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The effects of electron beam irradiation and infusion of L-ascorbic acid on the preservation of quality… Wong, Peter Yan Yung 2001

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THE EFFECTS OF ELECTRON BEAM IRRADIATION AND INFUSION OF L-ASCORBIC ACID ON THE PRESERVATION OF QUALITY OF FRESH GROUND BEEF PATTIES by Peter Yan Yung Wong B.Sc. (Agr.) The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES Department of (Food Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 2001 ©Peter Wong, 2001 UBC Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia,.I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of F o o d Sc ience. , The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada D a t e March 2 . 2001 ABSTRACT The objectives of this thesis were to assess the effectiveness of electron beam irradiation (e" beam) at 5, 10 and 20 kGy in reducing bacterial count in fresh ground beef patties of 4, 17 and 30% fat and to evaluate the effects of infusing L-ascorbic acid (LAA) into beef carcasses on the quality of irradiated fresh ground beef patties of 4, 17 and 30% fat. Aqueous and emulsion model systems, exposed to ultraviolet and electron beam irradiation, were first used to determine the catalytic activity of hemoglobin, the depletion of LAA and the effects of LAA and e" beam on the degree of lipid oxidation. Hemoglobin generated 50% less hydroxyl radicals than ferrous chloride in the Fenton reaction, indicating minor catalytic activity. An increase in irradiation dosage and storage time drastically decreased the concentration of lOmM L-ascorbic acid. Both ultraviolet and e" beam irradiation induced lipid oxidation in a dose-dependent manner, which was further enhanced by the prooxidant activity of 10 mM LAA. The 500 mM LAA exhibited the opposite effect. The effectiveness of electron beam irradiation in controlling microbial growth was then evaluated in ground beef patties made from LAA infused beef carcasses. Viable bacteria were not detected in ground beef patties receiving irradiation at dosages of 5, 10 and 20 kGy. The addition of tallow to increase total crude fat content in the beef patties led to an increase in bacterial counts. An increase in pH of patties was could have been attributed to the metabolic by-product generated by microorganisms. Finally, the physicochemical characteristics of the infused ground beef patties were evaluated. An increase in irradiation dosages resulted in an increase in lipid oxidation in the beef patties. LAA also increased lipid oxidation, as well as, deoxymyoglobin oxidation in the beef patties. Higher fat content resulted in higher lipid oxidation and pigment oxidation in the beef patties. Hardness attribute of the ground beef patty was significantly increased by the addition of i i fat. In general, the application of low dosage of e" beam to lean ground beef patties without the addition of LAA is optimum in preserving quality. TABLE OF CONTENTS Abstract n List of Tables vii List of Figures ix List of Abbreviations xi Acknowledgement xii 1. Introduction 1 2. Literature Review 5 2.1 Ground Beef 5 2.1.1 Production and Consumption 5 2.1.2 Incidence of Food Borne Illnesses 5 2.1.3 Sources of Contamination 6 2.2 Irradiation 7 2.2.1 Irradiation Process 7 2.2.2 Effects of Irradiation on Meats 8 2.2.2.1 Microbial Load 8 2.2.2.2 Quality Changes 12 2.3 Physiochemical Qualities of Ground Beef .14 2.3.1 Lipid Oxidation and Flavour 14 2.3.2 Colour Stability 15 2.3.3 Texture 17 2.4 Antioxidants 19 2.4.1 Ascorbic Acid in Meat 19 2.4.2 Protection Against Oxidative Reactions 20 2.4.3 Antioxidants and Microbial Growth 22 2.5 Experimental Objectives 22 3. Experiment 1: Model Studies 24 3.1 Materials and Methods 24 3.1.1 Hydroxyl Radical Generation 24 3.1.2 Ascorbic Acid Depletion Study 25 3.1.3 Ultraviolet Light-Induced Lipid Oxidation 26 3.1.4 Electron Beam Irradiation-Induced Lipid Oxidation 26 3.1.5 Statistical Analyses 27 iv 3.2 Results 28 3.2.1 Hydroxyl Radical Generation 28 3.2.2 Ascorbic Acid Depletion Study 28 3.2.3 Ultraviolet Light-Induced Lipid Oxidation 31 3.2.4 Electron Beam Irradiation-Induced Lipid Oxidation 34 3.3 Discussion 37 3.4 Conclusion 41 4. Experiment 2: Irradiation Effects on the Intrinsic Factors and Microbial Population in Ground Beef Patties 42 4.1 Materials and Methods 42 4.1.1 Cattle Raising and Ascorbic Acid Infusion 42 4.1.2 Production of Ground Beef Patties 42 4.1.3 Electron Beam Irradiation Procedure 43 4.1.4 pH of Ground Beef 45 4.1.5 Microbial Population 45 4.1.5.1 Aerobic Plate Count 45 4.1.5.2 Escherichia coli and Total Coliforms 45 4.1.5.3 Psychrotroph Count 45 4.1.6 Statistical Analyses 46 4.2 Results 47 4.2.1 pH of Ground Beef 47 4.2.2 Microbial Population 50 4.2.2.1 Aerobic Plate Count 50 4.2.2.2 Total Coliforms and Escherichia Coli 50 4.2.2.3 Psychrotroph Count 57 4.3 Discussion 62 4.4 Conclusion 68 5. Experiment 3: Irradiation Effects on the Physiochemical Attributes of Ground Beef Patties 69 5.1 Materials and Methods 69 5.1.1 Materials 69 5.1.2 Heme Iron Concentration 69 5.1.3 Lipid Oxidation 70 5.1.4 Colourimetric Determination 70 5.1.5 Metmyoglobin Content 71 5.1.6 Textural Analysis 71 5.1.7 Statistical Analyses 72 5.2 Results 73 5.2.1 Heme Iron Concentration and Lipid Oxidation 73 v 5.2.2 Colourimetric Determination and Metmyoglobin Content 78 5.2.3 Textural Analysis 87 5.2.4 Correlation 90 5.3 Discussion 93 5.4 Conclusion 107 6. General Discussion 109 7. General Conclusion 114 8. References 117 Appendix 1 - Standard curves derived for determining ascorbic acid concentration between the ranges from 0 m M to 25 m M (A), 25mM to lOOmM (B) and 125 m M to 500 m M (C) 127 Appendix 2 - The malondialdehyde content in relation to the absorbance reading as measured using the thiobarbituric acid test 128 Appendix 3 - Regression equations for best-fit lines depicting the malondialdehyde content in ground beef patties during storage 129 Appendix 4 - Regression equations for best-fit lines depicting the Hunterlab a value for redness in ground beef patties during storage . 