:. U ,. _ 3 . -. .‘I ". 3'13; 3; 4 f1 . 4 ‘ _ . 4; . ‘ . . . .. . . : . - J>r,-.1‘:§_.5~ I, . ,2 L3?) . . A .3 _, 3 . I. . , .7" .. , Ni. .4. ‘éiifi't' ‘ 1.3033.‘ 42 ‘ a ' , » 3 ,1 13.. ”,3 It I “N t 3.3. . i. ’h' _,| 15‘ : - : - “I 3 3 ' 3.933, 3 - . I33 my ‘3...3.7‘~,._;3 ' ,1, ~ g: ,. .. h » ‘9: '- Ir‘ I‘d-‘1" ‘ “ “ 'm ““‘~.§§‘;"73".c'j 5. O J} 3 '* :11; .¢:‘3 - 3‘ W. “'83!" ,,__: -v..d «x3?! ‘: 1-3": .. u a fit“: 13‘: ' WM}? -3 33‘ 3 ‘Rr‘lfw n $53!; '3 .«m- - K.- r I Ll“: 14 3‘ vii: .1. ~ gm. ‘t‘n (TL-“ .--— Maw- '53:." ....__- M”: ‘d ..'e usmlfis 333333 \llllilillllllllllilll 3 1293 02068 62 This is to certify that the thesis entitled Effects of Natural Antioxidants in Reducing Lipid and Cholesterol Oxidation in Irradiated Chicken Breast Meat presented by Keith M. Frosch has been accepted towards fulfillment of the requirements for Masters of Sci. degree in Food Science Major professor Date April 24, 1998 0.7639 MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE I DATE DUE DATE DUE F6832 {3 (206'! 6/01 c:/CIRC/DateDue.p65-p.15 EFFECTS OF NATURAL AN TIOXIDANT 8 IN REDUCING LIPID AND CHOLESTEROL OXIDATION IN IRRADIATED CHICKEN BREAST MEAT By Keith Frosch A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1998 ABSTRACT EFFECTS OF NATURAL ANTIOXIDANTS IN REDUCING LIPID AND CHOLESTEROL OXIDATION IN IRRADIATED CHICKEN BREAST MEAT By Keith F rosch It was postulated that irradiated chicken breast meat was more susceptible to lipid and cholesterol oxidation during refrigerated storage. The objectives of this study were: 1) to compare the use of a standard packaging material to a material enhanced with vitamin E, 2) to determine the effectiveness of dietary supplementation of a-tocopheryl-acetate on controlling oxidation, 3) to determine the effectiveness of the surface application of oleoresin rosemary on controlling oxidation, and 4) to determine if irradiation contributed to the level of oxidation in chicken breast muscle. No beneficial effects of vitamin E in reducing lipid and cholesterol oxidation were observed as a result of the use of a laminate film containing Surlyn-EVA (heat seal layer) incorporated with a-tocopherol, coextruded to high density polyethylene. Dietary supplementation of a—tocopheryl-acetate (200 IU / kg feed) significantly (P<0.05) reduced lipid and cholesterol oxidation in cooked breast muscle samples stored 3 days under retail display conditions. Surface application of oleoresin rosemary, however, did not cause a significant decrease in lipid and cholesterol oxidation levels. Irradiation with 3.0 kGy did not cause any significant (P>0.05) changes in lipid or cholesterol oxidation levels in either the raw or cooked samples. It was concluded, under the conditions used in this study, that: l) HOPE-vitamin E impregnated films and surface application of oleoresin rosemary were not effective in controlling oxidation, 2) dietary supplementation of a—tocopheryl-acetate proved to be an effective means for controlling oxidation, and 3) irradiation at 3.0 kGy did not cause significant oxidation in raw chicken / broiler breasts muscle. This thesis is dedicated to my family, especially to my father and mother, for without their support this would not be possible. iii ACKNOWLEDGMENTS I would like to express my gratitude to my major professor, Dr. Alden Booren, for his knowledge, guidance and support throughout my graduate studies at Michigan State University. I would also like to thank Dr. Theron Downes for his expertise and advice in helping me complete my thesis. A special thank you to Dr. J. Ian Gray for his generosity in allowing me to use his lab, and for his knowledge and support throughout my graduate career. My sincere thanks to Tom F orton for all his help and cheerfirlness. I would also like to express my gratitude to Debbie Beeuwsaert for all of the help she provided me and for going out of her way in doing it. I would like to thank Dr. Dennis Olson and Michael Holtzbaur at Iowa State University for helping me with the irradiation of my samples. Thanks to Dr. Robert Tempelman for his extraordinary support and guidance with SAS and the rest of my statistical analyses. I wish to extend my gratitude to all of my lab mates and friends for their support and encouragement, with a special thanks to Dr. Enayat Gomaa for all of her help and guidance in the lab. Finally, I would like to thank my family for all of their love and support, which allowed me the opportunity to achieve my goals. TABLE OF CONTENTS LIST OF TABLES .......................................................................................... viii LIST OF FIGURES ......................................................................................... x LIST OF APPENDICES ............................ ‘ ...................................................... xi INTRODUCTION ........................................................................................... 1 LITERATURE REVIEW ................................................................................. 3 Lipid Oxidation ..................................................................................... 3 Mechanism of Lipid Oxidation ................................................... 4 Factors Afi‘ecting Lipid Oxidation .............................................. 5 Lipid Oxidation Measurements ................................................... 6 Antioxidants .......................................................................................... 9 Free Radical Terminators ............................................................ 11 Oxygen Scavengers ..................................................................... 12 Chelating Agents ......................................................................... 12 Vitamin E .................................................................................... 12 Rosemary .................................................................................... 15 Cholesterol Oxidation ............................................................................. l7 Irradiation Induced Cholesterol Oxide Products ........................... 21 Irradiation ............................................................................................... 23 Timeline ...................................................................................... 23 Background ................................................................................. 24 Technique and Doshetry ............................................................ 26 Microbiological Efl'ects ............................................................... 27 Irradiation Induced Lipid Oxidation ............................................. 28 CHAPTER 1. Efi‘ects of Packaging Material on Lipid and Cholesterol Oxidation in Raw Chicken Breast Muscle .................................................... 30 Abstract .................................................................................................. 30 Introduction ............................................................................................ 3 1 Materials and Methods ............................................................................ 33 Preparation of chicken breast samples .......................................... 33 Lipid Extraction ........................................................................... 35 Lipid Oxidation Assessment ......................................................... 35 Cholesterol Oxide Standards ........................................................ 36 Cholesterol Oxide Clean-up ......................................................... 37 Cholesterol Oxidation Assessment ............................................... 37 Statistical Analysis ....................................................................... 39 Results and Discussion ............................................................................ 40 Lipid Oxidation ........................................................................... 40 Cholesterol Oxidation .................................................................. 44 Summary ..................................................................................... 61 CHAPTER 2. Effects of Dietary Supplementation of Vitamin E and Surface Application of Oleoresin Rosemary on Lipid and Cholesterol Oxidation in Cooked Broiler Breast Muscle ................................. 62 Abstract .................................................................................................. 62 Introduction ............................................................................................ 63 Materials and Methods ............................................................................ 65 Preparation of Broiler Breast Samples ......................................... 65 Preparation and Assessment for Lipid & Cholesterol Oxidation 68 Statistical Analysis ...................................................................... 69 Results and Discussion ............................................................................ 70 Growth Performance ................................................................... 70 Lipid Oxidation ........................................................................... 73 Cholesterol Oxidation ................................................................. 79 Summary .................................................................................... 99 CONCLUSIONS ............................................................................................... 100 FUTURERESEARCH ................................. 102 APPENDICES .................................................................................................... 104 REFERENCES ................................................................................................... 106 LIST OF TABLES Table 1. Methods used to detect and measure biological lipid oxidation ...................... 8 2. TBARS values for control chicken breasts and chicken breasts treated with a HDPE-vitamin E packaging material ............................................................... 41 3. Concentrations of total cholesterol oxide products in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material ............................................. 45 4. Total cholesterol levels for irradiated chicken breasts packaged with a HDPE-vitamin E packaging material .................................................................. 46 5. Percent oxidation of cholesterol for irradiated chicken breasts packaged with a HDPE-vitamin E packaging material ........................................................ 47 6. Concentrations of 7a-hydroxycholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material ............................................. 49 7. Concentrations of a-epoxide in irradiated chicken breasts packaged in a HDPE- vitamin E packaging material ................................................................................ 52 8. Concentrations of 7 B-hydroxycholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material ............................................. 55 9. Concentrations of B-epoxide in irradiated chicken breasts packaged in a HDPE- vitamin E packaging material .............................................................................. 57 10. Concentrations of 7-ketocholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material ................................................................. 59 11. Diets for control, control irradiated, oleoresin rosemary and oleoresin rosemary irradiated treatments .............................................................................. 66 12. Diets for vitamin E, vitamin E irradiated, vitamin E / oleoresin rosemary and vitamin E / oleoresin rosemary irradiated treatments ..................................... 66 viii 13. Average weights of broilers raised on control or or-tocopheryl acetate- supplemented diets .............................................................................................. 71 14. Growth performance of broilers raised on control or a-tocopheryl acetate- supplemented diets ............................................................................................. 72 15. TBARS values for cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on a-tocopheryl acetate-supplemented diet ........................... 74 16. Total cholesterol oxide products in cooked1 broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet .................... 80 17. Percent orddation of cholesterol in cookedl broiler breasts treated with an OR dip and/or raised on a-tocopheryl acetate-supplemented diet ....................... 82 18. Concentrations of 7-ketocholesterol in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on cr-tocopheryl acetate-supplemented diet .................................................................................................................... 85 19. Concentrations of 7a-hydroxycholesterol in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on or-tocopheryl acetate- supplemented diet ............................................................................................. 88 20. Concentrations of B-epoxide in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on ct-tocopheryl acetate-supplemented diet ................................................................................................................... 90 21. Concentrations of a-epoxide in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on or-tocopheryl acetate-supplemented diet .................................................................................................................. 93 22. Concentrations of 7B-hydroxycholesterol in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on a-tocopheryl acetate- supplemented diet ............................................................................................ 94 23. Concentrations of 20a-hydroxycholesterol in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on or-tocopheryl acetate- supplemented diet ............................................................................................ 96 24. Concentrations of 25-hydroxycholesterol in cooked broiler breasts treated with an oleoresin rosemary dip and / or raised on a-tocopheryl acetate- supplemented diet ............................................................................................ 97 ix LIST OF FIGURES Figure l. Mechanism for malonaldehyde formation ..................................................... 10 2. Cholesterol .................................................................................................. 18 3. Common cholesterol oxidation pathways ..................................................... 20 4. TBARS values for chicken breast meat over 20 days .................................... 43 5. Concentrations of 7a-hydroxycholesterol in raw chicken breast meat over time .................................................................................................................. 5 l 6. Concentrations of a-epoxide in raw chicken breast meat over 16 days ......... 53 7. Concentrations of 7B-hydroxycholesterol in raw chicken breast meat over 16 days ............................................................................................................. 56 8. Concentrations of B-epoxide in raw chicken breast meat over 16 days ............ 58 9. Concentrations of 7-ketocholesterol in raw chicken breast meat over 16 days .................................................................................................................. 60 10. TBARS values for cooked broiler breast meat over first 24 hr .................... 77 11. TBARS values for cooked broiler breast meat treated with dietary supplementation of or-tocpheryl acetate (200 IU / kg feed) over 4 days ................ 78 12. Total cholesterol oxide product values for cooked broiler breast meat over first 24 hr of storage ......................................................................................... 84 13. Concentrations of 7-ketocholesterol in cooked broiler breast meat over 4 days .................................................................................................................. 