JFS: Food Chemistry and Toxicology Effects of Antioxidants and Packaging on Lipid and Cholesterol Oxidation and Color Changes of Irradiated Egg Yolk Powder ABSTRACT: Electron-beam irradiation significantly increased the oxidation of docosahexaenoic, arachidonic and linolenic acids, and cholesterol in egg yolk powder. Arachidonic-acid content was reduced from 4.58% to 3.07%, and total cholesterol oxidation products increased from 11 g/g to 467g/g after 5.0 kGy irradiation. Further oxidation occurred during storage. Vacuum-packaging significantly reduced, but the use of antioxidants had no effect on the fatty acids and cholesterol oxidation during irradiation and storage. Irradiation caused color change in egg yolk powder. The Hunter color a- (redness) values decreased from 3.89 to 2.48 and 1.94, respectively, after 2.5 and 5.0 kGy irradiation. Hunter color a- and b-values were also decreased during storage. Vacuum-packaging and antioxidants significantly reduced color changes. Key Words: egg yolk powder, irradiation, antioxidants, lipid and cholesterol oxidation, color Introduction T HERE IS INCREASING CONCERN ABOUT the incidence of salmonellosis caused by eggs and other poultry products (Serrano and others 1997). Much effort has been made to control Salmonella in egg products, but irradiation treatment has been proposed as the most effective way of control (Radomyski and others 1994). With a dosage above 2.0 kGy, Salmonella and other bacteria in egg yolks can be successfully controlled (Narvaiz and others 1992). However, ionizing radiation generates free radicals that may cause lipid peroxidation and other chemical changes, which will deteriorate the quality of the egg products (Branka and others 1992; Lebovics and others 1992, Wong and others 1995). Branka and others (1992) reported that irradiation at 3 kGy is the threshold dose for organoleptic changes in dehydrated egg products, and that irradiation dosage and the presence of oxygen influenced the buildup of lipid hydroperoxides. Lebovics and others (1992) reported that the concentrations of cholesterol oxidation products (COPs) produced by 1 kGy ionizing radiation were similar to those formed by the autoxidation of nonirradiated egg powder stored under aerobic condition for 1 mo. Egg yolk lipids include polyunsaturated fatty acids (PUFAs) and cholesterol, which both can be oxidized easily following irradiation (Gardner 1989). Lipid oxidation causes the loss of PUFAs (Kaneda and Miyazawa 1987) and other nutrients and also causes carotenoids destruction and organoleptic changes (Katusin-Razem and others 1992). © 2000 Institute of Food Technologists Earlier studies have suggested that lipid oxidation products (LOPs) and COPs have negative effects on human health. Dietary COPs and LOPs have been shown to induce and accelerate the development of atherosclerosis, 1 of the main causes of death in the world (Staprans and others 1994, 1998). Staprans and others (1998) and Vine and others (1997) reported that LOPs in the diet were absorbed by the small intestines of humans and rodents and were incorporated into chylomicrons. With human studies, it has been demonstrated that the quantity of oxidized lipids in the diet directly correlated with the levels of oxidized lipids in serum postprandial chylomicrons (Staprans and others 1994). In rodents, dietary LOPs were also incorporated into serum very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) fractions (Staprans and others 1993), and the levels of oxidized chylomicrons and VLDL + LDL were directly correlated with the quantity of oxidized lipids in the diet. This indicated that LOPs were transported by LDL and VLDL (Krut and others 1997) and provided a mechanism by which