130 Appendix 5 - Correlation between physicochemical attributes, microbial and intrinsic factor in electron beam irradiated ground beef patties of all fat levels containing L-ascorbic acid 131 vi List of Tables Table 1 - Electron beam irradiation-induced depletion of L-ascorbic acid (10, 100 and 500 mM) in model systems with and without 3.13% (v/v) linoleic acid (LA) during 4°C storage Table 2a - Effects of electron beam irradiation dosage and fat level on the pH values of control ground beef patties during 4°C storage Table 2b - Effects of electron beam irradiation dosage and fat level on the pH values of ground beef patties made from L-ascorbic acid infused carcasses during 4°C storage Table 3a - Effects of electron beam irradiation dosage and fat level on the aerobic bacteria population (CFU/g beef) in control ground beef patties stored at 4°C for 14 days Table 3b - Effects of electron beam irradiation dosage and fat level on the aerobic bacteria population (CFU/g beef) in ground beef patties made from L-ascorbic acid infused carcasses stored at 4°C for 14 days Table 4a - Effects of electron beam irradiation dosage and fat level on the total coliform population (CFU/g beef) in control ground beef patties stored at 4°C for 14 days Table 4b - Effects of electron beam irradiation dosage and fat level on the total coliform population (CFU/g beef) in ground beef patties made from L-ascorbic acid infused carcasses stored at 4°C for 14 days Table 5a - Effects of electron beam irradiation dosage and fat level on the Escherichia coli population (CFU/g beef) in control ground beef patties stored at 4°C for 14 days Table 5b - Effects of electron beam irradiation dosage and fat level on the Escherichia coli population (CFU/g beef) in ground beef patties made from L-ascorbic acid infused carcasses stored at 4°C for 14 days Table 6a - Effects of electron beam irradiation dosage and fat level on the psychrotrophic bacteria population (CFU/g beef) in control ground beef patties stored at 4°C for 14 days Table 6b - Effects of electron beam irradiation dosage and fat level on the psychrotrophic bacteria population (CFU/g beef) in ground beef patties made from L-ascorbic acid infused carcasses stored at 4°C for 14 days Table 7 - Correlations between the intrinsic factors of ground beef patties and the various types of microorganisms present Table 8 - Effects of electron beam irradiation, L-ascorbic acid and fat level on heme iron concentration (ppm) in ground beef patties on day 14 of storage (A) and in 3 0 % ground beef patties on day 0 , 7, 14 of storage (B) at 4°C Table 9 - Effects of electron beam irradiation, L-ascorbic acid and fat level on metmyoglobin concentration (%) in ground beef patties on day 14 of storage (A) and in 3 0 % ground beef patties on day 0 , 7, 14 of storage (B) at 4°C Table 1 0 - Correlation between maximum shear forces, Hunterlab colour values, malondialdehyde concentration, heme iron content and percentage metmyoglobin for electron beam irradiated ground beef patties of 4%, 17% fat content (A) and 3 0 % fat content (B) containing L-ascorbic acid List of Figures Figure 1 - Interaction o f ( A ) gamma rays and (B) electron beam w i t h matter 9 F igure 2 - M e c h a n i s m o f autooxidation o f unsaturated fatty acids (adapted from Nawar , 1985) 16 Figure 3 - Deoxyr ibose degradation by h y d r o x y l radical generated from Fenton reaction init iated by Fe(II) and/or hemoglob in (Hb) under the influence o f heat incubat ion (open columns) or U V light i l lumina t ion (checkerboard columns) 29 Figure 4 - Quantitative analysis o f 10 m M L-ascorb ic acid during storage as influenced by ( A ) the absence o f l ino le ic acid and (B) the presence o f 3 .13% (v/v) l ino le ic acid w i t h an in i t ia l exposure to irradiat ion at 0 k G y (diamond), 2.5 k G y (triangle), 5 k G y (circle) and 10 k G y (square) 30 Figure 5 - Effects o f L-ascorb ic acid on U V - i n d u c e d photooxidat ion o f l ino le ic acid emuls ion system; 0 m M (diamond), 10 m M (square), 100 m M (circle) and 500 m M (triangle) ascorbic acid 33 Figure 6 - Effects o f 0 m M (diamond), 10 m M (square), 100 m M (triangle), and 500 m M (circle) L-ascorb ic acid and electron beam irradiat ion at 0 k G y , 5 k G y , 10 k G y and 20 k G y on oxida t ion o f l ino le ic ac id emuls ion system 35 Figure 7 - A regression analysis o f the relationship between irradiat ion dosages and oxida t ion o f l ino le ic acid emuls ion system without the influence o f L-ascorb ic acid 36 Figure 8 - F l o w diagram o f experimental design 44 Figure 9a - Effects o f fat level and electron beam irradiat ion dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on malondialdehyde content ( M D A ) in control ground beef patties during storage at 4 °C 76 Figure 9b - Effects o f fat level and electron beam irradiat ion dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on malondialdehyde content ( M D A ) i n ground beef patties made f rom L-ascorb ic acid infused carcasses during storage at 4 ° C 77 Figure 10a - Effects o f fat level and electron beam irradiat ion dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on Hunterlab a value for redness i n control ground beef patties during storage at 4 °C 79 Figure 10b - Effects o f fat level and electron beam irradiat ion dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on Hunter lab a value for redness i n ground beef patties made f rom L-ascorb ic acid infused carcasses during storage at 4 °C 80 Figure 1 l a - Effects o f fat level and electron beam irradiat ion dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on Hunterlab L value for redness in control ground beef patties dur ing storage at 4 °C 82 Figure 1 lb - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on Hunterlab L value for redness in ground beef patties made from L-ascorbic acid infused carcasses during storage at 4°C Figure 12 - Colour difference (delta E) between control patties and patties made from L-ascorbic acid infused carcasses during 14 days at 4°C storage as affected by fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy Figure 13a - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on maximum single blade shear force in control ground beef patties during storage at 4°C Figure 13b - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on maximum single blade shear force in ground beef patties made from L-ascorbic acid infused carcasses during storage at 4°C List of Abbreviations aw Water activity CDC Center for Disease Controls CFU Colony forming unit D A A Dehydroascorbic acid deoxyMb Deoxymyoglobin 6 aq