86 14. Concentrations of 7a-hydroxycholesterol in cooked broiler breast meat over 4 days ...................................................................................................... 89 15. Concentrations of B-epoxide in cooked broiler breast meat over 4 days ..... 91 16. Concentrations of 7 B-hydroxycholesterol in cooked broiler breast meat over 4 days ...................................................................................................... 95 LIST OF APPENDICES Appendix 1. Oleoresin rosemary pickup concentrations of dipped broiler breasts .............. 104 2. Total cholesterol concentrations for cooked broiler breasts treated with an OR dip and/or raised on a vitamin E-supplemented diet ................................... 105 xi INTRODUCTION Lipid and cholesterol oxidation is a major concern in meat products. It can lead to the development of ofl‘ odors and flavors, reduction in shelf-life, loss of quality and a decrease in nutritional value (Kanner et al, 1990). The presence of phospholipids, which contain high levels of unsaturated fatty acids, makes poultry susceptible to oxidation. Moreover, cholesterol is an integral part of the lipid bilayer and is closely associated with membranal phospholipids. Cholesterol, like phospholipid polyunsaturated fatty acids, undergoes oxidation by a free radical mechanism (Morrisey et al., 1995). Due to the closeness of the relationship between lipid and cholesterol, it is important to understand the great susceptibility to oxidation and how to possibly control it in poultry muscle. Oxidation can occur steadily over time or can be induced to occur more rapidly. Oxygen, light, chelating agents and irradiation are all possible contributing factors for inducing lipid oxidation. Irradiation has been shown to induce oxidation; however, the level at which this occurs is still under debate. Katta (1991) suggested that irradiation at a level of 3.0 kGy affected the palmitic and oleic fatty acids of broiler chickens. However, Heath et al ( 1990) and Murano (1995a) concluded that chicken irradiation at similar levels did not result in adverse effects. These discrepancies lead to uncertainty and indicate that firrther research is necessary. Regardless of whether irradiation induces oxidation, lipid and cholesterol oxidation will still occur naturally in meat products. Controlling its possible development is of utmost importance. Lipid and cholesterol oxidation can be effectively controlled through the use of packaging materials and/or antioxidants. The use of vitamin E and antioxidant compounds contained in the extracts from spices can provide an effective means for controlling oxidation. The overall goals of these studies were to: 1) determine if irradiation, at a level of 3.0 kGy, would induce oxidation in chicken / broiler breast muscle, and 2) evaluate the efi‘ectiveness of using natural antioxidants in controlling lipid and cholesterol oxidation in chicken / broiler breast meat. The research was divided into two studies, the first evaluating raw chicken breast muscle and the second evaluating cooked broiler breast muscle. The specific objective of the first study was to examine the efi‘ects of packaging materials in controlling lipid and cholesterol oxidation. The specific objectives for the second study were to: 1) determine the efl‘ects of a dietary supplementation of a- tocopheryl acetate, at a level of 200 IU / kg feed, and 2) determine the effects of a surface application of oleoresin rosemary in controlling lipid and cholesterol oxidation in cooked broiler breast meat. Both studies were designed to firrther examine the efl‘ects of irradiation, at a level of 3.0 kGy, and to determine if detectable levels of oxidation would be induced. LITERATURE REVIEW LIPID OXIDA TION Lipids are one of the principal structural components of all living cells. They play an important role in both the physical and chemical properties of food. They can react with other food constituents, producing both desirable and undesirable efi‘ects. A major concern associated with lipids in food is lipid oxidation. Lipid oxidation is one of the major causes of food spoilage. It can lead to development of off odors and flavors, reduction in shelf-life, loss of acceptability and a significant decrease in the nutritional value of foods (Sheehy et a1, 1993). It is important to identify the products associated with lipid oxidation, as well as the conditions that influencetheir production. A better understanding of the factors afi‘ecting lipid oxidation will help identify possible controls of its detrimental efi‘ects. The basic form of lipid is referred to as a triglyceride, which consists of glycerol attached to three fatty acid side chains. When the fatty acid contains single bonds between carbon atoms along its length, it is referred to as saturated fat. Ifit contains double bonds between carbon atoms, it is referred to as an unsaturated fatty acid. Phospholipids are any lipids that contain phosphoric acid as a mono- or diester. Saturated fatty acids tend to be more stable than unsaturated fatty acids and tend to resist oxidation induced by irradiation (Hackwood, 1991). The oxidation of fats occurs at the double bond sites. The degree of unsaturation also afl'ects the level of oxidation that occurs. The greater number of double bonds in a fatty acid side chain, the easier the removal of a hydrogen atom is. This phenomenon explains why polyunsaturated fatty acids (PUFA) are so susceptible to oxidation. Mechanism of lipid oxidation Lipid oxidation is a fi'ee radical chain reaction that can be described in steps of initiation, propagation and termination. Initiation: RH + Initiator ————> R. + H. Propagation: R. + 02 —-——> R00. R00. + RH ———-) ROOH + R. Termination: R. + R. ---—> R R. + ROO. -—-> ROOR ROO. + ROO. ———-> ROOR The mechanism for initiating oxidation is the removal of a hydrogen atom from a lipid (RH). Highly reactive radicals, such as hydroxyradicals (OIL), frequently attack lipids by abstracting hydrogen. This leaves behind an unpaired electron on the atom to which the hydrogen was originally attached. After the hydrogen is abstracted from the unsaturated lipid, the formation of a lipid alkyl radical (R.) occurs. The lipid alkyl radical will then react with oxygen to produce a peroxyl radical (ROO.). This is referred to as the propagation step. The abstraction of another hydrogen can occur, resulting in the formation of a hydroperoxide (ROOH) and another free alkyl radical capable of continuing the chain reaction. The chain reaction can be stopped by the interaction of free radicals which results in non-initiating and non-propagating products. This is referred to as the termination step. Factors affecting lipid oxidation One of the most important factors involved in the process of lipid oxidation is the source of the primary catalysts that initiate the process. Because lipid oxidation is autocatalytic in nature, determining what causes initiation and further propagation is very important. It has been shown that hydrogen peroxide (H202) plays a major role in the initiation of lipid oxidation. Hydrogen peroxide acts as the precursor for hydroxyl radicals and also as an activator of iron-containing heme proteins and enzymes (Harel and Kanner, 1985). Furthermore, it has also been shown that leukocytes may also initiate lipid oxidation. Kanner and Kinsella (1983) reported that leukocytes produce large amounts of superoxide radicals (02') and hydrogen peroxide, which can induce oxidation. Another major source for promotion of lipid oxidation include iron from hematin and hemeproteins, such as myoglobin, hemoglobin, cytochrome C and peroxidases. This concurs with Love and Pearson (1974), who stated that hemoglobin and other iron porphyrins act as the major pro-oxidants in meat and meat products. Transition metals are also known to be major catalysts of the initiation step. Fe”, for example, will reductively cleave lipid hydroperoxides to highly reactive alkoxyl radicals which in turn will attract a hydrogen atom fi'om lipids to form new lipid radicals (Wettasinghe and Shahidi, 1996). This is referred to as hydrop eroxide-dependent lipid oxidation. Lipid oxidation by heme proteins is thought to occur as a result of homolytic scission of preformed fatty acid hydroperoxides to flee radicals (Tappel, 1962 as cited in Hare] and Kanner, 1985). It is postulated, however, that heme proteins act as catalysts of the propagation step and are not truly initiators of lipid oxidation (Harel and Kanner, 1985). The autooxidation of oxyhemoglobin and oxymyoglobin leads to the formation of metrnyoglobin (Meth) or methemoglobin (MetHb) and 02', which dismutates to H202 (Harel and Kanner, 1985). It was suggested by Harel and Kanner (1985) that oxymyoglobin, which generates H202 during autooxidation, could activate its own molecule. The interaction of Meth with H202 has been reported to produce free radicals, which in turn were found to oxidize a series of phenols (Harel and Kanner, 1985). Furthermore, it has been demonstrated that small amounts of H202, generated continuously, could activate Meth and lipid oxidation more eficiently than incubation of large amounts of H202 with Meth (Harel and Kanner, 1985). In muscle tissues, one of the first steps leading to the initiation of lipid oxidation is the generation of endogenous H202. Harel and Kanner (1985) suggest that this could occur via the superoxide anion or directly. Moreover, these could be the precursors of the first catalysts {hydroxyl radicals and activated Meth (heme proteins)}, which appear to initiate lipid oxidation. Lipid Oxidation Measurements Lipid oxidation can be measured by assessing primary changes that can be described as the loss of reactants such as unsaturated fatty acids or oxygen. The formation of primary lipid oxidation products, such as hydroperoxides, can also be used to assess oxidation. Secondary changes occur when primary products decompose to form stable secondary products. These secondary products can also be used as a measure to determine the extent of lipid oxidation. Secondary changes can be described as the formation of carbonyls, including malonaldehyde, hydrocarbons and fluorescent products (Gray and Monahan, 1992). Halliwell and Chirico (1993) outlined some of the more popular methods to measure the extent of lipid oxidation in biological systems (Table 1). There are many methods available to measure lipid oxidation, however, no one method provides an accurate measure of the whole process. One of the more popular methods is the evaluating of secondary changes via the use of the thiobarbituric acid (TBA) test. Reacting TBA with malonaldehyde to detect rancidity in food has a long history. Kohn and Liversedge (1944) were the first to relate this reaction to oxidative changes. The TBA test is one of the most widely used method for determining changes in oxidative deterioration in muscle foods (Gray and Monahan, 1992). Color development of a red pigment is used to evaluate the extent of oxidation. The intensity of the color complex is a measure of the concentration of malonaldehyde and other products of lipid oxidation that react with TBA (Gray and Pearson, 1987). The formation of thiobarbituric acid-reactive substances (TBARS) can be quantified to represent the extent of lipid oxidation. It is believed that TBA-reactive material is produced in substantial amounts from fatty acids containing three or more double bonds. Dahle et al. ( 1962) suggested that radicals with a double bond B-S to the carbon bearing the peroxy groups cyclize to form peroxides with five-membered rings, which in turn decompose to produce malonaldehyde (Fennema, 1985). Table 1. Methods used to detect and measure biological lipid oxidation.l Method Analysis of fatty acids by GLC or HPLC Oxygen electrode Iodine liberation Heme degradation of peroxides Glutathione peroxidase Cyclooxygenase GC-mass spectrometry Spin trapping Hydrocarbon gases Light emission Fluorescmce TBA test GC-HPLC-antibody techniques Diene conjugation Measures Loss of unsaturated fatty acids Uptake Oz by carbon centered radicals Lipid peroxides Lipid peroxides Lipid peroxides Lipid peroxides Lipid peroxides and aldehydes Intermediate radicals Pentane and ethane Excited carbonyls, singlet oxygen Aldehydes TBA-reactive materials (TBARS) Cytotoxic aldehydes Diane-conjugated structures lHalliwell and Chirico (1993) This phenomenon is shown in Figure 1. It has also been suggested by Pryor et al (1976), that malonaldehyde may also be produced as a result of the decomposition of prostaglandin-like endoperoxides formed during the autooxidation of polyunsaturated fatty acids (Fennema, 1985). As indicated in Table 1, there are many techniques to assess lipid oxidation. Other popular methods presently being used inchrde hexanal measurement, peroxide value, diene conjugation and sensory evaluation. However, in this study only the TBARS test was used to assess lipid oxidation and will be the only technique discussed in detail. ANT IOXIDAN T S Antioxidants are used to preserve food by suppressing oxidation. Oxidative reactions occur within foods as a result of the removal of electrons from atoms or molecules. This, in turn, can lead to the development of off flavors and odors, discoloration of pigments, loss of texture and loss of nutritional value. Oxidation is catalyzed by several factors, including oxygen, light, heat, heavy metals and pigments. Other factors which can lead to oxidation include alkaline conditions and degree of unsaturation. Selecting the proper antioxidant is very important in achieving optimal effects in the desired food. Antioxidants can be classified into three categories, according to their mechanism: (1) free radical terminators, (2) oxygen scavengers and (3) chelating agents. 10 Figure l. Mechanism for malonaldehyde formation 1817 16 15 14 13 12 II 10 9 c-c-c-c-c-c-csc-c.c- o ¢C? c-c-g-c-c - c-c-c- c-c- 0 +0 c- c-cl c-c - sic-c-c-c - Q Q l C-C-C (i-C-C'Z C-C-C-C- prepane O O malonaldehyde H53 N\ H N 9-Cs.2 ‘3' 9‘" H St‘ ‘c" O O N.