a linear accelerator (Circe IIIR, Thomson CSF Linac, Saint- Aubin, France). The energy and power levels used were 10 MeV and 10 kw, respectively, and the average dose rate was 91.3 kgy/min. The max/min ratio was approximately 1.20 for 4.5 kgy. To con rm the target dose, two alanine dosimeters per cart were attached to the top and bottom surfaces of the sample. The alanine dosimeter was read using a 104 Electron Paramagnetic Resonance Instrument (Bruker Instruments, Inc., Billerica, MA). All samples were stored at 4 C for up to 7 days, and COPs and lipid oxidation in cooked meat were analyzed after 0 and 7 days of storage at 4 C. Lipid oxidation was determined by the uorometric thiobarbituric acid reactive substances (TBARS) method of Jo and Ahn (1998) Preparation of COPs Lipids were extracted from samples according to the method of Folch, Lees, and Sloan-Stanley (1957). Five grams of meat sample, butylated hydroxytoluene (50 ml, 7.2%), and 30 ml Folch 1 solution (chloroform: methanol=2:1) were added to a 50-ml test tube and homogenized using a Polytron (Brinkman Instruments, Inc., Westbury, NY) for 20 s at high speed. The homogenate was ltered through a Whatman No. 1 lter paper (Whatman Inc., Clifton, NJ) into a 100-ml graduated cylinder, and the lter paper was rinsed twice with 10 ml of Folch 1 solution. After addition of 8 ml of 0.88% NaCl solution to each cylinder, the cylinder was capped with a glass stopper and the content mixed. The inside of the cylinder was washed twice with 5 ml of Folch 2 solution (chloroform: methanol: water=3:47:48). After phase separation, the lipid layer volume was recorded, and the upper layer (methanol and water) of the solution was completely and carefully siphoned o to prevent contamination of the chloroform layer. The organic layer was put in a glass scintillation vial and dried in a block heater for 1 h at 50 C. The dried lipid was dissolved with an aliquot of hexane (with 2 drops of ethanol to improve the solubility of polar lipids) to make 0.1 g fat/ml hexane and used for the fatty acid and cholesterol analysis. A silicic acid (100 mesh), cellite-545, and CaHPO 4. 2H 2 O (10:9:1, w/w/w) mixture in chloroform was prepared and packed into a glass column (22 mm30 cm with a sintered glass frit at the bottom) to a height of 10 cm. The column was washed with 10 ml of Solvent I (hexane: ethyl acetate=9:1, v/v) before a sample was loaded. Lipid sample dissolved in hexane (0.2 g) was loaded onto the silicic acid column. Neutral lipids, cholesterol, and phospholipids were eluted by passing 40 ml of Solvent II (hexane: ethyl acetate=4:1, v/v) through the column. Then COPs were eluted with 40 ml of Solvent III (acetone: ethyl acetate: methanol=10:10:1, v/v/ v) and dried under nitrogen. The dried COPs were added with 200 ml pyridine and 100 ml bis-trimethylsilyltri uoroacetamide+1% trimethylchlorosilane and derivatized by heating in a dry bath (80 C) for 1 h.