Aqueous electron e" beam Electron beam EPR Electron pair resonance AE Delta E HACCP Hazard Analysis and Critical Control Point Hb Hemoglobin L A Linoleic acid L A A L-ascorbic acid LOO* Peroxyl radical Mb Myoglobin M D A Malondialdehyde metMb Metmyoglobin •OH Hydroxyl radical 0 2 « Superoxide radical oxyMb Oxymyoglobin T B A Thiobarbituric acid TBAR Thiobarbituric acid reactive substance WOF Warmed-overed-flavor USDA-FSIS • United States Department of Agriculture - Food Safety and Inspection Service xi Acknowledgement I would like to express my deepest gratitude to my supervisor and mentor, Dr. D. Kitts, for his dedication in preparing me for all the challenges that lies ahead in the field of Food Science, and all his encouragement and advice which was proven to be invaluable throughout my study in the Master's program. I would also like to extend my deepest appreciation to Dr. J. Thompson, Dr. T. Durance and Dr. C. Seaman for their time and effort spent in supervising my project. Many thanks to Dr. N. Wijiwickreme of Cantest Ltd. and Sherman Yee of Food Science for all their technical assistance, as well as Dr. A. Schaefer and Dr. Dubeski of Agriculture and Agri-Food Canada for their assistance in raising, infusing and dressing of beef cattle. An acknowledgement is given to the British Columbia Cattleman Association for their generous award of the W.N. Bostock Memorial Research Grant in 1999. 1. Introduction The preservation of raw ground beef quality is of great concern to the meat industry. Consumer perception of quality is based on the criteria of food safety, attractive appearance and wholesomeness of the beef. Because of the high moisture and nutrient rich environment of ground beef, it is prone to microbial spoilage by pathogenic bacteria and to oxidative deterioration such as colour change, off-flavour (both odour and taste) development and textural alterations. Inadequate control of spoilage or oxidation during retail display can therefore lead to financial losses to the meat industry through the reduction in price and the decline in sales of ground beef. To ensure food safety in meats, all abattoirs and meat processing plants must adhere to strict slaughtering and manufacturing procedures. Nevertheless, an outbreak of hamburger disease (i.e. Escherichia coli 0157:H7 contaminated ground beef) still occurred in 1993 at a Seattle fast-food restaurant, which resulted in the establishment of higher safety standards for ground beef by the United State Department of Agriculture's (USDA) Food Safety and Inspection Service (FSIS) (Wachsmuth, 1997). This included Zero-Tolerance for visible foreign matter on meat carcasses, higher pathogen reduction standards, Hazard Analysis and Critical Control Point (HACCP) program, and declaration of E.coli 0157:H7 contaminated products to be adulterated and the processor of the adulterated beef to be punishable by law (Dorsa, 1997). In order to comply with the new safety standards, various meat packers are now evaluating novel technologies (e.g. hot-water wash, steam pasteurization or organic acid wash) to decontaminate meat carcasses in their plants. Such technologies, used in conjunction with a HACCP program, can reduce or prevent the introduction of microorganisms on meat carcasses. However, these practices alone cannot prevent post-treatment contamination, nor ensure safety of further processed products such as ground beef or meat sausages. Presently, there is no 1 universally accepted method for the reduction of microbial load in meat products. The irradiation of meat has been, and continues to be, studied for effectiveness in reducing microbial load and ensuring food safety. Typically, irradiation is applied after the product, such as ground beef, has been packaged. This can prevent post-treatment contamination that can occur with other novel technologies. Therefore, pasteurization of meat by irradiation shows promise as an essential tool in the quality preservation of meats and meat products. Another criterion indicative of quality is the appearance of meat. The bright red colour of fresh beef is the single most important determinant of quality, as rated by the consumer (Shivas et al., 1984). Myoglobin (Mb), an oxygen carrier in the muscle, is responsible for the colour of beef. Oxygenation of the Mb results in the formation of oxymyoglobin (oxyMb), which provides the beef with the attractive bright red colour. On the other hand, oxidation of the Mb results in the formation of metmyoglobin (metMb), which produces the dull brown colour of beef. This oxidation-reduction cycling of Mb is oxygen dependent but may also be affected by free radicals produced from lipid peroxidation. Lipid peroxidation is the deterioration of unsaturated fatty acids into short-chain hydrocarbons via a free radical mediated reaction. The intermediate product of lipid peroxidation (i.e. lipid peroxyl radicals or lipid peroxides) oxidizes deoxymyoglobin (deoxyMb; deoxygenated form of oxyMb) to metMb, disrupts membrane structure resulting in increased drip-loss (i.e. exuded fluid), and induces protein aggregation in beef. Furthermore, the decomposition of lipid peroxides will produce short-chain aldehydes, ketones and alcohol intermediates that are responsible for the off-odour detected in chemically spoiled beef. Therefore, the oxidative reactions affecting the appearance of beef, as judged by colour and drip-lost, as well as off-odour and protein aggregation, must be controlled to enhance the shelf-life of beef products. Antioxidants are typically used to control oxidative reactions; although lowering the temperature and excluding oxygen from the immediate environment may also retard oxidation. 