‘ ’C heat [11‘ I I 0 0'0 11 Free Radical Terminators Free radical terminators work by donating a hydrogen from the phenolic hydroxyl group, which in turn forms stable free radicals which do not initiate nor propagate further oxidation of lipids (Sherwin, 1978). This type of antioxidant works by interrupting the - free radical chain of oxidative reactions. Bolland and ten Have (1947) have demonstrated this phenomenon (Fennema, 1985). Inhibitor: R02° + AH —-——) ROOH + A' Chain Propagation: R02° + RH —————> ROOH + R’ They concluded that antioxidants (AH) inhibit the chain reaction by acting as hydrogen donors or free radical acceptors and that AH reacts primarily with peroxy radicals (ROz') and not with lipid alkyl (R') radicals. This can be seen in the inhibitor step, shown above. Furthermore, they postulated that the basic mechanism behind fi'ee radical termination can be visualized as a competition between the inhibitor reaction and the chain propagation reaction. Hydroperoxide decomposition is the main initiation reaction of lipid oxidation, and hydroperoxide radical recombination is the main termination reaction of lipid oxidation. Free radical terminator antioxidants can prevent the formation of hydroperoxides and accelerate the termination of the chain reaction, making them very effective antioxidants. Some examples of free radical terminators include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone 12 (TBHQ), propyl gallate (PG) and tocopherols. Oxygen Scavengers (Reducing Agents) Oxygen is an essential component needed for lipid oxidation. The elimination of oxygen can help prevent this phenomenon. Oxygen scavengers eliminate the presence of oxygen by transferring hydrogen atoms. Generally, oxygen scavengers are reducing agents, which can react with oxygen. Examples include ascorbic acid, ascorbyl palmitate, sulfites, glucose oxidase and erythorbic acid. Chelating Agents Metals, such as iron and copper, act as pro-oxidants. Chelators form complexes with metals because they contain unshared pairs of electrons in their molecular structure. These resulting complexes are stable and reduce the opportunity for oxidation. Examples of chelating agents include citric acid, ethylenediaminetetraacetic acid (EDTA) and various polyphosphates. .Yita_mi_n_!3. Tocopherols are the most widely distributed antioxidants in nature and exist in seven structures. The most predominant include or, B, y, and 8. Tocopherols exert their maximum effectiveness at relatively low levels. If used at very high concentrations, they may have detrimental efi‘ects, acting as pro-oxidants. Tocopherols are located mainly in cell membranes and protect membrane fatty acids and cholesterol from peroxidative damage caused by highly reactive free radicals such as hydroxyl, peroxyl and superoxide 13 radicals (Ahn et al., 1995). The inclusion of or-tocopherol in the diet of broilers to delay lipid oxidation and extend the shelf-life of meat has been thoroughly investigated. Sklan et a1. (1983 ), Bartov et al (1983) and Sheldon (1984) all reported that dietary supplementation of or- tocopherol increased tissue tocopherol concentrations and decreased levels of lipid oxidation of poultry during storage. Selecting optimal tocopherol concentration is very important. The concentration of tocopherol in poultry meat is dependent upon the amount of tocopherol in the diet, as well as the length of time the broilers are fed the tocopherol supplemented diet (Lin et al., 1989). According to Sheehy et al (1995), concentration in the region of 100 to 200 mg a-tocopheryl acetate / kg feed is suficient to provide significant improvement in the oxidative stability of broiler meat. Alpha tocopherol is the most prominent antioxidant in meat (Dugan, 1976; Watts 1962). Furthermore, according to Bauerfeind (1980), alpha tocopherol is the most active form of vitamin E. It accounts for. almost all the vitamin E activity in foods of animal origin. Vitamin E is a donor antioxidant that reacts with the peroxyl radicals to inhibit the propagation cycle of lipid oxidation. According to Wagner et a1. (1996), vitamin E induces a concentration dependent slowing of the rate of iron-ascorbate stimulated lipid- derived radical production by cells. Furthermore, it was shown that vitamin E acts to slow the oxygen consumption of cells, resulting in a slower rate of oxidation. Vitamin E acts as a chain breaking antioxidant in membranes. The hydrogen atom of the phenolic hydroxyl group on the chromanol ring is easily removed. This leads to the reaction with lipid peroxy and alkoxy radicals by donating the labile hydrogen atom to l4 them Eventually, this leads to the termination of the chain reaction of oxidation by scavenging chain-propagating radicals (Deshpande et al, 1996). Machlin (1991) summarizes the autooxidation and antioxidant reactions involving vitamin E as follows: Initiation (formation of a free radical): RH Reaction of radical with oxygen: R' + 02 Propagation: ROz' + RH Antioxidant Reaction: R0; + E Regeneration: E' + C C' + NADPH E' + 2 GSH GSSG + NADPH Termination: E' + E' E' + R0; —___) ——__._) R0; R' + ROOH E + ROOH E+C° C +NADP E + GSSG 2 GSH + NADP E--E (dimer) EOOR Where R' = fatty acid radical, ROz' = peroxy radical, RH = fatty acid, E = tocopherol, E' = tocopheroxy radical, ROOH = hydroperoxide, C = ascorbic acid, C' = ascorbyl radical, GSH = reduced glutathione and GSSG = glutathione disulfide (oxidized GSH). 15 The tocopheroxy radical, which is formed during this reaction, is poorly reactive and unable to attack adjacent fatty acid side chains. The inability to abstract hydrogen from membrane lipids results fi'om the unpaired electron’s (on the oxygen atom) ability to delocalize into an aromatic ring structure. This leads to the increased stability of the tocopheroxy radical and its resulting antioxidant efl‘ects (Deshpande et al., 1996). Rosemag Rosemary (Rosmarinus oflicr‘nalis L.) is a naturally occurring spice with antioxidant properties. Approximately 90% of the antioxidant activity of rosemary can be attributed to camosol, a C20 isoprenoid with a phenolic structure. Other efl‘ective antioxidant components of rosemary include camosic acid (CA), rosmanol, rosmaridiphenol and rosmariquinone (Jadhav et al., 1996). Constituents of rosemary, such as camosal, rosmanol, rosmaridiphenol and rosmariquinone, function as free radical scavengers similar to BHA and BHT (St. Angelo et al., 1990). It has been shown that rosemary extracts have antioxidant properties greater than BHA and equal to BHT in turkey products (MacNeil et al., 1973). It has also been reported that oleoresin rosemary was comparable to a commercial blend of BHA / BHT/ citric acid in suppressing lipid oxidation in turkey sausage (Barbut et al, 1985). More specifically, camosal, the predominant active antioxidant compound found in rosemary, was isolated and shown to have effective antioxidant properties (Breiskom et al., 1964; Wu et aL, 1982). Furthermore, Nakatani and Inatani (1981) demonstrated that rosmanol, a phenolic diterpene, contained antioxidant activity to be similar to that of l6 camosal. Like other rosemary extracts, rosmaridiphenol, another diphenolic diterpene, has been shown to possess efiective antioxidant properties. Houlihan et a1. (1984) reported its effectiveness to be superior to BHA and approached the effectiveness of BHT . Furthermore, the antioxidant activity of pure CA was compared to BHT, BHA and TBHQ. It was determined that the antioxidant activity of CA was several times greater than BHT and BHA, but less effective than TBHQ (Chen et al., 1992; Richheimer et al., 1996) It has also been determined that rosemary is an effective antioxidant when used in conjimction with other compounds. Rosemary has been shown to have positive synergistic effects when combined with alpha tocopherol. Wada and Fang (1992) reported that an alpha tocopherol - rosemary mixture (0.05% + 0.02%) in sardine oil model system and frozen-crushed fish meat had stronger antioxidant activity than either one had alone. Spices are excellent sources for natural antioxidants. As stated above, rosemary has excellent antioxidant activity due to compounds such as camosal, rosmaridiphenol, rosmaric acid, carsonic acid and rosmanol. It is not, however, the only spice with such properties. Sage (Salvia officinalis L.) also contains similar compounds. The nature of this study, however, dealt only with the use of one spice, rosemary. Because of this, rosemary will be the only spice discussed in detail. l7 CHOLESTEROL OXIDA T ION Cholesterol is an essential part of the structure of all cell membranes and is necessary for the maintenance of life. Cholesterol molecules are an integral part of the lipid bilayer of cell membranes and are closely associated with membranal phospholipids (Morrisey et al, 1994). This relationship is important in understanding the mechanism by which cholesterol oxidizes. Like PUFAs, cholesterol contains unsaturated bonds (Figure 2). It is, therefore, prone to oxidation, and like PUFAs undergoes oxidation by a fiee radical mechanism (Morrisey et al., 1995). According to Paniangvait et al (1995), cholesterol oxidation should proceed in a way analogous to fatty acid oxidation. Cholesterol oxidation may be intermolecular or intramolecular. Intermolecular oxidation occurs as a result of hydrogen being extracted from cholesterol by peroxy or polyunsaturated fatty acids (phospholipids) in the membrane. Intramolecular oxidation results from the attack of the cholesteryl portion of the cholesteryl ester molecule by the oxidized fatty acyl portion of the same molecule (Paniangvait et al, 1995). It has been reported that the C7 position of cholesterol is the most sensitive to molecular attack by oxygen (Kumar and Singhal, 1991; Nourooz-Zadeh and Appelqvist, 1989). According to Maerker (1987) this results because the allylic C4 and C7 atoms are in the plane of the A5 double bond (Figure 2). However, the influence of the C3 hydroxyl group and the tertiary C5 atom protects the C4 atom fiom oxidative attack. This leaves the C7 atom most susceptible to oxidation, and results in the formation of A and B ring oxidation products. 18 Figure 2 - Cholesterol 19 Cholesterol is an important constituent in food products and is often implicated with averse biological activities, such as atherosclerosis and coronary heart disease (Kumar and Singhal, 1991). Furthermore, cholesterol oxide products (COP’s) have also been shown to contribute to adverse human health efiects. Eight common COP’s have been identified, as follows: cholesta-5-ene-3B,7A-diol(7a-hydroxycholesterol), 5,6-B- epoxy-SB-cholestan-3B—ol (B-epoxide), 5,6-a-epoxy-5a-cholestan-3B-ol (or-epoxide), cholesta-S-ene-3 B,7B-diol (7 B-hydroxycholesterol), cholestane-BB, Sor,6B-triol (cholestane—triol), 3B-hydroxycholest—5-ene—7-one (7-ketocholesterol) and cholest-S-ene- 3B,25-diol(25-hydroxycholesterol). The pathways of these eight COP’s are presented in Figure 3 (Paniangvait et al., 1995). The abstraction of allylic C-7 hydrogen will result in the formation of C-7 COP’s (7A-hydroxycholesterol, 7B-hydroxycholesterol and 7- ketocholesterol). The formation of the A-epoxide and B-epoxide result from the attack of cholesterol 7 hydroperoxide on the 5,6 double bond of cholesterol. These are referred to as secondary oxidation products. The formation of 25-hydroxycholesterol results from the oxidation of the side chain molecules of cholesterol. Moreover, the oxidation of 7- ketocholesterol results in the formation of cholestanetriol. As stated above, it has been suggested that cholesterol will undergo oxidation via free radical processes, in the same manner as polyunsaturated fatty acids (PUFAs) and their esters (Park and Addis, 1987). Furthermore, any events which may cause the formation of free radicals, are likely to initiate the oxidation of cholesterol (Smith, 1980). Because PUFAs are more likely tobe susceptible to oxidation than cholesterol, it was 20 IO Benin 83.38 £233 eufiefineeamé IO . O: envenoums . O. 0.. % Sbflfloflzxoaxm. ck. Banshee 0... x00 0: €R0bee.hmoh 0. No +- é .m .868 8X 01 93>»an eomumemxo Baggage 58800 - m oSwE 21 suggested that hydroperoxides formed fiom PUFAs might afi‘ect oxidation of cholesterol present in the same meat sample (Park and Addis, 1987). Irradiation-induced Cholesterol Oxide Products The formation of cholesterol oxides may also occur as a result of exposure to irradiation. The nature of these products is similar to those formed by autooxidation, however, the relative amounts formed by these two processes is substantially different (Maerker and Jones, 1992). According to Maerker and Jones (1992), the relatively stable derivatives of cholesterol autooxidation are the 7-hydroperoxycholesterols. Furthermore, the 7-keto and 7-hydroxy products are derived from the 7-hydr0peroxycholesterols intramolecularly and the 5,6-epoxides intermolecularly. By contrast, 7- hydroperoxycholesterol intermediates do not seem to be involved in the formation of cholesterol oxides fiom irradiated cholesterol The reason for this phenomenon may be a result of differences in radiolytic stability of the various products formed. The lack of 7-hydroperoxycholesterol intermediates may prove to be important by providing a possible marker for detection of irradiated materials. The use of 6- ketocholestanol, as an internal standard in gas chromatography (G—C) analysis of cholesterol oxide products, has occurred because it is not formed in the autooxidation of cholesterol However, it has been reported that the generation of 6-ketocholestanol has occurred as a result of irradiation-induced oxidation of cholesterol. According to Maerker and Jones (1992), 6-ketocholestanol was found to be a direct radiolysis product of the B- epoxide, and to a lesser extent, a-epoxide. This compound is otherwise not known to 22 occur in cholesterol containing foods, which leads to the possibility of its use as a potential marker for irradiated foods. Another potential source for a marker of irradiated foods is 7-ketocholestanol. According to Maerker and Jones (1992), irradiation of the isomeric 7-hydroxycholesterols causes the formation of 7-ketocholesterol as well as 7-ketocholestanol. The generation of 7-ketocholestanol was shown to have two sources of production, 7-hydroxycholesterol and 7-ketocholesterol The presence of 7-ketocholestanol is not known to occur in foods and is not formed in the autooxidation of cholesterol This also leads to the possibility for its use as a potential marker for irradiated foods. It was determined by Hwang and Maerker (1993) that three major cholesterol oxidation products, B.epoxide, a-epoxide and 7-ketocholesterol, increased substantially when irradiated (lOkGy). Furthermore, Maerker and Jones (1992) reported that the two epoxides (a and [3) gave rise to substantial amounts of trial when they were irradiated. Cholesterol was shown to produce6-ketocholestanol and 7-ketocholestanol upon irradiation. The production of these various compounds is an important aspect in determining the oxidation of cholesterol, whether it is the detection of compounds expected to be present or the use of compounds as potential markers for irradiation. Depending on which compounds arise and the levels at which they are detected, a better understanding of oxidation of cholesterol can be achieved. 23 IRRADIA TION M The concept of food irradiation has been around for almost a century, however, it is still considered a new technology. It was in 1896 when it was first suggested that ionizing radiation could be used to kill microorganisms in food. In 1921, this technology was first utilized by Schwartz who obtained a US. Patent on the use of x-rays to eliminate T richinella spiralis in pork (Hackwood, 1991). However, due to the extremely high costs of ionization sources, widespread commercial use of this technology was not feasible. In the 1950’s, a strong push for finding usefirl purposes for radioisotopes and radiation took place as a result of President Eisenhower’s “Atoms for Peace” program This provided an excellent opportunity for low cost ionization sources. However, the stigma attached to these sources prevented them from being utilized. As a result, extensive testing was conducted over the next two decades to determine the safety and wholesomeness of food irradiation. In 1965, the Surgeon General of the United States Army concluded that foods irradiated with doses up to 56 kiloGrays (kGy) were safe for human consumption. In 1970 a Joint Expert Committee was created consisting of members from the United Nations’ Food and Agriculture Organization (FAO), the International Atomic Energy Agency (IAEA) and the World Health Organization (WHO). This committee evaluated hundreds of studies which were conducted during the 1960’s and 70’s. According to Satin ( 1996), at the conclusion of these studies, the Joint Expert Committee assessed the 24 data and in 1980 stated that: “The irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard; hence toxicological testing of foods so treated is no longer required.” This conclusion was significant because it stated that irradiated foods were no different than foods processed under other ‘traditional” methods. Furthermore, food irradiation is the most extensively investigated food technology, and many of these “traditional” methods have safety standards nowhere near those established for food irradiation. In 1992, after extensively reviewing all of the existing data, the World Health Organization concluded that: Irradiated food produced under established Good Manufacturing Practices is safe Irradiation will not introduce changes in the composition of food which, from a toxicological point of view, would impose an adverse efl’ect on human health, will not introduce changes in the microflora of the food which would increase the microbiological risk to the consumer, will not introduce nutrient losses which would impose an adverse efl‘ect on the nutritional status of individuals or populations (Hayes et al., 1995). In 1993, the American Medical Association’s Council of Scientific Afl‘airs afirmed food irradiation as “a safe and effective process that increases the safety of food when applied according to governing regulations” (Hayes et al, 1995). Today, food irradiation is recognized as another method of food preservation. It is used in a limited capacity worldwide but ensures the safety and wholesomeness of the food which utilizes its technology. Backggound In order to understand how irradiation works, it is important to understand the terms radiate and irradiate. Radiate refers to the physical phenomenon in which energy 25 travels through space or matter, such as food. Irradiate refers to either emosure to, or illumination by, rays or waves of all types. It is also important to understand the relationship between these two terms. The term ionizing radiation is used to descnhe electromagnetic energy which lead to the formation of charged moieties (Glidewell et al., 1993). The principal primary reaction in food irradiation is that of ionizing radiation with water. Formation of the primary radical species occurs via the following reactions (Goodman et al, 1989 as cited in Glidewell et al, 1993): H20* _( —) H20+ + 8.