3 D.U. Ahn et al. / Meat Science 57 (2001) 413± Gas chromatograph (GC) analysis of COPs COPs were analyzed as described by Ahn, Lee, Jo, and Sell (1999) after 0 and 7 days of storage at 4 C. Analysis of COPs was performed with an HP 6890 GC (Hewlett Packard Co., Wilmington, DE) equipped with an on-column capillary injector and ame ionization detector (FID). An HP-5 capillary column of 0.25 mm i.d.30 m bonded phase 5% phenylsilicon with 0.25-mm lm thickness (Hewlett Packard Co.) was used. A splitless inlet was used to inject samples (0.5 ml) into the capillary column, and a ramped oven temperature was used (80 C for 0.25 min, increased to 230 Cat40 C/ min, increased to 270 C at 25 C /min, increased to 285 C at 1.5 C/min, and held for 8 min). Temperatures of both the inlet and detector were 280 C. Helium was the carrier gas at constant pressure of 18.5 psi. Detector (FID) air, H 2, and make-up gas (He) ows were 300, 30, and 28 ml/min, respectively. The area of each peak (pas) was integrated using the Chemstation software (Hewlett Packard Co.), and the amount of COPs was calculated using an internal standard GC analysis of fatty acid composition Fatty acid pro les of three meat species were determined by the method of Du and Ahn (2000). One milliliter of methylating reagent (boron-tri uoride methanol, Sigma Chemical Co.) was added to 50 ml of the lipid extract and incubated in a 90 C water bath for 1 h. After cooling to room temperature, 2 ml hexane and 5 ml water were added, mixed thoroughly, and left at room temperature overnight for phase separation. The top hexane layer containing methylated fatty acids was analyzed for fatty acid composition using a GC (HP 6890; Hewlett Packard Co.). An HP-5 (5%-diphenyl- 95%-dimethylsiloxane copolymer) capillary column (Hewlett Packard Co.) of 0.32 mm i.d.30m with mm lm thickness was used. A splitless inlet was used to inject samples (1 ml) into the capillary column. A ramped oven temperature condition (180 C for 2.5 min, increased to 230 C at 2.5 C/min, then held at 230 C for 7.5 min) was used. Temperatures of both the inlet and detector were 280 C. Helium was the carrier gas at linear ow of 1.1 ml/min. Detector (FID) air, H 2, and make-up gas (He) ows were 350, 35, and 43 ml/min, respectively. Fatty acids were identi ed by comparison of retention times to known standards. Relative quantities were expressed as weight percentage of total fatty acids Statistical analysis The experimental design was to determine the e ects of irradiation, packaging conditions, and storage on lipid oxidation and cholesterol oxidation. Data were analyzed using SAS software (SAS Institute, 1985) by the generalized linear model procedure, and the Student-Newman-Keuls' multiple range test was used to compare di erences among means. Mean values and standard error of the means were reported. Signi cance was de ned at P< Results and discussion At Day 0, vacuum-packaged cooked turkey meat produced more 7-ketocholesterol than aerobically packaged irradiated and nonirradiated meat. However, the amounts of other COPs, including total COPs, in cooked meat were not in uenced by irradiation and packaging during and after irradiation (Table 1). The amounts of total COPs in vacuum-packaged cooked turkey meat increased about two-fold, and those of aerobically packaged increased about 10-fold after 7 days of storage. Among the COPs, 7a-hydroxycholesterol, 7bhydroxycholesterol, b-epoxide, and 7-ketocholesterol were among the major COPs produced in cooked turkey meat, and their amounts increased dramatically during the 7-day storage in aerobic conditions. But, irradiation had no e ect on the amounts of any of the COPs found in cooked turkey meat. Hwang and Maerker (1993b) reported that irradiation of raw chicken meat at 10 kgy Table 1 The content of cholesterol oxidation products (COPs) in cooked turkey leg meat with di erent irradiation, packaging, and storage conditions a COPs Day 0 (mg COPs/g lipid) Day 7 (mg COPs/g lipid) V-C V-IR A-C A-IR S.E.M. V-C V-IR A-C A-IR S.E.M. 7a- and 7b-Hydroxycholesterol b 68.4 c a a 20.9 a-epoxide b-epoxide b 7.6 b 61.4 a 55.7 a a-Hydroxycholesterol Cholestantriol b 1.0 b 5.3 a 2.3 b Ketocholesterol 14.2a 9.5 a,b 5.4 b 4.9 b b 26.2 b a a 13.9 Total b b a a 39.3 a Sampled 2 h after cooking. Values with di erent letters within a row of the same storage time are di erent (P<0.05). A, aerobic packaging; V, vacuum packaging; C, nonirradiated control; IR, irradiated at 4.5 kgy dose; S.E.M., standard error of the mean.