dietary oxidized lipids can affect the oxidative state of endogenous lipoproteins. Staprans and others (1998) further showed that oxidized cholesterol in the diet could also be directly absorbed into the circulatory system. These results show that LOPs and COPs in the diet contribute to serum lipoprotein oxidation. Therefore, it is important to assess cholesterol and lipid oxidation of egg yolk powder by irradiation and during storage. Color is an im- Table 1—The change in color L-value of electron-beam irradiated yolk powder during storage Vacuum-packaging Irradiation Storage1 dose(kGy) (d) Control 0 0 45 90 SEM 90.32 aby 90.76ay 89.98bx 90.38ay 91.09ax 0.17 89.96ay 90.58bx 0.11 89.94ax 90.40bx 0.15 2.5 0 45 90 SEM 90.28by 91.59ax 92.02ax 0.23 91.11ay 91.17ay 92.01ax 0.12 5.0 0 45 90 SEM 90.86ay 91.88ax 90.82ay 0.09 90.84ay 91.36bx 91.01 axy 0.07 VE BHT Aerobic-packaging SEM Control VE 90.98ay BHT SEM 0.16 0.15 0.16 91.12ay 90.18by 91.40ax 91.99az 0.08 0.14 0.23 0.11 90.73ay 90.98ay 90.16by 0.14 90.95ay 90.47 abz 90.18by 0.16 94.31ax 94.31ax 94.02ax 0.08 0.12 0.13 0.13 90.59by 91.01bx 91.06ax 0.11 0.04 0.14 0.10 90.87az 90.94ay 93.56by 93.75bx 94.58ax 94.01bx 0.13 0.19 93.54ax 93.71ax 91.59ay 91.17ay 0.08 0.13 90.05by 0.18 93.42ax 0.13 az 91.40 0.23 0.20 90.66az 0.12 94.41ax 0.13 93.86by 0.11 0.10 a,bDifferent letters within a row of the same packaging method and irradiation dose are different (P < 0.05). n = 6. x-zDifferent letters within a column of the same irradiation dosage and storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 °C) with relative humidity of 73%. Vol. 65, No. 4, 2000—JOURNAL OF FOOD SCIENCE 625 FoodChemistryandToxicology M. DU AND D.U. AHN Antioxidants and Packaging on the Quality of Irradiated Yolk Powder . . . FoodChemistryandToxicology portant quality parameter of egg yolk. Carotenoids, the main pigments in yolk, can be oxidized by the same mechanism as lipids and cholesterol. Thus, the influence of irradiation on yolk color needs to be ascertained (Serrano and others 1997). BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), and vitamin E (VE) are widely used in the food industry to prevent lipid oxidation. They can terminate chain reactions of peroxides by scavenging chain-propagating radicals, and thus they can prevent lipids from oxidizing (Halliwell 1996; Halliwell and Gutteridge 1989). Li and others (1996) also indicated that the stability of lipids in chicken meat and egg yolk powder decreased with high contents of PUFAs but improved with dietary supplementation of tocopherol. The objective of this research was to determine the effect of antioxidants (VE or BHT) and packaging on color and lipids and cholesterol oxidation of irradiated egg yolk powder during storage. Table 2—The change in color a-value of electron-beam irradiated yolk powder during storage Irradiation Storage1 dose(kGy)2 (d) C ness) values of egg yolk powder were decreased by irradiation. The color of irradiated egg yolk powder looked paler than nonirradiated sample (Tables 1, 2, and 3). In the control treatment (no antioxidant added) with vacuum-packaging, the color a-value (redness) of egg yolk powder decreased significantly (from 3.89 to 2.48, p < 0.05) after 2.5 kGy irradiation and to 1.94 after 5.0 kGy irradiation (p < 0.05); b-value (yellowness) decreased significantly (from 26.23 to 21.12 and 20.36, p < 0.05) after 2.5 and 5.0 kGy irradiation, respectively. Although changes in color L values by irradiation were not consistent, significant decrease in color a- and b-values produced the color of egg yolk powder light. Egg yolk color is dependent on carotenoids that contain unsaturated double bonds and can be oxidized with the same mechanism as lipid