“. I'le"I ----——) OH. + H. H20+ + H20 —-—'--) I'I3()+ + OH. 02 + e-” -———) 02- o 202' ' + 2“ ---—-—> H202 + 02 02° '4' H202 ----) 02 + OH. + OH‘ Where HzO“ represents a water molecule in an excited state. Molecular oxygen dissolved in water may react with hydrated electrons (e'... ) to form a superoxide radical (02' '). This can, in turn, produce hydrogen peroxide (H202). The iron catalysed Haber-Weiss reaction can then act as a further source of hydroxyl radicals (O; + OH' + OH') (Glidewell et al, 1993) Ionizing radiation, also referred to as irradiation, contains energy at levels which 26 cause the ejection of electrons from their orbitals. This, in turn, results in the formation of charged, or ionized particles. The types of radiation that can induce ionization include gamma rays and x-rays. Likewise, electrons can be accelerated to high speeds to achieve high energy levels needed to induce ionization. The primary changes in irradiated materials are the creation of ions and radicals. High-energy radiation causes the ejection of electrons from chemical bonds. These ‘fast’ electrons are slowed during their passage through the food by interactions with the component molecules to form positively and negatively charged ions. These ions decompose very rapidly to form free radicals. Radicals formed as a result of irradiation react with other molecules in food to form other radicals (Glidewell et al, 1993 ). Technigue and Dosimetly The two major techniques used to irradiate food inchrde the use of gamma rays and accelerated electrons. Gamma rays are produced through the decay of certain :37. Moreover, high-energy electrons are produced isotopes such as Cobalt‘50 and Cesium by accelerating electrons to achieve a higher energy level via linear accelerators or Van de Graafi' generators. Both sources provide equally efl’ective ways to achieve an ionized state. In order to achieve optimal results fi‘om irradiation, selecting the proper dose is of utmost importance. The application of irradiation doses can be divided into three distinct categories: high doses(>10 kGy), medium doses (1-10 kGy) and low doses (<1 kGy). In the International System of Units, radiation is measured in Grays (Gy). One Gray is equal 27 to the absorption of one joule of energy per kilogram of food. Irradiation units are usually expressed in kiloGrays (kGy) or 1000 Gy. Depending upon the desired result, it is important to realize that each of the various levels of irradiation produce diiferent end results in the product being irradiated. The application of low doses is referred to as radurization. This can be defined as the treatment of foods with a dose of ionizing radiation suficient to improve shelflife by reducing substantial quantities of spoilage microorganisms. Medium doses can be descn’bed as the treatment of foods with a dose of ionizing radiation suficient to reduce the level of non-spore-forming pathogens, including parasites, to an undetectable level This is referred to as radicidation. Lastly, the application of high doses in the treatment of foods with a dose of radiation suficient to reduce the level of microorganisms to the point of sterility this is referred to as radappertization. Microbiological Effects Pathogens, such as Eschericia coli 0157:H7, Listeria monocytogenes, Salmonellae spp. and Staphylococcus aureus, can be eliminated or decreased significantly in number by treatment with pasteurizing ionizing radiation doses between 1.5 kGy and 10 kGy (Thayer et al, 1996). Prachasitthisakdi (1984) reported that a 4 kGy dose or less was suflicient to inactivate disease causing organisms in poultry (WHO, 1994). The primary mechanism in which irradiation destroys microorganisms is by breaking bonds on the DNA molecule, rendering the cells unable to replicate (Hayes et al, 1995). This phenomenon is logarithmic, which is important in predicting the proper dose needed to 28 destroy a certain number of microorganisms in a food. One of the most important factors for utilizing food irradiation is the resulting extension in shelf-life. It has been shown that chicken irradiated at 2.5 kGy resulted in a two-fold shelf-life extension at 4° C (392° F) from 6 days in unirradiated chicken to 15 days in irradiated samples (Varabiofl‘ et al, 1992). Furthermore, Prachasitthisakdi (1984) reported that chicken irradiated at a level of 4 kGy reduced non-spore-forming spoilage bacteria to sufficiently low levels and extended the shelf life of poultry by 1-2 weeks (WHO, 1994). In examining the efi‘ects of irradiation on reducing the number of microorganisms found in the normal flora of chicken, it was shown that 2.0 kGy reduced the microbial load from 10,000 to 100 cells per chicken (Sekhar et al, 1991). Irradiation induced lipid oxidation Irradiation induces lipid oxidation, which can lead to the development of lipid hydroperoxides. According to Murray (1990) the double bonds between certain carbon atoms in long chain fatty acids esterified with glycerol are selectively attacked by some free radicals produced by irradiation. When one oxygen atom is gained, a cyclic epoxide can form as a result of superoxide and hydroxyl radicals. These products are very reactive and can form new addition products that will add an oxygen or nitrogen to either side of the oxygen on the cyclic epoxide. When two linked oxygens are gained at an unsaturated carbon-carbon bond, it is possible for them to remain linked to one of the carbon atoms, thus forming a lipid peroxyl radical. This can in turn react with another carbon-carbon bond to become a lipid hydroperoxide, which can cause a chain reaction (Murray, 1990). 29 It is uncertain, however, the levels of irradiation at which this phenomenon occurs. It was shown that the palmitic and oleic fatty acids of broiler chickens were affected when irradiated at levels to 3.0 kGy (Katta, 1991). However, it was also reported by Heath et al. (1990), that chicken irradiated at levels of l, 2 and 3 kGy showed no observable differences between the irradiated and unirradiated samples (Murano 1995a). Furthermore, according to Murano (1995b) irradiation at doses approved for chicken does not adversely affect the organoleptic quality of poultry. CHAPTER 1 EFFECTS OF A PACKAGING MATERIAL CONTAINING VITAMIN E ON LIPID AND CHOLESTEROL OXIDATION IN IRRADIATED RAW CHICKEN BREAST MUSCLE ABSTRACT The effects of packaging material on controlling lipid and cholesterol oxidation in raw chicken breast muscle were studied by comparing a specialized HDPE-vitamin E material with a control material. It was determined that the effects of the vitamin E in the packaging material used in this study did not result in significant (P>0.05) differences, when compared to the control samples. TBARS values, as well as COPS, values were similar for all raw chicken breast muscle samples. In comparing the samples packaged in the specialized antioxidant material, to those in the control material, it was determined that the values did not change. Determining if irradiation, at a level of 3.0 kGy, caused detectable levels of oxidation, was assessed by comparing irradiated and non-irradiated samples. Irradiation, at a level of 3.0 kGy, did not cause detectable levels of oxidation in any of the samples. The TBARS and COPS values for irradiated samples were not significantly different from the unirradiated samples. It was concluded that HDPE-vitamin E impregnated films were not effective in controlling oxidation under these study conditions and that irradiation at 3.0 kGy would not cause significant oxidation in raw chicken breasts. 3O 31 INTRODUCTION Irradiation is effective in improving the quality and shelf stability of food. One concern, however, is that lipid oxidation can be induced as a result of free radicals introduced by irradiation (Murray, 1990). The extent to which this occurs, though, is still under debate. Determining if changes in oxidation are significant is essential in deciding if utilizing this technology is worthwhile. Irradiation for poultry in the United States is approved at a level of 3.0 kGy (Thayer et al., 1996). Determining the effects of irradiation on lipid and cholesterol oxidation at this level can provide some Valuable insight for practical applications of this technology. Lipid and cholesterol oxidation can be a major concern in meat if not properly controlled. Two effective ways to accomplish this include the use of packaging materials and antioxidants. Efl’ective packaging materials can significantly influence oxidation. However, as shown in a recent study by Ahn et al. (1995), the utilization of antioxidants in combination with packaging can be a more effective method for controlling the onset and extent of oxidation. Vitamin E has been proven to be an effective antioxidant (Lin et al, 1989; Asghar et al, 1990; Monahan et al, 1990; Asghar et al., 1991; Sheehy et al, 1993; Sheehy et al., 1995; Wagner et al., 1996). Its ability to act as a fi'ee radical quencher, as well as its reputation as a ‘natural’ antioxidant, makes it an ideal candidate for use in food products. Its incorporation into the product can be accomplished in different ways. The addition of vitamin E to packaging materials provides a practical way to utilize its activity. This 32 combination can provide a viable means for inhibiting and controlling the onset of oxidation. The objectives of this study include: 1) evaluating the effects of a specialized high density polyethylene (HDPE)-vitamin E packaging material on controlling lipid and cholesterol oxidation in chicken breast muscle, and 2) determining if irradiation, at a level of 3.0 kGy, induces detectable levels of oxidation in cooked broiler breast muscle. 33 MATERIALS AND METHODS Study 1 The effects of irradiation and package type on lipid and cholesterol oxidation were studied in chicken breasts. Each treatment was held fresh at 4-5°C, under fluorescent lights for 20 days. Analyses were done to determine lipid and cholesterol oxidation in the chicken breast meat. Preparation of chicken breast samples Chickens were purchased and humanely slaughtered at the Michigan State University Poultry Teaching and Research Facility. After slaughter, the birds were held at ambient air temperature (IS-17°C) for 60 min until rigor mortis developed. The breasts (pectoralis major and pectoralis minor) were removed, trimmed of excess fat and connective tissue, chilled on ice for approximately 3 hr and vacuum packaged. This study consisted of four treatments, control (C), control irradiated (CI), packaged (P) and packaged irradiated (PI). The packaged treatment refers to the use of a specialized packaging material which consisted of a heat seal layer (Surlyn-Ethelene vinyl acetate) incorporated with or-tocopherol (73 parts per million), coextruded to a high-density polyethylene (I-IDPE) layer (James River Corporation, Cincinnati, OH). The C breast samples were vacuum packaged in polyethylene-laminated nylon (3 mil) pouches (Koch, Kansas City, MO), which have an oxygen transmission rate of 9 ml / mz / 24 hr at 4°C. The samples were then kept frozen at -80°C for 5 days. The P samples were placed into the HDPE-vitamin E pouch, then were placed into vacuum packaging bags, vacuum 34 packaged and stored at - 80°C for 5 days. The CI and PI samples were prepared for storage in the same fashion as their non-irradiated counterparts. After vacuum packaging, the CI and PI samples were frozen and held at - 80°C for 18 hr. At the end of the 18 hr period they were placed in a Styrofoam cooler packed with dry ice. The samples were sent overnight to Ames, Iowa for irradiation using Federal Express and irradiated using an electron accelerator at a dose of 3.0 kGy at the Iowa State University Meat Laboratory. The irradiated samples were returned overnight to MSU in the same manner as described above. Prior to analysis, all samples were thawed for 12 hr at refiigerated conditions (4- 5°C). Breasts were removed from their packages and placed onto polystyrene trays. The trays which contained the control breasts were placed into unsealed vacuum packaging bags and stored under refrigerated conditions (4—5°C). The breasts which were packaged in the HDPE-Vitamin E pouch were removed from the pouches and placed onto polystyrene trays. The trays were then placed into pouches made from the HDPE-Vitamin E material and stored in refiigerated conditions (4-5°C) under fluorescent lights ( 30 fl- candles) to simulate retail holding conditions. Analyses were conducted on days 0, 4, 8, 12, 16 and 20 to assess oxidative stability, as determined by the 2-thiobarbituric acid (TBA) test, and days 0, 8 and 16 to determine formation of cholesterol oxide products (COPS). 35 Lipid extraction Lipids were extracted fiom 10 g sequential cross-sectional samples of chicken breast from each bird. Breast samples were placed into 250 ml Erlenmeyer flasks containing 100 ml hexanezisopropanol (3:2) and 30 ml distilled H20, as described by Fletcher et al (1984). Samples were then homogenized using a Polytron PCU-ll homogenizer for 30 sec on the number 6 setting. The top layer was then separated and any water present was removed using anhydrous sodium sulfate. The solution was passed through Whatman #1 filterpaper (Maidstone, KY) into a 500 ml round bottom flask. The solvent was then evaporated in a Buchi RE- 121 rotoevaporator at 45°C until dryness was attained. The lipid was transferred quantitatively to l dram vials using hexanezbutylatedhydroxytoluene (BHT) (0.005%). Vials were then placed under a Meyer N-Evap analytical evaporator (nitrogen flush) until all traces of solvent were removed. The vial was then capped and placed into frozen storage (-20°C) for 4-6 weeks, until subsequent analyses. Lipid oxidation assessment Lipid oxidation was determined using the 2-thiobarbituric acid (TBA) distillation procedure of Tarladgis et al. (1960), as modified by Crackel et al. (1988). Ten gram samples of finely cut breast meat were added to 100 ml beakerS containing 10 ml of an antioxidant solution (5 % n-propyl gallate, 5 % ethylenedinitrilo tetraacetic acid (EDTA), 20 % ethanol in distilled water). Ten ml distilled water was then added to the sample and homogenized using a polytron for 30 sec on the number 6 setting. The homogenate was 36 then transferred quantitatively to a 500 ml Kjedahl flask using 77.5 ml distilled water. The next step was to add 2.5 ml HCl : H20 (2:1), a small amount of antifoam agent (1 % silicone emulsion in water, Thomas Scientific, Swedesboro, NJ) and 4-5 Boileezer non- volatile granules (Fisher Scientific, Fair Lawn, NJ). The sample was distilled at medium heat until 50 ml of distillate were collected. A 5 m1 aliquot of the distillate was added to 5 ml of TBA reagent (0.2883 g TBA, 100 ml distilled H20) in a test tube and capped. The mixture was vigorously shaken and the test tube was placed into a boiling water bath for 30 min. The test tube was removed fiom the water bath and chilled under cold (12 °C) running tap water for 5 min. The absorbance of the solution was measured at 532 nm with a Bausch and Lomb Spectronic 2000 spectrophotometer (Bausch and Lomb, Rochester, NY). Absorbance was converted to mg malonaldehyde / kg tissue by multiplying by 6.2 (Crackel et