2 The addition of antioxidants to meat products is primarily for the prevention of metMb formation. However, the phospholipids in meats are readily oxidized when exposed to irradiation. Thus the antioxidant added to meat products may also retard the onset of lipid peroxidation by scavenging free radicals produced during irradiation. This is especially important because the build-up of lipid peroxidation products can lead to an excessive drip-loss by destruction of cellular membrane, protein aggregation (i.e. tougher meats) as a result of denaturation and contribute to the formation of metMb. Furthermore, lipid peroxidation products can reduce the wholesomeness of the beef through the destruction of nutrients (e.g. degradation of unsaturated lipids, decrease protein digestibility and loss of fat-soluble vitamins) and formation of cytotoxic products. With this aforementioned information in mind, the objectives of the following study were to assess the effectiveness of electron beam irradiation in reducing the microflora and the infusion of ascorbic acid in minimizing the. physiochemical quality changes in ground beef patties. Electron beam technology utilizes a non-radioactive source (i.e. linearly accelerated electrons) to irradiate foods. This electron beam carries more energy than gamma rays but has a shallow penetration depth in foods. Since raw ground patties are thin and of uniform thickness, it was hypothesized that electron beam irradiation would effectively reduce their microbial load. However, the use of electron beam irradiation may also induce oxidative changes in meat that can reduce shelf-life. Thus, it was further hypothesized that the infusion of ascorbic acid, as an antioxidant, may retard the oxidative changes induced by irradiation. To evaluate the above hypotheses, a simplified model system was used to identify the interactions between the antioxidant ascorbic acid and electron beam irradiation on oxidative reactions before assessing similar effects on a more complex system of ground beef. The aim of the model system was to determine the independent factors and interactions of electron beam irradiation and ascorbic acid on lipid oxidation using a linoleic acid model system containing hemo/myoglobin. Ground beef 3 was used to determine the effects of electron beam irradiation and ascorbic acid on microbial load and physiochemical qualities (i.e. lipid oxidation, colour, texture and pH) of ground beef patties containing various levels of crude fat. 4 2. L i t e ra tu re Rev iew 2.1 Ground Beef 2.1.1 Production and Consumption Commercially, ground beef is produced from skeletal muscles of utility or higher quality grades of both sexes of cattle. These muscles include the flank, short plate, shank, brisket, rib, chuck, loin, sirloin, neck and round of the cattle (Henrickson, 1978). Appropriate amounts of fats from good grade carcasses may be added to or removed from the trimmings during grinding to achieve the four levels of fat in ground beef; namely regular (maximum of 30% fat), medium (maximum of 23% fat), lean (maximum of 17% fat) and extra lean (maxiumum of 10% fat). Ground beef prepared for sale is either packaged in tubes or in styrofoam trays wrapped in oxygen permeable film. In Canada, the production of ground beef contributes approximately 42 to 45% of the $18 billion dollar industry (in farm cash receipts) specific to domestic meat products. Within the same year, the USDA reported that slightly more than 40% of the 25.4 billion lb (11.5 billion kg) of beef produced in the US was ground or used for hamburger (Murphy, 1999). The demand for ground beef continues to increase in both bulk and formulated hamburger patty form; with the majority of ground beef being served by the foodservice industry and the remaining volume being sold in supermarkets. 2.1.2 Incidence of Food Borne Illnesses A study conducted by the Center for Disease Control (CDC) in the United States reported that foodborne illness linked to Escherichia coli (E.coli) 0157:H7 was the third most frequent cause of food poisoning, claiming 2.9% of all foodborne related deaths (Anon., 1999). An example of this was the outbreak of hamburger disease that occurred at the Jack in the Box fast-5 food restaurant chain in Seattle in 1993, which claimed the lives of four children (Krizner, 1999). In 1997, E. coli 0157:H7 was isolated from the Hudson Foods' brand of ground beef and led to one of the largest single cases of ground beef recall (25 million lb. or 11.3 million kg) by a meat processor (Hollingsworth, 1998). Between 1997 and 1999, a total of twelve recalls resulting from E. coli 0157:H7 contamination in beef were recorded in the United States (Murphy, 1999). Since E. coli 0157:H7 was detected in approximately 3.7% of ground beef samples tested (Doyle and Schoeni, 1987), the likelihood of future outbreaks relating to ground beef remains high. This concern is supported by the findings of CDC released in March of 1999, stating that confirmed cases ofE.coli 0157:H7 rose 22% in 1998 (Murphy, 1999). Although not all cases of E.coli food poisoning were related to ground beef, it certainly represents a key vector for the spread ofE.coli derived food poisoning. 2.1.3 Sources of Contamination There are two major sources of contamination in ground beef; these being, from the animal itself or introduced during meat processing. Healthy cattle normally carry a heterogeneous load of pathogenic bacteria in the intestinal tract, or on hide and hair (Bolder, 1997; Dickson and Anderson, 1992). Pathogenic organisms, including E. coli 0157:H7, Salmonella spp., Camplylobacter spp., Listeria spp., and Staph, spp., were identified in 0.8 to 5% of all cattle tested in a US National Study (Bolder, 1997; Farrell et al., 1998). Poor slaughtering practice may result in the transfer of pathogen from the intestinal tract and/or the hide to the surface of sterile muscle. Occasionally, Clostridium spp. may be isolated in small numbers from the deep muscle tissue of healthy animals (Dickson and Anderson, 1992). Plant workers and equipment can contaminate pathogen-free meat during processing of ground beef. One example of this is the contamination of retail store ground beef by E. coli that occurred in 1995 via a meat grinder used in a supermarket (Farrell et al., 1998). Regardless of the sources of contamination, ground beef continues to be a key vector for the spread of foodborne illnesses. This situation 6 exists because grinding intact muscle will disturb the tissue fiber and allow the nutrient rich sarcoplasm to come in contact with pathogens, and support growth of these organisms (Henrickson, 1978). Furthermore, inadequate cooking may not satisfactorily destroy pathogens embedded in the center of the ground beef, as a result of grinding the meat into a homogenous mixture. 