oxidation. The color a- and b-values of egg yolk powder kept decreasing during storage, indicating possible destruction of pigments by oxidation. Ma and others (1992) also noted that yolk color was pale after irradiation. Branka and others (1992) found that the loss of carotenoids was positively correlated with irradiation dosage, which suggests that irradiation-dependent production of free radicals could have contributed to the destruction of carotenoids. Serrano and others (1997) suggested that there would be an irradiation-dose threshold for causing color changes in yolk. Tukey grouping analysis (SAS 1989) was employed to compare the difference between vacuum and aerobic packaging. VE Aerobic-packaging BHT SEM Control 0.06 0.05 0.03 3.69ax VE 3.50ax BHT SEM 2.30by 2.19by 0.05 2.42ay 2.46ay 0.04 0.07 2.31by 0.03 by 2.29 0.04 0.07 0 45 90 SEM 3.89ax 3.76ay 3.72ax 3.74bx 2.32by 0.05 3.94ax 2.57az 0.04 3.64bx 2.39by 0.03 2.5 0 45 90 SEM 2.48ax 2.19by 1.75bz 0.04 2.35ax 2.46ax 1.78by 0.05 2.45ax 2.29by 1.95az 0.03 0.04 0.04 0.03 2.26bx 1.74ay 1.65by 0.04 2.63ax 1.93ay 1.92ay 0.05 2.60ax 0.06 1.83ay 0.05 1.92ay 0.04 0.04 5.0 0 45 90 SEM 1.94bx 1.53ay 1.38bz 0.04 2.15ax 1.65ay 1.47az 0.03 2.16ax 1.60ay 1.53ay 0.03 0.03 0.04 0.03 2.12 cx 1.33by 1.17bz 0.05 2.44ax 1.60ay 1.30az 0.04 2.29bx 0.04 1.46 aby 0.05 1.19bz 0.02 0.03 0 3.50ax a-cDifferent letters within a row of the same packaging method and irradiation dose are different (P < 0.05). n = 6. x-zDifferent letters within a column of the same irradiation dosage and storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 ºC) with relative humidity of 73%. Table 3—The change in color b-value of electron-beam irradiated yolk powder during storage Irradiation Storage1 dose (kGy) (d) Vacuum-packaging Aerobic-packaging Control VE BHT SEM Control 0 0 45 90 SEM 26.23by 26.56bx 23.32az 0.07 26.65ay 26.85ax 23.51az 0.06 25.26cx 25.15cx 23.44ay 0.10 0.10 0.08 0.07 26.30ax 26.50ax 24.74ay 24.46ay 22.50az 21.63bz 0.12 0.11 24.88bx 0.14 24.47ay 0.09 21.81bz 0.18 0.08 2.5 0 45 90 SEM 21.12bz 22.50ax 21.99ay 0.08 21.69ay 21.63by 21.99ax 0.11 21.99ax 21.81bx 22.00ax 0.18 0.13 0.18 0.06 21.17bz 21.39bz 22.35 aby 22.56ay 23.33bx 24.38ax 0.12 0.08 21.19bz 0.09 21.97by 0.13 23.59bx 0.13 0.17 5.0 0 45 90 SEM 20.36bz 21.39ay 22.13ax 22.32ax 21.13 aby 20.87bz 0.11 0 .11 20.27by 21.47bx 21.53ax 0.13 0.10 0.06 0.17 21.51bx 22.68ax 20.88by 22.29ay 16.04bz 16.86az 0.09 0.10 21.35by 0.06 22.00ax 0.15 16.62az 0.11 0.12 Results and Discussion OLOR A - ( REDNESS ) AND B - ( YELLOW Vacuum-packaging Control VE BHT SEM a-cDifferent letters within a row of the same packaging method and irradiation dose are different (P < 0.05). n = 6. x-zDifferent letters within a column of the same irradiation dosage and storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 °C) with relative humidity of 73%. Table 4—The concentration of polyunsaturated fatty acids in vacuum-packaged yolk powder after electron-beam irradiation and storage1 0-d storage 90-d storage Irradiation dose (kGy) Control VE BHT SEM Control VE BHT Linolenic 0 2.5 5.0 SEM 2.62ax 2.12ay 1.88az 0.04 2.54ax 2.11ay 1.89az 0.06 2.61ax 2.12ay 1.95az 0.05 0.10 0.04 0.04 1.94bx 1.49ay 1.01az 0.02 2.13ax 1.53ay 1.07az 0.03 2.11ax 0.02 1.54ay 0.03 1.13az 0.04 0.04 Arachidonic 0 2.5 5.0 SEM 4.69ax 3.48ay 2.99az 0.10 4.49ax 3.54ay 3.22az 0.07 4.55ay 3.45ax 3.26az 0.08 0.16 0.11 0.07 3.75ax 2.45ay 1.73az 0.05 3.85ax 2.48ay 1.81az 0.05 3.87ax 0.04 2.51ay 0.05 1.79az 0.04 0.05 DHA 0 2.5 5.0 SEM 2.48ax 1.96ay 1.82az 0.04 2.45ax 1.99ay 1.93az 0.05 2.50ax 1.94ay 1.95az 0.07 0.08 0.06 0.07 2.10bx 1.01ay 0.80az 0.04 2.23abx 0.97ay 0.81az 0.04 2.34ax 0.04 0.97ay 0.04 0.85az 0.03 0.05 Fatty acid SEM a,bDifferent letters