al., 1988). Cholesterol Oxide Standards CholeSt-S-en-BB-ol (Cholesterol), cholest-S-ene-3B, 7a-diol (7a- hydroxycholesterol), cholest-S-ene-BB, 7B—diol (7 B-hydroxycholesterol), cholest-S-ene— 3B, 200t-diol (20a-hydroxycholesterol), cholest-S-ene-BB, 25-diol (ZS-hydroxy- cholesterol), cholesten-Sor, 6a-epoxy-3B-ol (or-epoxide), cholesten-SB, 6B-epoxy-3B-ol (Ii-epoxide), 5-cholesten-BB-ol-7-one (7-ketocholesterol) and 5a-cholesten-3B—ol— 6-one (6-ketocholestanol) were the nine cholesterol oxide standards used in this study. They were obtained from Steraloids Inc. (Wilton, NH). All standards, except 6- ketocholestanol, were prepared by accurately weighing 1.5 mg and dissolving it in 1 ml 37 ethyl acetate. The 6-ketocholestanol was prepared by weighing 7.5 mg and dissolving it in 5 ml ethyl acetate. Cholesterol oxide clean-up The clean-up procedure of Park and Addis (1987) was Started by prewetting a silica gel SuplecoClean LC-Si (Supleco Corporation, Bellefonte, PA) sep tube with 5 ml hexane. The extracted lipid was then added quantitatively using a disposable pipette and 5 ml hexane. The lipid sample was then ‘spiked’ with the internal standard by adding 10 ul of 6-ketocholestanol in hexane. The sep tube was then washed with 15 ml of a 95:5 hexane2diethyl ether mixture to elute the triglycerides from the column. To elute the remaining triglycerides and cholesterol, this step was followed by washing with 25 ml of 90:10 hexanezdiethyl ether and 15 ml of 80:20 hexanezdiethyl ether. The cholesterol oxides were eluted ofl” the column into a clean test tube using 10 ml of acetone. The acetone fi'action was evaporated to dryness under nitrogen. The sample was stored in 5 m1 hexane with .005% BHT to ensure stability. Cholesterol Oxidation Assessment Cholesterol oxidation was determined using the method of Monahan et al (1992). The first step in assessing cholesterol oxidation was the preparation of COP derivatives. Bis(trimethylsi1yl)triflouroacetamide (B STFA) + trimethylchlorosilane (TMCS) (99:1) and pyridine (Silyation grade) were obtained from Pierce Chemical Co. (Rockford, IL). The COPS were redissolved in a 50 ul BSTFA/ 50 3.11 pyridine mixture. The mixture was place he cl the seal in eqi II 118 pl 38 placed in the dark at room temperature for 1 hr to form trimethylsilyl ether derivatives of the cholesterol oxides. Alter 1 hr, the ether Sterols were evaporated under nitrogen. The ether Sterols were then redissolved in 100 pl hexane and transferred to a 1/2 dram crimp seal vial Ifnot used immediately, the vial was sealed and placed into a freezer (-20°C) for 1 to 7 hr until subsequent analysis. A Hewlett Packard 5890A gas chromatograph (Hewlett Packard, Avondale, PA), equipped with a flame ionization detector was used to quantify the COPS. Two 311 of TMS COP derivatives were injected and separated on a fused silica capillary column (DB- 1, 15m x .25 mm id, .25 um film thickness, J & W Scientific, Folsom, CA). Helium was used as a carried gas and was delivered at a rate of 1.2 ml / min. The oven was programmed to start at an initial temperature of 170°C and increase to 220°C at a rate of 10°C per minute. The temperature was held isothermally at 220°C for 5 min and was then increased to 234°C at a rate of 0.4°C per min. Next, the temperature raised at 1.5°C per min until a final temperature of 255°C was reached. This temperature was held isothermally for 15 min, creating a total run time of 68.40 min. The injector port temperature was 275°C, while the detector port was 330°C. A Hewlett Packard 3392A integrator was used to integrate the COPS. Retention times from the COPS were matched with retention times fi'om a mixture of standards in order to confirm the presence of each compormd. 39 Statistical Analysis Results were analyzed using analysis of variance in the Mixed Procedure of the Statistical Analysis System (SAS, 1996). A 2x2 factorial design was used with repeated measures over days. A least square means procedure was done to separate means at P<0.05. Twenty four birds were used, using samples from the breast muscle from each bird for analysis. Three replications were done with duplicate breasts analyzed in each treatment. All data underwent a log transformation prior to statistical analysis. Lam Tab the den HE re (“J RESULTS AND DISCUSSION Lipid Oxidation Lipid oxidation was assessed using the TBA procedure and values are presented in Table 2. Irradiated samples were not significantly difi’erent (P>0.05) when compared to the non-irradiated samples. It can be concludedthat irradiation at 3.0 kGy did not induce detectable lipid oxidation in chicken breast meat. This observation agrees with results of Heath et al. (1990) who demonstrated that irradiation at doses of 1, 2 and 3 kGy did not induce detectable levels of oxidation in raw chicken thigh meat. However, contradicting results were presented by Ahn et al (1997) who demonstated that irradiation at a level of 2.5 kGy caused significant (P<0.05) difl‘erences in thiobarbituric acid reactive substances (TBARS) values in ground raw turkey breast patty samples. This discrepancy may be a result of the difference in samples used in the two studies. Meat patty products tend to have higher levels of oxidation compared to whole muscle (Pearson et al, 1983 ). The TBARS values of the breast samples packaged in the vitamin E-HDPE packaging material were not significantly different (P>0.05) than those of the control samples. The lack of effectiveness of the vitamin E packaging material could have been a result of several problems associated with this form of packaging. One reason may be attributed to the interaction between the product and material. This particular material was not a good oxygen barrier and may not have been the most ideal choice for use with a meat product. Reports by Ahn et a1. (1997) suggest that limiting oxidation rates in raw 40 Table w 41 Table 2. TBARS values for irradiated chicken breasts packaged with a I-IDPE-vitamin E packaging material'. mg malonaldehyde / kg meat Day Day Day Day Day Day TRT 0 4 8 12 16 20 C2’3 0.5:0.12 0.5i0.14 07:01] 0.81.037 1 2:06] 1,310.68 C Irr4 0.5:006 0.6i0. l6 0.5i0. 12 0.8i0.37 1.510.60 2.2i1.21 P 0.6i0.10 0.6i0.21 0.6:034 0.9:t0.51 1,110.33 1.2i0.77 P In 0.5i0.17 0.7:002 0.8:029 l.2i0.32 1.9i0.59 2.510.79 ‘ Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Means i standard deviations in the same cohrmn are not significantly different (P>0.05). 4 Irr = Irradiated at a dose of3.0 kGy int pa OE Vi TI 42 l turkey is much more eflective in the absence of oxygen. They reported that patties stored in oxygen-permeable bags oxidized much faster than those stored utilizing vacuum packaging. Furthermore, the antioxidant effects of vitamin B would have been effective only if sufficient migration of the vitamin E from the package to the product occurred. Although the breast meat was not analyzed for vitamin E concentrations, the TBARS values may indicate that the amount of migration was too small to have an impact on oxidation. Because of the lack of significance (P>0.05) related to irradiation and treatment, neither will be discussed in further detail The only effect which indicated any significance among TBARS values was the efl‘ect of time. Figure 4 presents TBARS values for the control samples and HDPE-vitamin E packaged samples over the duration of this study (20 days). Because irradiation did not significantly afl‘ect TBARS values, the means and standard deviations in Figure 4 were calculated by combining all samples within the two treatment groups. Days which were significantly different are represented by an asterisk (*). As indicated in Figure 4, TBARS values increased (P>0.05) steadily over time, beginning after Day 8. This indicates that lipid oxidation was not significant over the first 8 days. This is similar to data reported by Ahn et a1. (1997) who reported that TBARS values in raw turkey breast patties remained unchanged over the first 7 days of storage at 4°C. These results are also similar to the data reported by Sheehy et al. (1993) who reported concentrations of TBARS in raw chicken muscle were low in all groups and increased slowly over a 7-day period. Furthermore, it was reported that mean values did not differ significantly. These findings were attributed to the fact that in raw muscle iron 43 3:39: m EESS E coomxomm 39$ 22.5.. 2282.3.» 2.. 89 E. o 79 N 3 £5 . 30.53: «>3 ca .350 32: 53.5 5:25 .2 ao:_a> 92m... 6 2:9“. .0380 RHNO 3.0 85.0 mm 6). I apmomtuo'w 5m is bound to heme proteins and is compartmentalized from membrane polyunsaturated fatty acids. Consequently, the rate of iron-catalyzed lipid peroxidation was low (Sheehy et al., 1993 ). In this study, the first indication of significant changes (P<0.01) in TBARS values occurred between days 8- 12 and continued to occur thereafier. These results are comparable to those reported by Ahn et al. (1997), who stated that after 14 days of storage at 4°C, TBARS values of raw turkey patties were two times higher than those at O or 7 days. Cholesterol Oxidation Total cholesterol oxide products were calculated as the sum of detectable levels of COPs for each treatment and are presented in Table 3. Total cholesterol levels for the chicken breasts utilized in this study averaged 52.25 mg / 100 g meat (Table 4). Percent oxidation of cholesterol was determined by dividing the total COPS for each treatment by the amount of total cholesterol in that treatment (Table 5). It can be seen that an increase % oxidation occurred over time. This is consistent with Pie et al. (1991) who reported an increase in % oxidation of cholesterol in fiesh beef, veal and pork after 3 months of storage at -20°C. In examining the COP data, it should be noted that a lack of repeatability existed. Each sample was replicated three times, however, the resulting data did not necessarily provide three detectable values. Some replications produced levels which were below the level of detectability (0.1 pg / g meat). Analysis was, however, conducted on these values. The average and standard deviation on the samples which did not produce 45 Table 3. Concentrations of total cholesterol oxide products in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material]. ug / g meat TRT 0 a 16 C2 0.63 2.77 7.59 C 1113 0.88 2.15 1.62 P 1.12 4.25 14.23 P Irr 3.10 7.13 7.55 ' Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 In = Irradiated at a dose of3.0 kGy 46 Table 4. Total cholesterol levels for irradiated chicken breasts packaged with a HDPE- vitamin E packaging material‘. Treatment mg/ 100 g meat Control2 52.0 Control Irradiated3 52.0 Packaged 52.5 Packaged Irradiated 52.5 ' Packaged in a pouch made fi'om a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy Means i standard deviations in the same column are not significantly different (P>0.05). 47 Table 5. Percent oxidation of cholesterol for irradiated chicken breasts packaged with a HDPE-vitamin E packaging materiall. Treatment % Day Day Day TRT 0 8 16 Controlz 0.12 0.53 1.46 Control m3 0.17 0.41 0.31 Packaged 0.22 0.82 g 2.71 Packaged In 0.60 1.36 1.44 ‘ Packaged in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy 48 detectable levels were calculated by using 0.00 for the undetectable values. This information should be noted when assessing the resulting data which are presented in Tables 3, 6-10 and Figures 5-9. The conclusions drawn were done based on the data which were calculated with undetectable values. Cholesterol oxides, including 7a-hydroxycholesterol, a-epoxide, 7 B- hydroxycholesterol, B-epoxide and 7-ketocholesterol are presented in Tables 8-12. The data for all the resulting total COPS indicated that irradiation and the vitamin E packaging treatment had no significant (P>0.05) effect on their formation. As a result, values which are presented in Figures 5-9, were calculated by disregarding treatment and combining the concentrations of all resulting data to form an average daily value, for each compound. The results which indicate that irradiation did not have a significant effect on the formation of COPS supports the findings of Sheehy et al. (1995), who reported that irradiation, at levels of 2.5 kGy and 4.0 kGy, did not influence the concentrations of 25- hydroxycholesterol, 7-ketocholesterol, cholestane triol and total COPS in broiler leg muscle stored for 5 days at refiigerated temperatures. These observations conflict with that of Hwang and Maerker (1993) who reported that irradiation had a significant effect on several COPS (or-epoxide, B-epoxide and 7-ketocholesterol) in raw beef; pork and veal. However, a 10 kGY dose of irradiation was used in the experiments carried out by Hwang and Maerker (1993), 2-4 times the levels used by Sheehy et a1 (1995) and in the current study. The only Signifcant differences observed in this Study (P<0.05), were the effects of time. 49 Table 6. Concentrations of 7a-hydroxycholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material]. pg / g meat Day Day Day TRT 0 8 16 C2 0.22:0.382 l.68i2.037 2.29:0.745 C m3 0.11i0.183 0.67i0.294 0.69:1:1.186 P ND4 l.08i0.430 2.31:0.491 P Irr 0.29:0.502 0.72:0.787 2.03:0.296 ‘ Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy 4ND - Not Detectable - Minimum level of detection equals 0.1 pg / g meat. Means :1: standard deviations in the same cohrmn are not significantly difl‘erent (P>0.05). 50 The results for 7a-hydroxycholesterol are presented in Table 6. A significant difl‘erence exists between days, as indicated in Figure 5. A Significant change (P<0.01) occurred between days 0 and 8, indicating that cholesterol oxidized to 701- hydroxycholesterol over this time. Between days 8 tol6, no significant change occurred between values. This suggests that oxidation has reached a point of leveling off for this compound after day 8 or this compon may fiuther oxidize to form other compounds (epoxides) and thus remain constant. Previous research conducted on cholesterol oxidation which examined the effects of time on raw meat samples (Engeseth and Gray, 1993; Monahan et al, 1992; Hwang and Maerker, 1993) did not report the presence of 7a-hydroxycholesterol. The results for a-epoxide are presented in Table 7. As indicated in Figure 6, a- epoxide followed the same trend as seen for 7a-hydroxycholesterol A significant (P<0.01) change occurred between the amounts of this cholesterol oxide for days 0 to 8, however, not for days 8 through 16. Engeseth and Gray (1993) reported the presence of (at-epoxide in cooked veal muscle after 0 and 4 days of storage at 4°C. In assessing these data, a trend appears to exist for increasing levels of a-epoxide over the 4 day period. Drawing comparisons is difficult because of the differences in meat specie, treatments and storage time. Hwang and Maerker (1993) also reported a significant increase in or- epoxide over time in beef, pork and not for veal; however, values were only reported for :odva. 322% 2.2855.” 26 3 >8 . recurs/6d «>2. 3 .83 52: 33.5 case—co 32 E _o._3uo_o:u>x2>:.d~. no ”coast-.350 .u 959". 52 Table 7. Concentrations of a-epoxide in irradiated chicken breasts packaged in a HDPE- vitamin E packaging material]. pg / g meat Day Day Day TRT 0 8 16 C2 ND 02610.447 05910.511 C m3 ND“ 0.4510073 0.28:|:0.482 P ND 0.5410286 1.0511625 P In ND ND ND ‘ Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy ‘ND - Not Detectable - Minimum level of detection equals 0.1 pg / g meat. Means i Standard deviations in the same column are not significantly different (P>0.05). 53 gay": 522% 268586 26 o «>2. 3 .83 30:. .32.. 53.25 38 5 0233.6 3 acozahcoucoo .o 2:2". 0 £60 .. 