2.2 Irradiation 2.2.1 Irradiation Process Radiation may be defined as the emission and propagation of energy through space or a material medium (Jay, 1996). Thus, ionizing radiation describes radiation capable of ejecting an electron from an atom in a material medium, resulting in the formation of a charged or ionized particle (Olson, 1998). The absorbed dose of irradiation is typically measured in grays (Gy) (one Gy equals to one joule per kilogram) or kilograys (kGy). Ionizing radiation has been proposed for the preservation of food since the early 1930s. As of Dec. 23, 1999, USDA FSIS approved the use of irradiation of fresh and frozen ground beef at 4.5 and 7.0 kGy, respectively, which took effect on Feb. 22 of 2000 (Anon, 2000). Today, irradiation processing of foods is primarily accomplished by the use of gamma rays or an electron beam. Gamma rays (y-ray) are electromagnetic radiation emitted from an excited nucleus of elements such as 6 0 Co and 1 3 7 Cs (Jay, 1996). The process of y-ray irradiation is merely exposing a product to either 6 0 Co or 1 3 7 Cs radiation at a pre-determined distance and time to achieve a specific dose. Electron beam (e" beam) radiation makes use of a linear electron accelerator that generates and accelerates electrons in a vacuum. The food sample is carried on a conveyor belt to the accelerator where the e" beam is magnetically scanned across the entire product. Duration of exposure to the e" beam determines the dose of irradiation. 7 The deposition of energy from the irradiating beam to a food medium follows two basic principles: photoelectric effect and Compton effect. In the photoelectric effect, a low-energy (below 60 keV) photon from a y-ray or e" from an e" beam is applied to a food, causing the ejection of an e" from the inner orbital of an atom and the transfer of remaining energy of the incident ray to the ejected electron in the form of kinetic energy (Rosenthal, 1992). Thus, an ionization of an atom is produced. These photoejected e" may carry considerable kinetic energy and are, therefore, capable of ionizing other molecules in the food. With an increase in the energy level of the incident ray to 1 MeV, a Compton effect is generated in the food (Fig. 1). The high energy photon or e" gives up only part of its energy in ejecting an orbital e" (i.e Compton recoil e") and is deflected with a longer wavelnegth (Rosenthal, 1992). The less energetic secondary incident ray will continue to penetrate through the food medium to ionize other atoms, until all the energy of the incident ray has been transferred to e". The ejected e" will in turn ionize other molecules in the food. Therefore, the deposition of energy to a food by irradiation relies on both the energy deposited by the incident ray and the ejected e" that are set in motion by the primary beam from either source. However, e" beam irradiation is different from y-ray in that it has a shorter penetrate depth into the food due to the friction generated between the e" in the incident beam and the molecules in foods. In either case, food samples can be irradiated fresh or frozen in respective packaging. 2.2.2 E f f e c t s o f I r r a d i a t i o n o n M e a t s 2.2.2.1 M i c r o b i a l L o a d Irradiation by y-rays or an e" beam can reduce the microbial population and thereby improve food safety and extend shelf-life of fresh meats. Reduction or destruction of microorganisms is primarily the result of DNA and/or cellular membrane damage caused by the absorbed energy (Lopez-Gonzalez et al., 1999; Roberts and Weese, 1998). In addition, radiolysis of water in meats, which produces free radicals with antimicrobial effects, can contribute to the 8 (a) Gamma rays (b) Electron beam | material | | Figure 1- Interaction of (a) gamma rays and (b) electron beam with matter. Note: 1, 2, 3 denotes three independent energy waves f r o m both sources of i r rad ia t ion and the relative distance t raveled by each wave into the mater ia l represent the depth of penetrat ion into a food sample. 9 overall destruction of microorganisms during irradiation processing (Cabedo et al., 1998; Jay, 1996; Murano, 1995b). Irradiation dosages can influence the reduction of microorganism in beef. Low dosages of irradiation between 2 and 3 kGy effectively reduce the number of spoilage organisms (e.g. pseudomonads, enterobacteriaceae or lactic acid bacteria) and eliminate up to 99% of the pathogenic bacteria in ground beef (Radomyski et al., 1994). This result is in agreement with the findings of Roberts and Weese (1998), who reported a 1- and 3-log reduction in the microbial counts of irradiated ground beef at 1 and 3 kGy, respectively. On average, a D value (dose required to inactivate 90% of a microbial population) which varies from 0.27 to 0.63 kGy, can be achieved by irradiating ground beef at low temperatures and with oxygen-permeable packaging material (Lopez-Gonzalez et al., 1999). Specifically, a minimum dose of 1.5 kGy of irradiation would destroy at least 6 logs of E. coli 0157:H7, which has a D value of about 0.24 kGy (Olson, 1998). The reduction in the microbial population by irradiation can greatly enhance the shelf-life of fresh meats. Fresh ground beef has a limited shelf-life of less than 7 d under refrigerated storage (Roberts and Weese, 1998). Earlier studies have reported that the refrigerated shelf-life of aerobically irradiated fresh ground beef at 2 kGy was extended to 8 d and a further extension up to 60 d was observed when frozen samples were irradiated anaerobically (Murano, 1995a). In particular, Roberts and Weese (1998) reported the refrigerated shelf-life of vacuum-packaged frozen ground beef patties exposed to 1, 3, 5 and 7 kGy was acceptable up to 14, 21, 42 and 42 d of storage, respectively. This stability was higher than the results of Lefebvre et al. (1992) who reported that 1 and 5 kGy of irradiation merely extended the refrigerated shelf-life 4 and 15 d, respectively. In addition to dosage, two other major factors can also affect the efficiency of irradiation in reducing of microbial load. These two important factors are the bacteria composition and the 10 physical conditions of the food. The initial bacteria load can have the most significant impact on the reduction of population by irradiation (Jay, 1996; Roberts and Weese, 1998). Furthermore, individual microorganisms exhibit relatively different sensitivities to irradiation. Gram-positive bacteria are more resistant to irradiation than Gram-negative bacteria, and