4 416 D.U. Ahn et al. / Meat Science 57 (2001) 413±418 increased the content of 6-ketoxholestanol to about four times the level of nonirradiated chicken. However, no 6- ketoxholestanol was detected in the irradiated cooked turkey in this study. The composition of COPs in cooked beef at Day 0 was quite di erent from that of the cooked turkey where 7aplus 7b-hydroxycholesterol and 7-ketocholesterol were the major COPs. The amounts of a-and b-epoxides, 20ahydroxycholesterol, and triol found in cooked beef were greater than those of the cooked turkey at Day 0. The e ects of packaging and/or irradiation on the contents of COPs were not consistent (Table 2). After 7 days of storage, signi cant increases in 7a- plus 7b-hydroxycholesterol and 7-ketocholesterol were observed in beef with aerobic packaging. Total COPs also increased in cooked beef after 7 days of storage in aerobic conditions. However, other COPs such as a-epoxide, b-epoxide, 20ahydroxycholesterol, and triol remained unchanged or decreased after 7 days of storage, especially with vacuum packaging. Hwang and Maerker (1993a) reported that irradiation of raw beef and pork increased the content of COPs. However, irradiation had no e ect on the formation of COPs in cooked beef as in cooked turkey meat (Tables 1 and 2). The decrease of COPs in vacuumpackaged cooked beef after 7 days of storage cannot be explained. As in turkey meat, a- plus 7b-hydroxycholesterol and 7-ketocholesterol were the major COPs in cooked pork at Day 0 (Table 3). The amounts of a- plus 7b-hydroxycholesterol and total COPs in aerobically packaged cooked pork were higher than those of the vacuumpackaged pork at Day 0, but irradiation had no e ect on the content of COPs in cooked pork. After 7 days of storage, aerobically packaged pork produced 10- to 15- fold higher amounts of total COPs than the vacuumpackaged pork. Irradiation signi cantly increased the amounts of a- plus 7b-hydroxycholesterol, b-epoxide, 7- ketocholesterol, and total COPs in aerobically packaged cooked pork. Table 4 indicates that packaging of cooked meat is more important than irradiation on the development of lipid oxidation in cooked meats during storage. With vacuum packaging, all meats except irradiated cooked turkey showed little changes in TBARS during the 7-day storage. With aerobic packaging, three to ve-fold increases in TBARS were observed in all cooked meats. Irradiation decreased the TBARS of vacuum- and aerobically packaged cooked turkey meat at Day 0 and aerobically packaged turkey and beef at Day 7. The TBARS of cooked turkey meat was the highest, beef was the lowest, and pork was intermediate at both Day 0 and Day 7. Table 2 The content of cholesterol oxidation products (COPs) in cooked pork with di erent irradiation, packaging, and storage conditions a COPs Day (mg COPs/g lipid) Day 7 (mg COPs/g lipid) V-C V-IR A-C A-IR S.E.M. V-C V-IR A-C A-IR S.E.M. 7a- and 7b-Hydroxycholesterol b 13.7b a 88.7 a 7.7 a-epoxide a 3.3 b 4.7 b 7.7 b 1.7 b-epoxide a-Hydroxycholesterol 15.9 a 8.4 b 5.2 b 5.2 b b 4.4 a 3.9 a 3.1a 0.9 Cholestantriol 4.9 a,b 3.4 b 5.0 a,b 6.2 a b 0 b 4.0 a 4.2 a Ketocholesterol 12.6 a,b 9.5 b 20.1 a 19.2 a b 7.3 b 45.6 a 44.8 a 5.5 Total b 28.6 b a a 12.4 a Sampled 2 h after cooking. Values with di erent letters within a row of the same storage time are di erent (P<0.05). A, aerobic packaging; V, vacuum packaging; C, nonirradiated control; IR, irradiated at 4.5 kgy dose; S.E.M., standard error of the mean. Table 3 The content of cholesterol oxidation products (COPs) in cooked beef with di erent irradiation, packaging and storage conditions a COPs Day 0 (mg COPs/g lipid) Day 7 (mg COPs/g lipid) V-C V-IR A-C A-IR S.E.M. V-C V-IR A-C A-IR S.E.M. 7a- and 7b-Hydroxycholesterol 14.0 b 11.0 b 26.3 a 27.5 a c 7.3 c b a 9.2 a-epoxide b 2.4 b 16.2 a 17.1 a 0.9 b-epoxide b 4.6 b 7.3 b 37.8 a 2.2 Cholestantriol Ketocholesterol c 5.9 c a c 7.5 Total 29.9 a,b 25.2 b 49.8 a 42.5 a c 22.3 c b a 17.3 a Sampled 2 h after cooking. Values with di erent letters within a row of the same storage time are di erent (P<0.05). A, aerobic packaging; V, vacuum packaging; C, nonirradiated control; IR, irradiated at 4.5 kgy dose; S.E.M., standard error of the mean.