within a row of the same packaging method and irradiation dose are different (P < 0.05). n = 6. x-zDifferent letters within a column of the same irradiation dosage and storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 °C) with relative humidity of 73%. Abbreviation: DHA, docosahexanoic acid There were significant differences for aand b-value (p < 0.05), indicating that the color change of egg yolk powder in aerobic-packaging was faster than that in the vacuum-packaging. Thus, vacuum-pack- 626 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 4, 2000 aging is effective in reducing oxidative changes during storage. The color a-values in antioxidant (VE or BHT) treatments were higher than the control treatment, suggesting that antioxidants reduced the Table 5—The concentration of polyunsaturated fatty acids in yolk powder of aerobic-packaging after electron-beam irradiation and storage1 Fatty acid 0-d storage Irradiation dose (kGy) Control 90-d storage VE BHT 0 2.5 5.0 SEM 2.59ax 2.65ax 2.66ax 2.02ay 1.55az 0.08 2.13ay 1.65az 0.06 2.09ay 1.61az 0.06 Arachidonic 0 2.5 5.0 SEM 4.58ax 3.49ay 3.07az 0.12 4.22ax 3.69ay 3.22az 0.11 DHA 0 2.5 5.0 SEM 2.46ax 1.96by 1.77az 0.07 2.39ax 2.06 aby 1.80az 0.07 Linolenic SEM Control 0.07 0.04 0.03 1.79bx VE 2.00ax BHT SEM 1.46ay 1.06az 0.02 1.53ay 1.02az 0.03 0.03 1.54ay 0.03 az 0.99 0.04 0.04 4.21ax 3.58ay 3.26az 0.12 0.10 0.09 0.06 3.10bx 2.27ay 1.62az 0.05 3.15 abx 2.28ay 1.67az 0.04 3.25ay 0.04 2.21ax 0.05 1.69az 0.04 0.07 2.45ax 2.13ay 1.83az 0.08 0.07 0.04 0.05 1.53bx 0.98ay 0.62az 0.06 1.82ax 0.97ay 0.77az 0.05 1.92ax 0.05 0.97ay 0.04 0.75az 0.04 0.04 1.99ax a,bDifferent letters within a row of the same storage time are different (P < 0.05). n = 6. x- zDifferent letters within a column of the same irradiation dosage are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 ºC) with relative humidity of 73%. Abbreviation: DHA, docosahexaenoic acid Table 6—The content of COPs (g/g) in vacuum-packaged yolk powder after electron-beam irradiation and 90 d of storage1 Dose (kGy) 0-d storage COPs Control 90-d storage VE BHT SEM Control VE BHT SEM 0 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 2a 5a 0a 2a 1a 1a 2a 4a 0a 3a 0a 0a 1a 4a 0a 2a 0a 0a 1.1 0.6 0.0 0.7 0.2 0.0 442a 97a 0a 49a 0a 28a 142b 73b 0a 22c 0a 14b 117b 64b 0a 32b 0a 0a 10.7 4.8 0.0 3.3 0.0 4.4 2.5 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 85a 57a 13b 93a 21a 41a 49b 36b 15a 78a 11b 84b 54b 49a 16a 66a 10b 42b 2.7 2.7 1.2 8.6 2.1 4.5 522a 121a 2a 134a 0a 168a 430b 94b 0a 48b 0a 142a 475ab 82b 1a 40b 0a 59b 20.1 4.0 0.4 12.3 0.0 12.9 5.0 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 108a 74a 12b 122a 12a 139a 89b 59b 20a 112a 15a 91b 104a 60b 23a 139a 15a 71b 3.7 4.0 2.8 7.4 1.3 7.4 516a 199a 17b 176a 0a 196a 499a 76c 0b 100b 0a 217a 557a 114b 87a 61b 0a 179a 18.0 5.6 14.6 17.7 0.0 21.4 a-cDifferent letters within a row of the same storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 ºC) with relative humidity of 73%. Abbreviation: COPs, cholesterol oxidation products Table 7—The content of COPs (g/g) in aerobic-packaged yolk powder after electron-beam irradiation and 90 d of storage1 Dose (kGy) 0-d storage 90-d storage COPs Control VE BHT 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 10a 5b 4b 18a 1a 10a 0b 4a 10a 2a 9a 3b 1b 12a 0a 10a 12a 0b 1.5 1.0 1.0 2.7 1.6 1.0 2.5 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 184a 110a 23a 187a 27a 119a 114b 81b 23a 152b 7b 85b 148ab 73b 20a 162ab 26a 110a 5.0 7␣-hydroxycholesterol 7-hydroxycholesterol 25-hydroxycholesterol ␣- plus -epoxide 6-ketocholesterol 7-ketocholesterol 390a 135a 43a 240a 45a 369a 380a 112b 34b 169b 27b 187c 365a 90c 46a 196ab 23b 266b 0 SEM Control VE BHT SEM 383b 545a 105a 3a 41a 0a 38a 86b 0a 62b 0a 52a 50.6 4.6 2.1 4.9 0.0 9.8 18.4 5.0 2.3 18.8 4.5 5.5 1,503 a 1,249a 1,470 a 73a 67a 60a 0a 1a 0a 74b 58b 57a 0a 0a 0a 303a 180b 194b 116.0 15.6 0.4 14.7 0.0 20.5 27.7 6.9 1.7 15.4 3.1 14.7 1,994 a 1,790b 1,859 b 160a 157a 70b 51a 9b 10b 215a 217a 220a 0a 0a 0a 514a 390b 270c 32.6 5.9 8.0 22.1 0.0 14.8 653a 95ab 3a 41a 0a 36a a-cDifferent letters within a row of the same storage time are different (P < 0.05). n = 6. 