the same fact applies to spore forming bacteria versus non-spore forming bacteria (Jay, 1996; Lebepe et al., 1990). The observed difference in irradiation sensitivity may be explained partially by the microorganism's cellular envelope, the ability to inactivate free radicals and the effectiveness of the DNA repair mechanisms of the organism (Jay, 1996). The physical environment of the bacteria can offer protection against irradiation. Sensitivity of bacteria to irradiation-induced damage or death is much greater in buffer than in a protein or a food matrix (Jay, 1996; Murano, 1995b). Also, bacteria exhibit relatively higher resistance to irradiation when the food is frozen or dried. The D value for Yersinia enterocolitica in irradiated ground beef was approximately 0.2 kGy when irradiated at 25°C but is increased to 0.4 kGy at -30°C (Murano, 1995b). This occurs because the radiolysis of water is retarded when frozen (Cabedo et al., 1998; Jay, 1996; Murano, 1995b). Free radicals produced during radiolysis, which are mostly •OH, e'aq and hydrogen atoms, exhibit weak antimicrobial effects (Lawrie, 1985). In the presence of air, these free radicals react with oxygen to produce the more unstable peroxyl radical, hydroperoxides and superoxide anion. These oxygenated or oxygen centered free radicals will be more effective in the destruction of microorganisms. Inactivation of microorganisms in food by substerilizing doses of irradiation can leave a fraction of the surviving population in a damaged but repairable state (Lucht et al., 1998; Ray, 1986, 1979). Damaged microorganisms are temporarily unable to proliferate or divide and may exhibit different nutritional requirements (Lucht et al., 1998, Lucht, 1997). For example, the survival of post-irradiated E. coli is dependent on the rapid activation of repair mechanisms, whereby reconstruction of damaged DNA, cellular components or membranes occurs at 11 temperatures of up to 18°C (Lucht et al., 1998). Cell death occurs when repair protocol is not activated within a given time, or when conditions (e.g. oxygen availability or temperature) are unfavorable (Lucht et al., 1998, 1997). Moreover, irradiated and injured microorganisms may exhibit mutations (Lucht et al., 1998; Diehl, 1990). Therefore, the ability of damaged microorganisms to repair and adapt to changes in physiological state and changes in taxonomically relevant characteristics may lead to an underestimation of viable bacterial count thereby overestimating the food safety and product shelf-life of irradiated foods. 2.2.2.2 Quality Changes Ionizing radiation can induce quality changes in meat. These changes are primarily achieved by the oxidation of molecules by free radicals generated during radiolysis of water. In ground beef, irradiation-induced changes of concerns include discolouration, lipid oxidation and protein aggregation. In the presence of air, Mb combines with oxygen to form oxyMb, which gives the beef a bright red colour (Renerre, 1990). Upon irradiation, Mb in general tends to oxidize to metMb, which gives the dull brown colour (Olson, 1998; Lawrie, 1985; Doty, 1965). The conversion of oxyMb to metMb is caused by the deoxygenation of oxyMb to deoxyMb, followed by the subsequent oxidation of ferrous heme iron in deoxyMb to the ferric heme iron in metMb (Renerre, 1990; Lanari et al., 1996). Oxidation of the heme iron can be accomplished in two different, but synergistic ways. First, deoxyMb, can be autoxidized to the metMb form in the presence of air, regardless of irradiation (Lanari et al., 1996). The accumulation of metMb is then further increased by the oxidation of deoxyMb by free radicals and hydrogen peroxide (Chen et al., 1999; Lanari et al., 1996). Therefore, radiolysis of water and lipid oxidation in irradiated ground beef has been postulated be the second pathway for the increase in the formation of metMb via oxidation of deoxyMb by free radicals (Lanari, 1996; Yin and Faustman, 1993; Harel and Kanner, 1985a). 12 The mechanism of irradiation-induced lipid oxidation in meats resembles those of oxidative rancidity (Lawrie, 1985; Doty, 1965). Oxidative rancidity is the destruction of unsaturated fatty acids by oxygen. Lipid oxidation of irradiated meats is thought to be initiated by hydroxyl radicals generated from the radiolysis of water (Chen et al. 1999, Thakur and Singh, 1994). Oxygen dissolved in meat tissue, or in the immediate surrounding surface medium is subjected to activation by irradiation to singlet oxygen (Diehl, 1995). The combination of generated hydroxyl radical and singlet oxygen can attack lipid to form lipid hydroperoxides (Chen et al., 1999). Subsequent breakdown of these hydroperoxides generates volatile compounds that contribute to the typical off-odour associated with lipid oxidation. Nevertheless, gamma irradiation of frozen beef, lamb, pork, turkey breast and leg with a maximum dose of 9.43 kGy does not significantly increase lipid oxidation, as measured by the end-point indicator thiobarbituric acid reactive substance (TBAR), in comparison to non-irradiated samples (Hampson et al., 1996). However, irradiation of refrigerated raw chicken, turkey and pork meats between 0 to 5 kGy has been shown to generate lipid oxidation volatiles (e.g. hexanal, propanol, pentanal and their corresponding ketones and alcohols) and other volatile compounds derived from protein oxidation including methyl mercaptan, sulfur dioxide, dimethyltrisulfide (Ahn et al., 1999; Ahn et al., 1998; Chen et al, 1999). Similar to lipid oxidation, protein oxidation in meat is also catalyzed by free radicals (i.e. hydroxyl radical) from radiolysis of water (Giroux and Lacroix, 1998). Irradiation induced changes to protein are all free radical-mediated reactions, including primary deamination (cleaving amino group at the end terminus of the protein chain) and secondary deamination (cleaving amino group at the middle of the protein chain), decarboxylation, reduction of disulfide linkages, oxidation of sulfydryl groups, breakage of peptide bonds by alpha-amidation mechanisms and changes of valency states of the coordinated metal ions in enzymes (Diehl, 1990; Delincee, 1983). With increasing irradiation dosages, these physiochemical changes lead 13 to the denaturation and cross-linking of protein radical intermediates to form dimers, trimers and