54 days 0 and 14. This time span does not allow accurate comparisons of the data in this Study. Concentrations of 7 B-hydroxycholesterol are presented in Table 8. AS indicated in Figure 7, similar trends as seen for 7a-hydroxycholesterol and a-epoxide, hold true for 7 B—hydroxycholesterol. The results provide a good example of data which contains concentrations below the level of detectability. The concentrations of B-epoxide are presented in Table 9. A Significant effect of time was not seen for this compound (Figure 8). It was determined that no significant changes occurred at any point over time. This indicates that no change in oxidation occurred for this compound. One reason for these observations may be attributed to the relative stability of B-epoxide, compared to a-epoxide. The concentration of B-epoxide is approximately 2-3 times greater than a-epoxide. This supports the findings of Moriarity and Maerker (1990) who reported the B-isomer to be 2-3 times the concentration of the a-isomer in an aqueous sodium stearate dispersion, irradiated at a level of 50 kGy. Lai et al. (1995) reported a range of ratios for [3:01 in egg powders of 4:1 to 4.921 afler 3 and 6 months of storage. These ratios may be slightly higher than those reported in this study because of the longer storage time. As indicated in Figure 9, 7-ketocholesterol, on the other hand, showed a significant effect (P<0.01) at day 16. In assessing the results, variation in the values for 7- ketocholesterol existed (Table 10). Two of the treatments obtained detectable values at day 0, then undetectable levels at day 8 and detectable levels again at day 16. As indicated in Table 10, the levels detected for this compound are relatively close to the lower limit of 55 Table 8. Concentrations of 7B-hydroxycholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material]. pg / g meat Day Day Day TRT 0 8 16 C2 ND‘ ND‘ 1.8311601" c In3 ND“ ND3 ND‘ P NDa 1.6611502" 971116.812c P In 1.6112792" 4.8214336" 3.2410734" ' Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy 4ND - Not Detectable - Minimum level of detection equals 0.1 pg / g meat. "° Means i standard deviations in the same column followed by the same letter are not Significantly different (P>0.05). 56 $0.90. 32615 2.18: {an EBm Em w - o 960 . 96v 3 .03 «no... $3.5 :oono 32 E _o.8um_ozu>xo._u>: .nn no 22355050 K 2:9“. reamfilfi'rl 57 Table 9. Concentrations of B-epoxide in irradiated chicken breasts packaged in a HDPE- vitamin E packaging materiall. pg / g meat Day Day Day TRT 0 8 16 C2 04110.220 0.8111126 0.5510617 C In3 0.5910339“ 1.0211027 0.3010515 P 0.2610268 0.9710837 0.7610299 P In 0.6910204 09711.426 0.7610081 1 Packaged (P) in a pouch made from a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy Means :1: standard deviations in the same column are not significantly different (P>0.05). S8 23 u 2. .63 “no... .32.. 53.25 >2: 5 onion? a .0 82353050 .a 2:2“. 59 Table 10. Concentrations of 7-ketocholesterol in irradiated chicken breasts packaged in a HDPE-vitamin E packaging material]. pg/ g meat Day Day Day TRT 0 8 16 C2 ND ND 2.3311656 C In3 01810.314 ND4 0.3610617 P 0.8611498 ND 04110.713 P In 0.5110521 0.6111055 1.5310128 ‘ Packaged (P) in a pouch made fi'om a material consisting of a heat seal layer (Surlyn- Ethylene vinyl acetate) incorporated with alpha-tocopherol, co-extruded to a high-density polyethylene (HDPE) layer. 2 C = Control samples overwrapped using an unsealed plastic bag. 3 Irradiated at a dose of 3.0 kGy 4ND - Not Detectable - Minimum level of detection equals 0.1 pg / g meat. Means i standard deviations in the same column are not significantly different (P>0.05). 60 :3 A 5.9% 286% 1.1.8.1126 26 2 - o :3 . .n B I B m a w. name 3 .26 32: $35 :oquu 32 :. 3.23.05834. .o 32.22350 .a San.“— 61 detection (0.1pg / g meat). The loss of detectable levels for this compound at day 8 may be attributed to the extremely low values associated with this compound. Summary The effects of irradiation and the use of a vitamin E packaging material were investigated in raw chicken breast muscle samples. It was determined that irradiation at a level of 3.0 kGy did not result in increased levels of oxidation. TBARS values and COPS indicated no Significant difl‘erences between the irradiated and non-irradiated samples. Furthermore, the potential beneficial efl‘ects of the vitamin E packaging material were not observed. Lipid and cholesterol oxidation, as measured by TBARS values and COPS, were not Significantly difl‘erent when comparing samples packaged in the control material versus samples packaged in the vitamin E enhanced material. CHAPTER 2 EFFECTS OF DIETARY SUPPLEMENTATION 0F VITAMIN E AND SURFACE APPLICATION OF OLEORESIN ROSEMARY ON LIPID AND CHOLESTEROL OXIDATION IN IRRADIATED COOKED BROILER BREAST MUSCLE ABSTRACT This study was designed to observe the efi‘ect of two treatments in cooked broiler breast muscle; dietary supplementation of a-tocopherol acetate (200 IU / kg feed) and the surface application of oleoresin rosemary (applied by dipping, at a target level of 400 parts per million based upon 70% activity, after breasts were removed fi'om carcass). Dietary supplementation of a-tocopheryl acetate significantly (P<0.01) affected thiobarbituric acid reactive substances (TBARS) values and cholesterol oxide products (COPS) during refiigerated storage. It was determined that 7B—hydroxycholesterol, 2001- hydroxycholesterol and 25-hydroxycholesterol were found in samples from broilers not fed the vitamin E supplemented diet. However, they were not detectable in cooked broiler breasts from broilers fed the vitamin E supplemented diet. The effects of the surface application of oleoresin rosemary were also examined. No Significant (P>0.05) difi‘erences in the TBARS values and COPS of these samples were observed. Irradiation at a dose of 3.0 kGy prior to cooking resulted in no significant (P>0.05) differences in either TBARS values or COPS. It was concluded that vitamin E supplementation was very effective in controlling lipid and cholesterol oxidation in cooked broiler breast meat. 62 63 INTRODUCTION Lipid and cholesterol oxidation are closely associated. Understanding this relationship and the mechanisms involved can help in controlling oxidation. Lipid and cholesterol both undergo oxidation by a free radical mechanism. The application of antioxidants can provide an effective means for controlling both lipid and cholesterol oxidation. Two antioxidants which have been shown to possess excellent potential include vitamin E and oleoresin rosemary. These two antioxidants work by acting as free radical terminators. The inclusion of al.-tocopherol in the diet has a long history in delaying lipid oxidation and extending the shelf-life in meat. Studies have demonstrated the effectiveness of dietary supplementation of vitamin E in broilers (Sheehy et al., 1993; Asghar et al., 1990; Lin et al, 1989), pigs (Buckley et al, 1995; Monahan et al., 1992; Buckley et al., 1989) and veal (Engeseth et al., 1993) on increasing membranal lipid stability. Beneficial effects associated with use of vitamin E include decreased oxidation of lipid and cholesterol, increased shelf-life, and improved quality. The use of Spices to preserve the quality of food has been utilized for centuries. The use of rosemary is becoming increasingly popular for providing the beneficial antioxidant effects associated with its use in meat. Studies have shown the efl‘ectiveness of oleoresin rosemary in poultry (Lai et al., 1991; Barbut et al., 1985), fish (Akhtar, 1996; Wada and Fang, 1992) and pork (Liu et al, 1992). Because of the concern associated with the use of synthetic food additives, the use of naturally occurring compounds is becoming increasingly popular. The application of these antioxidants can provide an effective method for controlling oxidation without the stigma associated with synthetic chemicals. Furthermore, as reported by Wada and Fang (1992) a-tocopherol and rosemary, if used in conjunction with one another, may result in possible synergistic efl‘ects. They reported that a treatment of a-tocopherol-rosemary mixture provided the strongest antioxidant activity of several different antioxidants tested. It was concluded that a synergistic effect between a-tocopherol and rosemary occurred. Irradiation can provide an effective means for improving the quality and shelf stability of food. However, it is uncertain if lipid and cholesterol oxidation can be induced as a result of fiee radicals introduced by irradiation, at a level of 3.0 kGy. The Food and Drug Administration allows a maximum irradiated dose of 3.0 kGy for poultry (Thayer et al., 1996). This level was selected as the treatment dose in this study to provide data which would represent practical applications in the food industry. Determining the efl‘ects of irradiation on lipid and cholesterol oxidation at this level can provide some valuable insight for utilization of this technolgy. The objectives of this study include: 1) evaluating the effectiveness of a dietary treatment of vitamin E and the surface application of oleoresin rosemary dip in controlling lipid and cholesterol oxidation in cooked broiler breast meat, 2) examining the possibility of synergistic efieas between vitamin E and rosemary, and 3) determining if irradiation, at a level of 3.0 kGy, induces detectable levels of oxidation in cooked broiler breast meat during storage. 65 MATERIALS AND METHODS Study 2 Feeding dL-alpha-tocopheryl acetate at 200 IU / kg feed and surface application of oleoresin rosemary (400 parts per million) were investigated in cooked broiler breasts. Analyses were done to determine lipid and cholesterol oxidation of broiler breast meat. Preparation of broiler breast samples Ninety-Six baby chicks were obtained from Hoover Hatchery (Rudd, IA) and divided into 6 pens. The pens were randomly selected to either have a control diet or a or- tocopherol acetate supplemented diet. The feed, mixed at MSU Swine Teaching and Research Facility, was enhanced with dL-alpha-tocopheryl acetate (BASF Corporation, Wyandotte, M1) at a level of 200 IU per kg feed. The respective diets consisted of a Starter and finisher diet. The composition is shown below in Tables 2 and 3. The birds were fed the starter diet for 4 weeks and the finisher diet until the time of Slaughter. Bird weights, feed consumption and growth performance were monitored periodically. An average of seventeen birds per pen were used to determine the bird weights. Dead birds were removed from the pens, and averages were adjusted accordingly. Daily feed consumption represents the average daily feed intake per broiler, for each of the Six pens, during the study. The average daily intake was calculated for each bird over days 0 to 6, 6 to 24 and 24 to 41. Daily weight gain also represents the average, taken from seventeen birds, for each of the six pens. A ratio of feed per gain was calculated by dividing averages over time. After assessing this information, the birds were humanely slaughtered 66 Table 11. Diets for control, control irradiated, oleoresin rosemary and oleoresin rosemary irradiated treatments. Ingredient Starter Diet ' Finisher Diet 1 Corn 53.0 57.6 Soy 44% 35.8 30.15 Meat and Bone 50% 3.0 3.0 Alfalfa 17% --- 3.0 Whey 2. 5 --- Fat 3.0 4.0 Limestone 1.0 .75 Dicalcium phosphate .9 .75 DL Methionine .1 .1 Salt .3 .25 Premix (layer premix)2 .4 .4 I All numbers shown represent percentages 2 Obtained from Dawe’s Laboratories (Ft. Dodge, IA) Table 12. Diets for vitamin E, vitamin E irradiated, vitamin E / oleoresin rosemary and vitamin E / oleoresin rosemary irradiated treatments. Ingredient Starter Diet ' Finisher Diet ' Corn 53.0 57.6 Soy 44% 35.8 30.15 Meat and Bone 50% 3.0 3.0 Alfalfa 17% --- 3.0 Whey 2.5 --- Fat 3.0 4.0 Limestone 1.0 .75 Dicalcium phosphate .9 .75 DL Methionine .l .1 Salt .3 .25 Premix (layer premix)2 .4 .4 Vitamin E (BASF) .04 .04 I All numbers shown represent percentages 2 Obtained from Dawe’s Laboratories (Ft. Dodge, IA) 67 at the end of week six at the MSU Poultry Research and Teaching facility. The carcasses were Skinned, eviscerated and held in a chilled water bath (2-3°C) for 30 min until rigor mortis developed. After chilling, the birds raised on the control diet were randomly assigned to one of four treatment groups, control (C) and control irradiated (CI), oleoresin rosemary (O) and oleoresin rosemary irradiated (OI). The breast muscles were then removed, trimmed of excess fat, connective tissue, and chilled on ice for approximately 3 hr. The C breast samples were then immediately vacuum packaged, fiozen and held at -80°C for 5, 10 or 15 days, until subsequent analyses. The CI breast samples were vacuum packaged, frozen, held at -80°C for 18 hr, then shipped, irradiated at Iowa State University and returned in the same manner as described for the breast samples in Chapter 1. Oleoresin rosemary was applied to both the irradiated and non-irradiated samples. The samples were kept under refiigerated conditions (4-5°C) before and during the application of the dip. Herbalox Seasoning Type W was obtained from Kalsec, Inc. (Kalamazoo, MI) and added to distilled water to attain a 50:1 vzv concentration. The breasts were weighed prior to and after dipping, in order ensure an accurate 3 % pickup of the 2% solution. This was done to achieve an antioxidant activity of 400 parts per million as recommended by the manufacturer (based upon 70% activity). Afler the breasts were dipped and weighed, they were vacuum packaged, frozen and held at -80°C for 18 hr. The breast samples were then prepared for Shipment, shipped, irradiated and returned in the same manner as described above. 68 The birds raised on the a-tocopheryl acetate-enhanced diet were randomly assigned to the remaining four treatment groups, vitamin E (E), vitamin E irradiated (El), vitamin E / oleoresin rosemary (E0) and vitamin E / oleoresin rosemary irradiated (E01). After removal and preparation of the breast, the E samples were handled in the same manner as their control (C) counterparts. Furthermore, the E0 samples were treated and handled in the same manner as the 0 samples. Afler irradiation breast samples were placed in a freezer (-80° C) and held with all other samples for 18 hr. Samples were removed from the freezer, placed in refiigerated conditions (4-5°C) and allowed to thaw for 12 hr, until the time of cooking. Samples were cooked by immersing them into water heated to 200°C using_steam jacketed kettles. They remained in the water bath until an internal temperature of 71°C was achieved. After the desired temperature was reached, samples were remOved fi'om their packages and placed onto polystyrene trays. Samples were wrapped with Sysco Disposables plastic food wrap (Sysco Corporation, Houston, TX) and placed in a cooler (4-5°C), Imder fluorescent light ( 30 ft-candles) to simulate retail conditions. Analyses were conducted at days 0, l, 2 and 3 to determine oxidative stability and changes in cholesterol oxides. Preparation and assessment for lipid and cholesterol oxidation Lipid extraction, lipid oxidation assessment, cholesterol oxide standards, cholesterol oxide clean-up, and cholesterol oxidation assessment were performed in the same manner as described in Study 1. The only exception was the determination of 25- hydroxycholesterol The method which was used to determine the levels in the other 69 compounds was not utilized for this compound. An alternative method was used as a result of an error which was occurring in calculating the response factor for this compound. In order to avoid the source of error, the levels were calculated by comparing the area of the internal standard and relating it to the area for 25-hydroxycholesterol. The concentration was calculated and adjusted accordingly by multiplying it by its determined percent recovery (86.8%). Statistical Analysis Results were analyzed using analysis of variance in the Mixed Procedure of the Statistical Analysis System (SAS Institute, Inc., 1996). A Split plot design with repeated measures over days was used. A least square means procedure was employed to separate means at P<0.05. The main plot was the application of irradiation versus non-irradiation. The subplot factors include the use of vitamin E versus no vitamin E and the inclusion or exclusion of the application of the oleoresin rosemary dip. Forty eight birds were used. Subsamples from two breasts were used for each replication. Samples were analyzed using three replications per treatment. All data underwent a log transformation prior to statistical analysis. RESULTS AND DISCUSSION 91W Broiler weights were recorded on days 0, 6, 24 and 41 and are presented in Table 13. It can be concluded that typical body weights were achieved, as reported by Nutrient Requirements of Poultry (Anonymous, 1994). There were no significant differences (P>0.05) between the six pens, indicating no pen to pen variation. These results indicate that a-tocopheryl acetate had no efl‘ect on the weights of the birds when compared to the control pens. These findings support those of Sheehy et a1 (1993) who reported that dietary supplementation with a-tocopheryl acetate had no significant effect on body weights of chicks. Furthermore, this data supports the observations of Monahan et a1 (1990) who observed no efl‘ect of dietary vitamin E supplementation on the body weights of pigs. The grth performance of the broilers during this study (41 days) is summarized in Table 14. Daily feed consumption represents the average daily intake, calculated for each bird over days 0 to 6, 6 to 24 and 24 to 41. There were no Significant differences (P>0.05) between pens for each of the three time periods. As indicated by the bird weight data, this suggests no pen to pen variation. Furthermore, no Significant difl‘erences occurred between pens for daily weight gain or feed per gain, remaining consistent with previous data. This supports the findings of Monahan et al. (1990) and Engeseth (1990), who reported no significant changes with respect to growth performance for pigs and veal, respectively, fed vitamin E-Supplemented diets. Furthermore, it was determined 70 71 Table 13. Average weights of broilers raised on control or a-tocopheryl acetate- supplemented dietsl 8 Pen2 Day 0 Day 6 Day 24 Day 41 1 38.213.09 122.611228 9754111562 22900126064 2 38.513.35 123.512660 9737111339 22218125173 3 37.313.89 122911274 9146118430 23413120078 4 37.01207 125011729 992.811 15. 