1Samples were stored at room temperature (22 ºC) with relative humidity of 73%. Abbreviation: COPs, cholesterol oxidation products Vol. 65, No. 4, 2000—JOURNAL OF FOOD SCIENCE 627 FoodChemistryandToxicology destruction of pigments both by irradiation and storage. Fatty-acid composition of egg yolk powder indicated that irradiation significantly increased the oxidation of PUFAs (Tables 4 and 5). In vacuum-packaging at d 0, docosahexaenoic-acid (DHA) content of egg yolk powder decreased from 2.48% of total egg yolk lipid to 1.96% after 2.5 kGy and 1.82% after 5.0 kGy irradiation. In aerobic-packaging at d 0, DHA content of egg yolk powder decreased to 1.96% and 1.77% of total lipid content after 2.5 and 5.0 kGy of irradiation, respectively. The contents of arachidonic and linolenic acid also decreased after irradiation, with a greater decrease after 5 kGy than 2.5 kGy irradiation (Table 4). Tukey grouping analysis showed that there was significant difference between vacuum- and aerobicpackaging. The decrease of PUFAs was smaller in vacuum-packaging compared with aerobic-packaging because the presence of oxygen could stimulate oxidative changes in irradiated yolk powder. During storage, the PUFAs were further decreased with increasing storage time (Tables 4 and 5). After 90 d of storage, the content of DHA in egg yolk powder irradiated at 5.0 kGy was reduced to 1/3 of its original content to about 1/2 for the 2.5 kGy group and to about 1/4 for nonirradiated group. The reason for this accelerated lipid oxidation could be the free radicals formed by irradiation. Irradiation induced the build-up of lipid hydroperoxides, which could further accelerate lipid oxidation (Branka and others 1992) in egg yolk powder. The concentrations of the 3 selected PUFAs in the control, VE- and BHT-treated egg yolk powders showed that antioxidant treatments prevented the loss of PUFAs in nonirradiated powder but could not prevent the loss of PUFAs induced by irradiation (Tables 4 and 5). Several kinds of COPs were formed in egg yolk powder after irradiation, but significantly more COPs (p < 0.05, data not shown) were formed after 90 d of storage (Tables 6 and 7). The formation of COPs was positively correlated to irradiation dosage. Data analysis (data not showed) indicated that there were significant differences between vacuum- and aerobicpackaging in total amount of COPS formed both immediately after irradiation and after storage. Vacuum-packaging reduced irradiation-induced oxidation of cholesterol in egg yolk powder as with color and fatty-acid composition changes. The composition of COPs in egg yolk powder was also changed during storage; immediately after irradiation, 7␣- and 7-hydroxycholesterol, ␣- and - epoxides, and 7-ketocholesterol were the main COPs formed. This result was the same as previ- Antioxidants and Packaging on the Quality of Irradiated Yolk Powder . . . FoodChemistryandToxicology ous reports (Lebovics and others 1992; Paniangvait and others 1995). After 3 mo of storage, the amounts of 7␣-hydroxycholesterol and 7-ketocholesterol were increased. Tocopherol or BHT treatment significantly reduced the formation of COPs in nonirradiated egg yolk powder during storage but had no effect in irradiated groups. More