even tetramers in the absence of oxygen. Analyses have indicated that almost 90% of aggregates formed are held together by new covalent bonds and not by disulfide linkages (Davie and Delsignore, 1987). In the presence of oxygen, little or no aggregates are formed. Instead, protein will denature and fragment into an array of low molecular weight protein fragments (Giroux and Lacroix, 1998). In the presence of an oxidized lipid, lipid-protein complexes, instead of the usual protein aggregate or protein fragment will form (Delincee, 1983; Delincee and Paul, 1981; Urbain, 1986), resulting in loss of protein solubility. The irradiation of myosin at dosages up to 10 kGy has been shown to result in the formation of protein aggregates, and loss of water-holding capacity (Zabielsky et al., 1984). Aggregation and loss of solubility of fibrillar proteins has been known to increase the tensile strength of meats. Moreover, the freezing of meat can also decrease both the water holding capacity and solubility of fibrillar protein, thereby enhancing the tensile strength of meats. Therefore, the irradiation of frozen meats should have higher tensile strengths than irradiated fresh meats, and this was confirmed the findings of Segars etal. (1981). 2.3 Physiochemical Qualities of Ground Beef 2.3.1 Lipid Autoxidation and Flavour Autoxidation of unsaturated fatty acids occurs via a free radical mediated chain-reaction, which is commonly represented in a simplified three-step mechanism shown in Figure 2. A catalyst is usually required to initiate autoxidation, and is typically a transition metal ion (e.g. iron or copper) or a free radical (e.g. hydroxyl radical or superoxide anion). However, autoxidation can also be initiated by exposure to a high energy source such as heat, light, U V radiation or ionizing radiation. Once initiated, a lipid free radical is formed and in the presence oxygen, the reaction is propagated by the formation of a peroxide and a second lipid free radical. 14 Autoxidation is terminated when two free radials randomly collide to form a stable product, complete oxidation of all PUFA occurs, or free radicals are scavenged by an antioxidant. The ground beef fatty acids consist of 46% saturated, 51% monounsaturated and 3% polyunsaturated (Johnson et al., 1984). In the unsaturated fatty acid fraction, 14% are of linoleic acid (18:2), in addition to oleic acid (18:1), palmitoleic acid (16:1), linolenic (18:3) and arachidonic acid (20:4) (Giroux and Lacroix, 1998; Dugan, 1987). Membrane phospholipids containing PUFA account for only 0.5 to 1.0% of the overall lipid content in meat (Giroux and Lacroix, 1998), but are the major contributing factors for the development of rancidity in beef (Meynier et al., 1999; King et al, 1995; Schaefer et al, 1995; Buckley et al., 1989). The oxidation of phospholipids has been widely accepted as the cause of off-odour in raw meat and warm-over-flavour (WOF) generated in cooked and refrigerated meats. Oxidation of beef lipids during storage was found to be higher than that of chicken and pork lipids (Rhee et al, 1996). These researchers explained that their findings are based on the larger proportion of heme iron present in beef compared to pork or chicken. Heme iron acts as a catalyst of lipid oxidation (Brown et al., 1963;Verma et al., 1985; Apte and Morrissey, 1987; Yin and Faustman, 1993) and subsequent release of the heme iron to free iron during processing can increase lipid oxidation in meats (Morrissey et al., 1998; Monahan et al., 1993; Rhee and Ziprin, 1987). Furthermore, the production of superoxide anion during the oxidation of deoxyMb to metMb can be dismutated to hydrogen peroxide, which in turn, is further oxidized to a «OH by ferrous ion. These sources of free radicals are potent prooxidants of phospholipids (Yin and Faustman, 1993). Therefore, the initiation of lipid oxidation in meats can be attributed to both the heme iron concentration and the oxidative reactions of Mb. 2.3.2 Colour Stability The redox state of the heme iron and the presence and nature of the ligand bound to Mb accounts for the colour of beef. In general, ground beef turns brown more rapidly than unground 1 5 1. Initiation: L i H + Catalysts Lr + FT where L = lipid moiety H = hydrogen atom Catalysts = transition metals (e.g., Fe and Cu), free radicals (e.g., •OH) or radiation (e.g., U V light, y-ray or electron beam irradiation) 2. Propagation: r* ^ 1 * - L i « + 0 2 -> L i O O * L i O O ' + L j H -> L i O O H + L 2 - - > where L O O * = lipid peroxyl radical L O O H = lipid hydroperoxide 3. Termination: Li/2* + Li/2* —> Non-radical product Li/2* + Li /200* —> Non-radical product L i / 2 0 0 * + Li /200* —» Non-radical product Figure 2 - Mechanism of autoxidation of unsaturated fatty acids (adapted from Nawar, 1985). 16 beef, a consequence of the grinding process which increases exposure to air and enhances the chance for microbial contamination (Mitsumoto et al., 1993). In addition, grinding meat also increases the loss of intracellular reductants such as N A D H (Renerre, 1990). Under an aerobic environment, autoxidation of the heme iron in the deoxyMb complex and the reduction of oxygen tension on the surface by bacteria growth results in an accumulation of metMb (Renerre, 1990). Mitsumoto et al. (1993) reported that ground beef stored at 4°C under aerobic conditions for 9 d contained 86.8% of Mb as metMb on the surface. This result was supported by the findings of O'Grady et al. (1998) where 89.7% metMb was detected after 8 d under similar storage conditions. It is generally accepted that the redox reaction involving the interconversion of deoxyMb and metMb dependts upon the degree of lipid oxidation. An increase in lipid oxidation in ground beef has been associated with an increase in the development of brown colour (Lanari et al., 1996; Schaefer et al., 1995; Yin and Faustman, 1993; Harel and Kanner, 1985a). Peroxyl radicals generated from lipid oxidation have been suggested to act as promoters of deoxyMb oxidation (Lanari et al., 1996; Mitsumoto et al, 1993). Furthermore, the production of superoxide anion during the oxidation of deoxyMb to metMb can initiate lipid oxidation (Schaefar et al., 1995). Superoxide anion can also be dismutated to hydrogen peroxide, and when in the presence of heme or free iron, can be further degraded into hydroxyl radicals, thereby providing a source for initiating unsaturated fatty acid oxidation (Harel and Kanner, 1985b; Verma et al, 1985; Brown et al., 1963). 