18 2364.11272. 19 5 37.312.96 124311682 931.9119965 23207136558 6 37,212.27 122711417 9284110243 22169128682 1Feed containing 200 IU / kg feed a-tocopheryl acetate in addition to Standard diet 2 Pen 1, 2, 5 = or-tocopheryl acetate-supplemented; Pen 3, 4, 6 = Control Means 2*: standard deviations in the same column are not significantly different (P>0.05). 72 Table 14. Growth performance of broilers raised on control or a-tocopheryl acetate- supplemented dietsl'2 Pen Days 0-6 Days 6 -24 Days 24-41 Daily feed intake (g / day) 1 31.9 87.9 188.1 2 32.6 81.2 172.9 3 31.9 78.8 187.3 4 35.4 88.9 184.3 5 33.7 80.0 175.7 6 34.9 81.8 182.9 Daily weight gain (g / day) 1 14.1 47.4 77.3 2 14.2 47.2 73.4 3 14.3 44.0 83.9 4 14.7 48.2 80.7 5 14.5 44.9 81.7 6 14.2 44.8 75.8 Daily gain / feed (g / g) l 0.44 0.54 0.41 2 0.44 0.58 0.42 3 0.45 0.56 0.45 4 0.42 0.54 0.44 5 0.43 0.56 0.46 6 0.41 0.55 0.41 1Feed containing 200 IU / kg feed a-tocopheryl acetate in addition to standard diet 2Means i standard deviations in the same column are not significantly different (P>0.05). 73 that no significant diflerences occurred between pens for daily weight gain or ratios of feed per gain (Table 14) remaining consistent with the previous data. Lipid Oxidation Lipid oxidation in cooked broiler breasts was assessed using TBARS values (Table 15). All samples were held at 4° C throughout the duration of this study. It was determined that the TBARS values for the irradiated samples were not significantly diflerent than those for the non-irradiated samples. Thus, irradiation at a level of 3.0 kGy did not induce detectable lipid oxidation in broiler breast meat as measured by TBARS. This supports the observations of Heath et al. (1990) who irradiated broilers at 1, 2 and 3 kGy and reported no differences in TBARS values between irradiated and non-irradiated samples. Oleoresin rosemary can provide excellent antioxidant properties in certain foods. Akhtar (1996) demonstrated the efl‘ectiveness of oleoresin rosemary in cooked rainbow trout muscle. He reported that the surface application of oleoresin rosemary had a Significant effect (P<0.05) on the TBARS values for cooked samples stored over a three day period. It was determined that the oleoresin rosemary was most effective in combination with other antioxidant treatments. Highly signifith interactions (P<0.01) were shown between the surface application of oleoresin rosemary and dietary treatments containing supranutritional levels (500 mg / kg feed) of a-tocopheryl acetate (Akhtar, 1996). The results for cooked breast samples treated with the oleoresin rosemary (OR) 74 Table 15. TBARS values for cookedl broiler breasts treated with an OR dip and/or raised on an a-tocopheryl acetate-supplemented diet. mg malonaldehyde / kg meat Treatment Day 0 Day 1 Day 2 Day 3 Control 0810.30 9611.20" 10.710.84‘ 10.510.80' Control Irradiated2 0,910.213 9,011.12" 10.912.03‘ 12.211.15a W E3 0,310.21" 0.510.02" 0,510.06" 0,510.10" Vit E Irradiated 0.310.21" 0.510.09" 0.410.11" 0.310.09" 08‘ 0.51016“ 6.412.23a 8811.36" 9.612.73a on Irradiated 0.510.38a 8.712.15‘ 9.811.07a 10212.29" Vit E/OR 0.310.34" 0,410.24" 0.410.07" 0,410.22" Vit E/OR Irradiated 0310.30" 0.410.06" 0.410.07" 0.410.06" ‘ Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU a-tocopheryl acetate / kg feed in addition to standard diet. 4 Dipped to pick up 3% solution targeting 400 ppm OR/ g based upon 70% activity. ‘4’ Means 1 standard deviations in the same column followed by the same letter are not Significantly diflerent (P>0.05). 75 dip indicated no Significant diflerences (P>0.05) in TBARS values when compared to the control samples (Table 15). The lack of effectiveness of the oleoresin rosemary may be attributed to the depth of penetration. Dipping produces only a surface application, which may not have resulted in the oleoresin rosemary equihhrating throughout the breast samples. Another reason may be attributed to the physical differences between the meat species. Trout fillets can be much thinner and more absorptive than broiler breasts, thus leading to deeper penetration of the oleoresin rosemary into the sample. Because very little work has been done with application of oleoresin rosemary to broiler breast meat, its inefl‘ectiveness may be because of the lack of understanding of the optimum technique for its application. No Significant (P>0.05) synergistic efl‘ects were observed when applying the oleoresin rosemary dip to the samples raised on a-tocopheryl acetate-supplemented diets, which is contrary to the results of Akhtar (1996). This is supported by comparing the results for the dietary a-tocopheryl acetate-supplemented (Vit E) and dietary a-tocopheryl acetate-supplemented / oleoresin rosemary dipped (Vit E / OR) samples. Although there was a trend for dietary a-tocopheryl acetate / oleoresin rosemary dipped samples to have lower TBARS values when compared to dietary a-tocopheryl acetate-supplemented samples, the differences were not Significant (P>0.05). As presented in Table 15, the a- tocopheryl acetate-supplemented samples had highly significant difl‘erences (P<0.01) in TBARS values when compared to the control samples. This was seen consistently over the time of the study. The beneficial effects of dietary supplementation of a-tocopherol 76 has been shown to occur at different concentrations in various types of cooked meats. Lin et a1 (1989) demonstrated increased oxidative stability in cooked breast and thigh muscle of broilers raised on diets containing or-tocopheryl acetate concentrations of 100 mg / kg feed. Sheehy et a1 (1993) reported Significant protective effects of a-tocopherol in broiler thigh muscle stored for 2 months under frozen conditions. TBARS values, after 2 months, for chicks given 180 mg / kg feed were similar to values measured immediately after cooking (Sheehy et al., 1993 ). Moreover, Monahan et a1 (1990) showed the eflemiveness of dietary supplementation at 200 mg / kg feed in the oxidative stability of cooked pork The effectiveness of this treatment may be attributed to the fact that vitamin E is deposited in the bilayer of cell membranes (Buckley et al., 1995). Because vitamin E fimctions as a free radical quencher, its location is essential for optimizing its potential for antioxidant activity. This may explain the difl‘erences demonstrated between vitamin E and the surface application of oleoresin rosemary as effective methods for antioxidant activity. Figure 10 indicates that there were Significant difl‘erences (P<0.01) in the Day 0 values compared to the Day 1 values for the control and OR samples. This increase in TBARS values indicates that the oxidation of the lipid in these samples occurred during the first 24 hr. This change was not, however, seen in the dietary a-tocopheryl-acetate- supplemented samples. Moreover, there were no significant changes seen over the 4 days ill the TBARS values for the dietary or-tocopheryl-acetate-supplemented samples (Figure 11). This further supports the effectiveness of vitamin E as an antioxidant. 77 8:85 86% mod notoEoEnau m :_Ea-> .596. torso 1:85:96 2. 2 >5 2. c an . E0 53F “83qu 86% mod 86% 86% 3%.... 3225.693 >553... r2232 greasing 3.5.5.33 volt-t. m 1.5-g 58.8.0 1.8.86 m =_E.~> m 1.5-g .228 952. cm «on: .53 30E 33.5 3:95 $9.08 .8 32.; 922. .3 2:9... 0.. team 61' I apAqepluuq-ul Bur 78 .5 .53.... Bing. 8&5 mo \ 8&5 mo ‘ 232:. 3225.33 920E039.» 320E053» uoEoEoEaau w 5585 m 5525 m sci; m 5823 gnu v 33 :38 9. ~ 2 3% 383a Each—82.5 ho 538560.33» E5»? 5.3 3.3.: .35 «235 3:03 U933 .8 was: 925... .3 2:9”. nan: 6x I opkqaplwollm Bu: 79 MIME The concentrations of total and individual cholesterol oxide products were determined. Both irradiation and the OR dip treatment had no significant effects (P>0.05) on the formation of the COPs which were quantitated. This followed the same trend as seen for lipid oxidation. Four major COPs were present in detectable quantities in all samples. Concentrations of 7a-hydroxycholesterol, B-epoxide, (at-epoxide and 7-ketocholesterol were detected in all of the samples, regardless of treatment. The concentration of each COP, however, varied from treatment to treatment. Furthermore, the extent of oxidation over time varied for each compound. Three COPs, 7B—hydroxycholesterol, 20a- hydroxycholesterol and 25-hydroxycholesterol, were present in detectable concentrations in some of the samples. These compounds, however, were never found at detectable concentrations in any of the dietary vitamin E-supplemented samples. Total cholesterol oxide products were calculated as the sum of the concentrations of the detectable COPs for each treatment (Table 16). The control and OR samples were higher in total COPs than the dietary a-tocopheryl-acetate—supplemented samples. Furthermore, an increase in total COPs occurred between days 0 and 1 for the control and OR samples. This indicates oxidation of cholesterol occurring within the first 24 hr. Total COPs for the dietary a-tocoheryl acetate samples, however, remained constant over time. This indicates that a-tocopheryl acetate, when fed at a concentration of 200 IU per kg Table 16. Total cholesterol oxide products in cookedl broiler breasts treated with an OR 80 dip and/or raised on a a-tocopheryl acetate-supplemented diet. ug / g meat Treatment Day 0 Day 1 Day 2 Day 3 Control 6.23 25.44 25.07 28.22 Control Irradiated2 6.99 21.32 24.30 27.77 Vitamin E3 2.24 2.79 2.56 1.67 Vitamin E Irradiated 1.84 2.54 2.37 3.73 OR‘ 4.20 19.99 24.61 26.37 OR Irradiated 4.88 12.54 26.36 23.63 Vitamin E / OR 2.42 2.61 2.82 2.66 Vitamin E / OR Irradiated 2.98 2.47 2.20 2.87 ' Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed a-tocopheryl acetate in addition to standard diet. 4 Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. 81 feed, acts as an antioxidant for reducing cholesterol oxidation in cooked broiler breast muscle. This supports the results of previous studies which indicate the effectiveness of vitamin E in controlling cholesterol oxidation in cooked meat. Engeseth and Gray (1994) reported a 65% reduction in cholesterol oxide concentrations after 4 days of storage at 4°C in raw veal as a result of dietary supplementation of vitamin E. Moreover, Monahan et a1 (1992) demonstrated the effectiveness of vitamin E by comparing basal levels (10 IU / kg feed) versus supplemented diets (200 IU / kg feed) in cooked pork Total COPs for basal levels represented 2.7% of the total cholesterol content, while the supplemented diets represented 1.6%. This trend also remains consistent with the data in this study (Table 17). Buckley et al. (1995) also reported the efi‘ectiveness of dietary supplementation of vitamin E (100 or 200 mg / kg feed) in reducing individual COPs (B- epoxide, 7B-hydroxycholesterol and 7-ketocholesterol) as well as total COPs in cooked pork. Percent oxidation of cholesterol (Table 17) was determined by dividing the total COPs for each treatment (Table 16) by the amount of total cholesterol for that treatment (Appendix 2). It can be seen that an increase % oxidation occurred over time for the control samples, while the dietary a-tocopheryl acetate-supplemented samples remained constant. This remains consistent with previous data. Furthermore, this supports the findings of Pie et al. (1991) who reported an increase in % oxidation of cholesterol in control cooked beef; veal and pork after 3 months of storage at -20°C. 82 Table 17. Percent oxidation of cholesterol in cookedl broiler breasts treated with an OR dip and/or raised on a-tocopheryl acetate-supplemented diet. % Treatment Day 0 Day 1 Day 2 Day 3 Control 1.52 6.20 6.11 6.88 Control Irradiatedz 1.77 5.40 6.15 7.03 Vitamin E3 0.37 0.47 0.43 0.42 Vitamin E Irradiated 0.35 0.48 0.45 0.71 OR‘ 0.88 4.21 5.18 5.55 OR Irradiated 0.94 2.41 5.07 4.54 Vitamin E / OR 0.38 0.42 0.45 0.43 Vitamin E / OR Irradiated 0.43 0.35 0.31 0.41 lCooked in a 93-95°C water bath to an internal temperature of 71°C. 2Irradiated at a dose of 3.0 kGy. 3Fed diets containing 200 IU vitamin E in addition to standard diet. ‘Dipped to pick up 3% solution targeting 400 ppm OR / g. 83 As shown in Figure 12, COPs increased over the first 24 hr in storage at 4°C for cooked control and OR-dipped broiler breast samples. According to. Pie et al. (1991), the efiect of cooking caused an 81% increase in C-7 compounds (701- hydroxycholesterol, 7 Bchydroxycholesterol and 7-ketocholesterol), a 100% increase in C20-25 compounds (20-hydroxycholesterol and 25-hydroxycholesterol) and a 58% increase in epoxide compounds (oz-epoxide, B-epoxide and cholestanetriol) in pork samples. The primary oxysterols (oxidized at the C—7 or side chain) had the largest increase compared to the secondary oxysterols (cholesterol epoxides and cholestanetriol). This is further supported by Buckley et al. (1995) who stated that cholesterol oxides in pork increased rapidly to significantly elevated levels following cooking. According to Park and Addis (1987), the predominant. cholesterol oxide product is 7-ketocholesterol. In this study, 7-ketocholesterol was found to have high concentrations compared to the other COPs. However, the concentrations were not neccesarily the predominant COP over time. The concentrations of 7-ketocholesterol were shown to be significantly (P<0.01) affected by the presence of vitamin B (Table 18). This supports the findings of Engeseth et al. (1993) who reported the positive efi‘ects of dietary supplementation of vitamin E on the concentrations of 7-ketocholesterol in cooked veal muscle. Furthermore, as indicated in Figure 13, the concentrations of 7-ketocholesterol in the vitamin E samples did not change (P>0.05) fiom day to day. The control samples, on the other hand, showed significant (P<0.01) change between days 0 to 1, but not after. This may be a result of 7—ketocholesterol further oxidizing to form other COPs. 84 I? o El :8an 222% man-8.8.» 2o 25 mo o... o to mo . 25.9“: 2.5% 228588 2. . >8 .226". on. o sec .228 . «:0 .53: 2&6 mo 2 88... 3.580.958 b-Eooo. untoEoEnam w c_Eua> 58810 m 5.5.; .2Eoo o e an 3965 .0 952. Va .2: coca :35 $35 . 