COPS were formed in egg yolk powder after irradiation and storage. Irradiation also increased the oxidation of PUFAs and pigments in yolk. Antioxidants (VE and BHT) were not effective in preventing chemical changes (lipid and cho- Materials and Methods Sample preparation Three different batches of fresh liquid egg yolk (solid content, 49%) were obtained from a commercial egg processor. Each batch was separated into 3 groups, respectively, and an antioxidant was added (none, 0.01% BHT, or 0.01% vitamin E) before spray-drying (APV Crepaco, Inc., Dryer Division, Attleborofalls, Mass., U.S.A.). This level of antioxidant (0.01%) was selected because 0.02% BHT can prevent oxidative changes in food. The level of antioxidant in egg yolk powder becomes approximately 0.02% after drying because the solid content in liquid egg is 49%. The inlet temperature of the spray dryer was set at 185 °C, and the exhaust temperature was maintained at 85 °C by adjusting the flow rate of liquid yolk to the atomizer. Dried egg yolk powder was either vacuum-packaged in oxygen-impermeable bags (9.3 mL O2 /m2 /24 h at 0 °C, clear; Koch) or aerobic-packaged in oxygenpermeable plastic clear bags and stored at 4 °C before irradiation. Samples packaged in bags were irradiated using the Linear Accelerator Facility (Circle III Linear Electron Accelerator, Thomson CSF Linac, Saint-Aubin, France) at 0, 2.5, or 5 kGy of average absorbed dose (true absorbed doses were measured by an alanine dosimetry system). After irradiation, samples were stored at room temperature (22 °C, dark with relative humidity of 73%) for 3 mo. Samples were prepared 3 times (3 replications) at intervals of 2 to 3 mo. Fatty-acid composition, cholesterol oxides, and the color of irradiated egg yolk powders were determined at 0, 45, and 90 d of storage. The color of egg yolk powder was measured using a Hunter LabScan Colorimeter (Hunter Laboratory, Inc., Reston, Va., U.S.A.) and expressed as color L (lightness), a- (redness), and b- (yellowness) values. lesterol oxidation and color changes) caused by irradiation in egg yolk powders but were effective in nonirradiated groups. Compared with aerobic-packaging, vacuum-packaging was effective in preventing adverse changes in egg yolk powders caused by irradiation. Conclusions R ESULTS FROM THIS STUDY SHOWED that the lipids of egg yolk powder were oxidized during storage, and irradiation accelerated the changes greatly. After Lipid extraction Lipids were extracted from egg yolk powder according to the method of Folch and others (1957). Egg yolk powder (3 g), 50 mL BHT (7.2%), and 30 mL Folch I solution (CHCl3 :CH3 OH = 2:1) were added to a 50-mL test tube, which was then capped and mixed well. The homogenate was filtered into a 100-mL graduated cylinder through Whatman no. 1 filter paper (Whatman Inc., Maidstone, England), and the filter paper was rinsed twice with 10 mL Folch I solution. After adding 8 mL of 0.88% NaCl solution, the cylinder was capped with a glass stopper, and the content was mixed. The inside wall of the cylinder was washed twice with 5 mL of Folch 2 solution (3:47:48/ CHCl 3:CH3 OH:H 2O). After phase separation, the top layer (methanol and water) of the solution was completely and carefully siphoned off so as not to contaminate the CHCl3 layer. The bottom organic layer was transferred to a glass scintillation vial and dried in a block heater (1 h at 50 ºC) under a nitrogen stream. The dried lipid material was dissolved with an aliquot of hexane to make 0.2 g fat/ mL hexane, which was used for the next step. Separation of cholesterol oxidation products A silicic acid (100 mesh), celite-545, and CaHPO4 .2H 2O (10:9:1, w/w/w) mixture in chloroform was prepared and packed