5 D.U. Ahn et al. / Meat Science 57 (2001) 413± Table 4 E ect of irradiation, storage, and packaging conditions on TBARS values of cooked turkey leg, beef, and pork a Treatment TBARS (mg MDA/kg meat) Turkey meat Beef Pork Day 0 Day 7 S.E.M. Day 0 Day 7 S.E.M. Day 0 Day 7 S.E.M. V-C 3.95 a,y 4.44 c,x c b,y 1.71 b,x 0.08 V-IR 2.86 b,y 3.63 c,x c b 0.97 b 0.08 A-C 3.55 a,y a,x y 5.06 a,x a,y 6.95 a,x 0.73 A-IR 2.85 b,y 8.95 b,x y 3.69 b,x a,y 5.58 a,x 0.63 S.E.M a Sampled 2 h after cooking. Di erent letters (a±c) within a column of the same storage time are signi cantly di erent (P<0.05). Values with di erent letters (x and y) within a row of the same meat are di erent (P <0.05). A, aerobic-packaging; V, vacuum-packaging; C, nonirradiated control; IR, irradiated at 4.5 kgy dose; S.E.M., standard error of the mean Because polyunsaturated fatty acids tended to be oxidized by a free radical mechanism, the oxidation rates of lipids and cholesterol for those di erent meats were in uenced by the composition of fats in meat as reported by Li, Ahn, Cherian, Chung, and Sim (1996). The total fat contents of turkey meat, pork and beef were 6.65, 8.27 and 9.38%, respectively. The fatty acid compositions of meats from the three animal species showed that turkey meat contained 26.16% of linoleic and 4.02% of arachidonic acid, pork had 16.96% of linoleic and 3.26% of arachidonic acid, and beef had 5.19% of linoleic and 1.19% of arachidonic acid. The TBARS of meats correlated well with the proportions of polyunsaturated fatty acids and amounts of total COPs in meat, and were in good agreement with those of others (Angulo, Romero, Ramirez, & Gil 1997; Galvin, Morrissey, & Buckley, 1998). 4. Conclusion Large amounts of COPs can be formed in meat after cooking and storage. Packaging was far more important than irradiation in the formation of COPs and lipid oxidation in cooked meat. The COPs produced in turkey, beef, and pork during cooking and storage were mainly 7a-hydroxycholesterol, 7b-hydroxycholesterol, and 7- ketocholesterol, which have signi cant health implications because these COPs can be absorbed in guts and are directly associated with the initiation and development of atherosclerosis in animals (Lyons, Samman, Gatto, & Brown 1999; Vine, Mamo, Beilin, Mori, & Croft 1998). References Ahn, D. U., Olson, D. G., Lee, J. I., Jo, C., Chen, X., & Wu, C. (1998). 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