3.35 39.98 .8 8252.. «2.8 3.83.23 .58 no wee—255950 .Nw 239". "311151611 85 Table 18. Concentrations of 7-ketocholesterol in cookedl broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet. ug/gmeat Treatment Day 0 Day 1 Day 2 Day 3 Control 1.98:1.950‘ 6.8711555" 4.6610462' 5.1813480‘1 Control Irradiated2 1.5610851' 5.83i2.902' 4.5912738" 6.1212251" Vit E3 07510.187" 1.4310894" 1.1810354" 0.6210231" VitEIrradiated 1.0610644" 1.1010536" 0.9310567" 1.1611004" on4 1.3310480' 5.6914261" 9.4911414" 3.2311429' ORIrradiated 1.0210405" 3.7011931" 5.1213649' 4.0611985" VitE/OR 0.8710229" 1.1610500" 1.2910048" 0.8510301" VitE/OR Irradiated 1.1710393" 0.9510121" 1.2610324" 1.1310710" ‘ Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed a-tocopheryl acetate in addition to standard diet. ‘ Dipped to pick up 3% solution targeting 400 ppm OR/ g based upon 70% activity. "" Means 1' standard deviations in the same column followed by the same letter are not significantly different (P>0.05). 86 :0.an teen... €855» 2. F .5 .280 on. o in .228 . {an untoEoEaam $3338. 5 l .Stoa «>2. v .52. «3:. «use... 3:2: 38.03 c. 3.23.3023; no ace—255230 .2. 2:2... 87 Compared to the control samples, concentrations of 7a-hydroxycholesterol were lower for the dietary a-tocopheryl-acetate supplemented samples over time (Table 19). Furthermore, there were no significant differences (P>0.05) between dietary vitamin E- supplemented samples fiom day to day. Engeseth et al. (1993) did not detect the presence of 7a-hydroxycholesterol in cooked veal held at 4°C for 4 days. However, other COPs such as 7B-hydroxycholesterol, followed similar trends and did not significantly increase after storage, in dietary a-tocopheryl-acetate supplemented veal samples. The control samples, on the other hand, had significant increases in concentrations (P<0.01) between days 0 to l and l to 2 (Figure 14). There were no significant changes between days 2 to 3. The increase over days 0 to 2 suggests that detectable oxidation of cholesterol took place during the first 48 hr of refiigerated storage. Engeseth Vet al. (1993) reported similar trends in cooked control veal samples for COPs including a-epoxide, 7B- hydroxycholesterol and 7-ketocholesterol, over a 4 day storage period. Although detectable concentrations were not seen, this trend may be a result of 7-ketocholesterol further oxidizing to form cholestanetriol, thus causing the values to level ofl‘ after day 2. Compared to the control samples, the dietary a-tocopheryl-supplemented samples had lower concentrations of B-epoxide (Table 20). Like 7a-hydroxycholesterol, no significant difi‘erences occurred over time for the samples raised on the a-tocopheryl- acetate supplemented diets. The control samples had significantly higher concentrations afler day l, but not between days 1 to 2 or 2 to 3 (Figure 15). This suggests that detectable oxidation occurred over the first 24 hr and amounts leveled out over the next 88 Table 19. Concentrations of 701- hydroxycholesterol in cookedl broiler breasts treated with an OR dip and/or raised on a a-tocopheryl-acetate-supplemented diet. ug/ gmeat Treatment Day 0 Day 1 Day 2 Day 3 Control l.58i0.769‘ 4.4610081' 5.4711830a 5.8011947" Control Irradiated2 2.12:0.657‘ 3.9811642a 6.15i0.335‘ 6.0312690" Vit 153 0.8210358" 0.7510233" 0.9310097" 0.6010244" VitEIrradiated 1.1310416" 0.9110610" 0.9410244" 1.6310944" OR“ 1.6110401" 4.3711312" 5.2910648“ 6.7613023a OR Irradiated l.60:t0.826‘ 2.58:1.410‘ 6.9814159' 6.16128848 VitE/OR 0.8810379" 0.9810477" 1.1010626" 1.2311152" VitE/OR Irradiated 1.1910185" 0.8210299" 0.9610314" 1.1410703" ‘ Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed a-tocopheryl acetate in addition to standard diet. 4 Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. “'b Means i standard deviations in the same column followed by the same letter are not significantly different (P>0.05). 89 28.9... 22...... 228518 2. u in .228 o... r 18 .228 . 25.9.0. 222.... >285er 2. . >8 .2218 o5 0 >8 .228 . 8.52283 .3. .8108? a I 2.88. uaurByBfi «>3 v .26 So... .235 3:93 noses .8 _o..o.uo_o:u>xo._u>zreh no 22.22850 .3. 2:2". 90 Table 20. Concentrations of B-epoxide in cookedl broiler breasts treated with an OR dip and/or raised on a a-tocopheryl-acetate supplemented diet. ug/gmeat Treatment Day 0 Day 1 Day 2 Day 3 Control 1.1310377" 4.5711505" 2.8810624" 4.4410508" Control Irradiated2 1.4310551" 4.1112568" 3.7310832" 3.6911224" Vitra3 0.56fl.234" 0.4510205" 0.3010056" 0.3010297" VitEIrradiated 0.5610273" 0.4310191" 0.3810227" 0.6210239" 08‘ 10610.117" 3.1711123" 2.8211451" 4.6311419" ORIrradiated 0.9110352" 19211.038" 3.2512012" 3.5211118" VitE/OR 0.4810219" 0.3810084" 0.3310257" 0.3910310" Vit E/OR Irradiated 04810.074" 0.3810069" 0.1910066" 0.3510086" l Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed a-tocopheryl-acetate in addition to standard diet. 2 Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. "2 Means i standard deviations in the same column followed by the same letter are not significantly different (P>0.05). 91 .89... 22...... 1.2.8.8.. 2. . .5 .228 .2. o .8 .228 . {an N '0. N «>3. v .02. .52: .32.. 3:03 29.03 2. 82.253... 822.22.22.90 .3 9590 “311181511 92 72 hr. This leveling ofl‘ effect may also be a result of B-epoxide further oxidizing to form other COPs. Concentrations of oz-epoxide are presented in Table 21. The trends over time as demonstrated with B-epoxide were also seen for a-epoxide. However, the major difference was the concentrations at which each occurs (Tables 20 and 21). On average, the ratio of (It-epoxide to B-epoxide is approximately 1:4. This may be a result of the thermodynamic stability of the B-epoxide compared to its A-isomer (Smith, 1981). The presence of 7B-hydroxycholesterol did not occur at detectable concentrations in dietary a-tocopheryl-acetate-supplemented samples (Table 22). The minimum concentration of detection for every compound was determined to be 0.1 ug / g meat. If the detection of any compound did not occur, the response was indicated by ND. The reason for this phenomenon may be explained by the sensitivity of this compound to the antioxidant activity of vitamin E. The control samples showed significant differences (P<0.01) between days 0 to l, but no differences between days 2 and 3 (Figure 16). This demonstrates early oxidation occurring from day 0 to day l. The reason for this phenomenon is uncertain, however, it may be a result of this compound oxidizing into other COPs. The concentrations of Nor-hydroxycholesterol and 25-hydroxycholesterol are presented in Table 23 and Table 24, respectively. Neither compound had detectable concentrations in the dietary vitamin E-supplemented samples. As described above, these 93 Table 21. Concentrations of (It-epoxide in cookedl broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet. ug/ gmeat Treatment Day 0 Day 1 Day 2 Day 3 Control 0.2110079" 0.9210380" 0.9510364" 0.9910280" Control Irradiated2 0.2310071" 0.5610082" 1.0210308" 0.9110129" VitIi3 0.1210052" 01510.142" 0.1610103" 01610.091" VitEIrradiated 0.0910091" 0.0910031" 01110.132" 0.3210229" OR“ 0.2110079" 0.5510155" 1.2610514" 1.0810062" OR Irradiated 0.1610020" 0.5810219" 0.9710382" 1.1210046" VitE/OR 0.1910060" 00810.021" 0.0910011" 0.1910149" VitE/OR Irradiated 0.1910099" 0.0910049" 0.1110023" 0.1210080" l Cooked in a 93-95°C water bath to an internal temperature of 7 1°C. 2 Irradiated at a dose of 3.0 kGy. 2 Fed diets containing 200 IU / kg feed a-toc0pheryl acetate in addition to standard diet. 4 Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. '2’ Means i standard deviations in the same column followed by the same letter are not significantly difi‘erent (P>0.05). 94 Table 22. Concentrations of 7B-hydroxycholesterol concentrations in cooked1 broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet. 11g / g meat Treatment Day 0 Day 1 Day 2 Day 3 Control 1.3410487" 6.9713528" 8.8811926" 9.1711821" Control Irradiated2 1.6510921" 5.2211723" 7.5810403" 9.0910444" Vit E2 ND ND ND ND Vit E Irradiated ND ND ND ND 08‘ ND 6.2211309" 4.2111233" 8.5112725" OR Irradiated 1.1910474" 3.7610476" 9.1815500" 6.1113003" Vit E/OR ND ND ND ND Vit E/OR Irradiated ND ND ND ND ‘ Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 2 Fed diets containing 200 IU a-tocopheryl acetate in addition to standard diet. 2 Dipped to pick up 3% solution targeting 400 ppm OR/ g. ‘ Means 1' standard deviations in the same column followed by the same letter are not significantly difl‘erent (P>0.05). ND = Not detectable = 0.1 ug / g meat Figure 16. Concentrations of 713- hydroxycholesterol in cooked broiler breast meat over 4 days re ’. z -.< Mm .‘I (I ”$56 I 4’ iv . . "III ’ me 95 l cr/}/}I (<(€{<( IMIH av.” .- >I/(1/ an; ’ _a. l/.‘// ~ e v‘ ‘5‘ .l” .\ ”a NIrJ-ri.~'r:~$ (I. ffi I. IN$I$ NW! team 6 I 6'1 Days ‘ Control Day 0 and Comoi Dey1 are significartiy differed (P4101) 96 Table 23. Concentrations of Nor-hydroxycholesterol in cooked1 broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet. pg / g meat Treatment Day 0 Day 1 Day 2 Day 3 Control ND 0.61:0.173' 1.04:0.495‘ 1.17:t0.236‘ Control Irradiated2 ND 0.66:0.249' 03210.29 1‘ 0.9110078“ Vit 153 ND ND ND ND Vit E Irradiated ND ND ND ND 011‘ ND ND ND 0.8610204" OR Irradiated ND ND ND 1.1110376a Vit E/OR ND ND ND ND Vit E/OR Irradiated ND ND ND ND 1 Cooked in a 93-95°C water bath to an internal temperature of 71°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed a-tocopheryl acetate in addition to standard diet. ‘ Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. ' Means i standard deviations in the same column folloWed by the same letter are not significantly different (P>0.05). ND=0.l pig/gmeat 97 Table 24. Concentrations of 25-hydroxycholesterol in cookedl broiler breasts treated with an OR dip and/or raised on a a-tocopheryl acetate-supplemented diet. pg / g meat Treatment Day 0 Day 1 Day 2 Day 3 Control ND 1.0410885‘ l.19:t0.721‘ 1.4710745”I Control Irradiated2 ND 0.97 $0656" 0.9010561" l.03:i:0.261' Vit E3 ND ND ND ND Vit E Irradiated ND ND ND ND OR“ ND ND 1.5510874" 1.2910399" OR Irradiated ND ND 0.86i0.654‘ 1.54:0.552‘ Vit E/OR ND ND ND ND Vit E/OR Irradiated ND ND ND ND l Cooked in a 93-95°C water bath to an internal temperature of 7 1°C. 2 Irradiated at a dose of 3.0 kGy. 3 Fed diets containing 200 IU / kg feed or-tocopheryl acetate in addition to standard diet. ‘ Dipped to pick up 3% solution targeting 400 ppm OR / g based upon 70% activity. ‘ Means i standard deviations in the same column followed by the same letter are not significantly different (P>0.05). ND=0.1 ug/gmeat 98 compounds may also be sensitive to the antioxidant efi‘ects of vitamin E, thus resulting in their absence, or may also be oxidizing, at very low concentrations, to form other COPs. As indicated in Table 23, the concentrations of Nor-hydroxycholesterol were low compared to other compounds, regardless of treatment. As can be seen, most treatments did not result in any detectable concentrations. The control samples, after day 0 had low concentrations of this compound. This may be common for this compound, whose presence is not often detected until after long periods of storage and extreme oxidative conditions. This is supported by Lai et al. (1995) who reported that the products of side chain oxidation of cholesterol (Na-hydroxycholesterol and 25-hydroxycholesterol) were detected in egg powders afier 5 years of storage. Moreover, Sheehy et al. (1995) reported the formation of Nor-hydroxycholesterol in cooked chicken breast and thigh muscle after boiling for 4 hr, deep fat frying for 10 min and microwave heating for 15-25 min. Another compound which is not usually present at detectable concentrations is 25- hydroxycholesterol As demonstrated in Table 24, 25-hydroxycholesterol had relatively low concentrations over the four days. As previously stated, this supports the finding of Lai et al. (1995). Furthermore, Engeseth et aL (1993) reported concentrations of this compound below the detection limit of 1 ng in control cooked veal samples at day 0 and in dietary vitamin E-supplemented veal at days 0 and 4. 99 Summagy The effects of irradiation, dietary supplementation of or-tocopheryl acetate and the surface application of oleoresin rosemary were investigated in cooked broiler breast muscle samples. Irradiation, at a level of 3.0 kGy, did not significantly affect oxidation, as measured by TBARS values and COPs. Dietary supplementation of a-tocopheryl acetate at a level of 200 TU / kg feed, resulted in significantly lower levels of lipid and cholesterol oxidation. The surface application of oleoresin rosemary at a level of 400 parts per million (based upon 70% activity) did not have any significant effect on reducing lipid and cholesterol oxidation. Furthermore, results from samples treated with both or-tocopheryl acetate and oleoresin rosemary indicated that no synergistic effects were observed as a result of the combination of these two treatments. CONCLUSIONS The influence of irradiation on inducing detectable levels of oxidation was examined in raw and cooked chicken breast muscle. Furthermore, the effects of several antioxidants on the oxidative stability of lipid and cholesterol were also investigated. TBARS values and COPs were determined to study the various effects of these treatments. Study 1 investigated the efi‘ects of a vitamin E packaging material on raw chicken breast muscle samples. It was determined that the potential beneficial effects of this treatment were not observed. Lipid and cholesterol oxidation levels were shown to be not significantly different when comparing samples packaged in a control material with samples treated with the vitamin E enhanced material. Results indicated that irradiation, at a level of 3.0 kGy, did not result in increased levels of lipid oxidation. TBARS values and COPs from irradiated samples had no significant differences when compared to non- irradiated samples. Study 2 investigated the effects of two antioxidants, or-tocopheryl acetate and rosemary, in irradiated cooked chicken breast muscle. Dietary supplementation of a- tocpheryl acetate, at a level of 200 IU / kg feed, resulted in samples with significantly lower TBARS and COPs values compared to control samples. Lipid and cholesterol oxidation was significantly altered as a result of this treatment effect. The surface application of oleoresin rosemary did not, however, have any significant effect on reducing lipid and cholesterol oxidation. Results from samples treated with both or-tocopherol and rosemary demonstrated that no synergistic effects were observed as a result of a 100 101 combination of these two treatments. It was also observed that irradiation, at a level of 3.0 kGy, did not induce detectable levels of oxidation in any of the samples in this study. This confirms data fiom study 1. Similar values were detected when comparing the irradiated samples with the non-irradiated samples. FUTURE RESEARCH The objectives of this study were to investigate the effects of: l) irradiation, at a level of 3.0 kGy, 2) vitamin B, through the use of packaging and dietary supplementation, and 3) oleoresin rosemary by surface application, in raw and cooked chicken breast muscle. Further research is warranted as described below. Determining the effects of irradiation at higher doses needs to be conducted to determine its effects on inducing oxidation. The effect of irradiation at sterilization and pasteurization doses should be examined. Furthermore, the application of irradiation to frozen samples versus raw samples should be examined. This may play a significant role in the resulting level of oxidation. The dietary supplementation of vitamin E proved to be very efl‘ective. Rosemary supplementation should be considered as an individual treatment as well as in conjunction with vitamin E. The utilization of other spices containing antioxidant compounds contained in their extracts, such as sage, may also prove to be a potentially beneficial treatment effect. Dietary supplementation, as well as surface application, may prove to be beneficial. The beneficial efi‘ects of a surface application of oleoresin rosemary were not observed in this study. Its potential as an effective antioxidant needs to be re-evaluated at varying concentrations, as well as exposure times. The potential for this treatment is dependent upon obtaining ideal pickup levels. Determining these levels should be 102 103 considered. Furthermore, the efl‘ects of this treatment may be species related. Examm' m’ g the effects of the surface application of oleoresin rosemary should also be carried out in other animals, such as beef, pork and veal.