into a glass column (22 mm X 30 cm with sintered glass frit at the bottom) to a height of 5 cm. The column was washed with 10 mL of hexane:ethyl acetate (9:1 vol/vol; solvent I) before loading the sample. Cholesterol oxides were separated from egg yolk powder by the method of Ahn and others (1999). The lipid sample dissolved in hexane (0.2 g) was loaded onto the silicic-acid column. Neutral lipids, cholesterol, and phos- 628 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 4, 2000 irradiation, high levels of COPs were formed, and PUFAs and pigments were partly destroyed. Thus, pasteurizing egg yolk powder using irradiation is not highly recommended. Addition of antioxidants such as VE and BHT at the 0.01% level significantly reduced the oxidative changes in nonirradiated egg yolk powders but was not effective in protecting quality changes in irradiated egg yolk powders. Vacuumpackaging was more effective in preventing oxidative changes in egg yolk powders than antioxidant treatments. pholipids were eluted by passing 40 mL of solvent I (hexane:ethyl acetate = 9:1, vol/vol) and 40 mL of solvent II (hexane:ethyl acetate:ethyl ether = 4:1:2, vol/vol/vol) through the column. Cholesterol oxides were then eluted with 40 mL of solvent III (acetone:ethyl acetate:methanol=10:10:1, 1 mL/min flow rate) and dried under nitrogen gas. The dried cholesterol oxides were derivatized in a 60 °C water bath for 30 min after adding 50 mL Sylon BFT [99% bis(trimethylsillyl)trifluoroacetamide+1% trimethylchlorosilane; Supelco) and 50 mL pyridine. The derivatized samples were dried under nitrogen, redissolved in 200 L ethyl acetate, and then used for gas chromatographic (GC) analysis. GC analysis of cholesterol oxides Analysis of cholesterol oxides was performed with a gas chromatograph (GC HP 6890; Hewlett Packard Co., Wilmington, Del., U.S.A.) equipped with an autosampler and flame ionization detector (FID). A capillary column (HP-5, 0.25 mm i.d. ∞ 30 m, 0.25 m nominal; Hewlett Packard Co.) was used. A splitless inlet was used to inject samples (0.5 L) into the column, and a ramped oven temperature was used (260 °C held for 2 min, increased to 290 °C at 3 ºC/min, and then kept at 290 °C for 18 min). Temperatures of the inlet and detector were 280 °C. Helium was the carrier gas at a constant flow of 1.1 mL/min. Detector (FID) air, H2, and make-up gas (He) flows were 350 mL/ min, 40mL/min, and 43.9 ml/min, respectively. The peaks were identified by standards (Sigma Chemical Co., St. Louis, Mo., U.S.A.) and also by a Mass Selective (MS) Detector (Model 5973; Hewlett Packard Co.) The GC-MS system was used with the same column conditions as described above. The ionization potential of the MS was 70 eV, and the scan range was 45 to 550. Tentative identification of Fatty-acid composition analysis One mL of methylating reagent (boron-trifluoride methanol, Sigma Chemical Co.) was added to 50 L of lipid extract and incubated in a 90 ºC 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 con- References Ahn DU, Lee JI, Jo C, Sell JL. 1999. Analysis of cholesterol oxides in egg yolk and turkey meat. Poultry Sci 78:10601064. Branka K, Branka M, Dusan R. 1992. Radiation-induced oxidative chemical changes in dehydrated egg products. J Agri Food Chem 40:662-666. 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Arterioscler Thromb Vasc Biol 17:778785. taining methylated fatty acids was analyzed for fatty-acid composition using a GC (HP 6890; Hewlett Packard Co.) A ramped oven temperature (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 inlet and detector were 280 °C. Fatty acids were identified using a Mass Selective (MS) Detector (Model 5973; Hewlett Packard Co.) as described above. The GC-MS was performed using the same column conditions as described above. The area of each peak was integrated using the ChemStation software (Hewlett Packard Co.), and total peak area was used to calculate fatty-acid composition. The fatty acids were reported as a percentage of Lebovics VK, Gaal OE, Somogyi L, Farkas J. 1992. Cholesterol oxides in gamma-irradiated spray-dried egg powder. J Sci Food Agri 60:251-254. Li SX, Ahn DU, Cherian G, Chung TY, Sim JS. 1996. Dietary oils and tocopherol supplementation on cholesterol oxide formation in freeze-dried chicken meat during storage. J Food Lipids 3:27-42. Ma CY, Sahasrabudhe MR, Poste LM, Harwalkar VR, Chamber JR, OÕ Hara KPJ. 1992. Gamma irradiation of shell eggs. Internal and sensory quality, physicochemical characteristics, and functional properties. Can Inst Food Sci Tech J 23:226-232. Narvaiz P, Lescano G, Kairiyama E. 1992. Physicochemical and sensory analyses on egg powder irradiated to inactivate Salmonella and reduce microbial load. J Food Safety 12:263-282. Paniangvait P, King AJ, Jones AD, German BG. 1995. Cholesterol oxidates in foods of animal origin. J Food Sci 60:11591174. Radomyski T, Murano EA, Olson DG, Murano PS. 1994. Elimination of pathogens of significance in food by low-dose irradiation. J Food Prot 57:73-86. SAS. 1989. SAS User’s Guide. 6 th Ed. Cary, N.C.: Statistical Analysis Systems Institute. Serrano LE, Murano EA, Shenoy K, Olson DG. 1997. D values of Salmonella enteritidis isolates and quality attributes of shell eggs and liquid whole eggs treated with irradiation. Poultry Sci 76:202-205. total lipids. Statistical analysis The effects of irradiation and antioxidants, packaging, and storage time on the COPs, color, and fatty-acid composition of egg yolk powder were analyzed by SAS (1989) software. Analysis of variance was conducted to test the difference in various treatments. The Student-Newman-Keul’s multiple range test was used to compare differences among mean values, and the Tukey group test was used to compare the difference between packaging effects. Mean values and standard errors of the mean (SEM) were reported. Values of p < 0.05 were considered statistically significant. Staprans I, Rapp JH, Pan XM, Feingold KR. 1993. The effect of oxidized lipids in the diet on serum lipoprotein peroxides in control and diabetic rats. J Clin Invest 92:638-643. Staprans I, Rapp JH, Pan XM, Kim KY, Feingold KR. 1994. Oxidized lipids in the diet are a source of oxidized lipids in chylomicrons of human serum. Arterioscler Thromb Vasc Biol 14:1900-1905. Staprans I, Pan XM, Rapp JH. 1998. Oxidized cholesterol in the diet accelerates the development of aortic atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol 18:977-983. Vine DF, Croft KD, Beilin LJ, Mamo JC. 1997. Absorption of dietary cholesterol oxidation products and incorporation into rat lymph chylomicrons. Lipids 32:887-893. Wong YC, Herald TJ, Hachmeister KA. 1995. Comparison between irradiation and thermally pasteurized liquid egg white on functional, physical, and microbiological properties. Poultry Sci 75:803-808. Journal paper J-18531 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011-3150. Project No. 3322, and supported by the Iowa Egg Council. Authors are with the Department of Animal Science, Iowa State University, Ames, Iowa 500113150, U.S.A. Direct correspondence to D.U. Ahn (E-mail: duahn@iastate.edu). Vol. 65, No. 4, 2000—JOURNAL OF FOOD SCIENCE 629 FoodChemistryandToxicology COPs was achieved by comparing mass spectral data with those of the Wiley library (Hewlett Packard Co.) The area of each peak was integrated using ChemStation software (Hewlett Packard Co.), and the amount of COPs was calculated using an internal standard.