2.3.3 Texture Meat structure can be considered, in its simplest form, as a collection of parallel fibers, with a myofibrillar structure, that are bound together by a connective tissue network. Therefore, the main factors affecting texture of meat will involve the condition of the myofibrillar proteins, muscle cytoskeleton, intramuscular connective tissue and the intrafibre water bound to the 17 protein components (Palka and Daun, 1999). In the case of ground beef, the levels of fat and mechanical processing are additional factors that will affect the final texturaf properties of the product. The proportion of connective tissue to muscle fiber can influence meat texture. A large proportion of connective tissue distributed within the muscle decreases the rate and extent of myofibril disintegration during aging. The presence of larger fibers is correlated with a lower level of connective tissue (Henrickson, 1978). Since water is bound to protein muscle fibers, extremely low pH that induces denaturation of sarcoplasmic proteins and their aggregation onto the myofilaments will reduce the available spaces for water-binding. As a result, meat will tend to be more palatable due to an increased release of exudate when pressure is applied during chewing. Additional factors influencing meat texture, including the age of the cattle, the breed of the cattle, the physiological status of the cattle before slaughtering and the activity of proteolytic enzymes such as cathepsin. By far, the level of fat in ground beef imparts the greatest influence on texture. Increasing fat levels from 14 to 24% produced ground beef patties with a decreasing perception of hardness as measured by the single blade shear test (Berry and Leddy, 1984; Troutt et al., 1992). A decrease in shear force, which reflects textural quality, can be attributed to a reduction in the protein content of ground beef. Reducing the diameter size of a grinding plate has been reported to create greater softness of ground beef, faster sample breakdown in chewing and a greater number of smaller size chewed pieces (Berry et al., 1999). Since smaller meat pieces were correlated to greater tenderness, Berry et al. (1999) further stated that fully hydrated muscle fibers similar with those of hot-processed beef produced smaller pieces during grinding. This effect of grinding was attributed to the more hydrated fibers (typical of pre-rigor or high pH muscle) that are rather immovable in the grinder and therefore resisted grinding. It is conceivable that when muscle resists motion through the meat grinder assembly, greater grinder 18 blade contact/unit of muscle occurs along with repeated cutting on the same area of meat, which produces smaller pieces of meat. Smaller pieces of ground meat, in conjunction with a high fat content, will produce a softer texture in ground beef. 2.4 Antioxidants 2.4.1 Ascorbic Acid in Meat In cattle, ascorbic acid is synthesized in the liver to meet the metabolic demands of the animal. Under certain environmental and physiological conditions, the amount of endogenous ascorbic acid may be insufficient to meet requirements. Lactating cows are especially prone to subclinical ascorbic acid deficiency because of the huge drain on glucose, the precursor for ascorbic acid for lactose production (Macleod et al., 1999). Non-lactating ruminants under stress may also exhibit a similar deficiency (Hirdoglou et al., 1977). This situation arises because ascorbic acid has a possible role in the production and regulation of adrenal corticosteriods; the production of which has been reported to reduce circulating plasma levels of ascorbic acid (Fin and Johns, 1980). Infection by pathogenic organisms may also drain the endogenous ascorbic acid pool that is associated with immune function. Ascorbic acid, in vivo, facilitates the migration of neutrophils to the site of inflammation (Macleod et al., 1999). Furthermore, the antioxidant capacity of ascorbic acid can scavenge free radicals and protect the structural integrity of in cells of the immune system. The concentration of surplus ascorbic acid in meat is minimal, ranging from 0 to 17.6 mg/kg (Anderson and Hoke, 1990; Jaffe, 1984). For example, plasma and longissimus concentrations of ascorbic acid in Holstein steers were reported to be 8.8 and 5.3 mg/kg, respectively (Brubacher et al., 1985). Dietary supplementation of ruminant animals with ascorbic acid is not of much value since it is rapidly matabolized by the rumen microorganisms (Macleod et al., 1999). Studies with ascorbic acid analogues fed to Hostein heifers (e.g. 80 g of 19 ascrobyl-2-polyphosphate/head/day for 31 d) resulted in an increase of ascorbic acid in both plasma (4.56 mg/L) and biceps femoris (10.11 mg/kg) over that of control (3.58 mg/L in plasma and 8.32 mg/kg in biceps femoris) (Macleod et al., 1999). Alternatively, pre-slaughter intravenous injections of ascorbic acid into the jugular vein (50% w/v solution of sodium ascorbate) have resulted in deposition of ascorbic acid in muscle at 100-200 mg/kg tissue, and residual levels of ascorbic acid were detectable after 10 d storage at 0°C under vacuum (Hood, 1975). Wheeler et al. (1996) also attempted to inject a sodium ascorbate solution intramuscularly, into cattle at concentrations of 0.25 to 4% and obtained some success in deposition. Post-mortem addition of ascorbic acid to meat can also be achieved by either mixing it with an ascorbic acid solution of 0.01 to 1% (v/w) to muscle (Lee et al., 1999; Shivas et al., 1984; Mitsumoto et al., 1991a), or by applying a topical/dip treatment of an ascorbic solution at concentrations ranging from 1 to 10% (Harbers et al., 1981; Mitsumoto et al., 1991a; Mistumoto etal., 1991b). 2.4.2 Protection Against Oxidative Reactions The primarily role of ascorbic acid in meat is as an antioxidant against peroxidation reactions. The mechanism of protection comes from its ability to scavenge free radicals and singlet oxygen by donating either one or two electrons to the oxidized species (Schaefer et al., 1995). Ascorbic acid can also protect the red colour of meat by preventing autoxidation of deoxyMb to metMb (Faustman and Cassens, 1990; Harbers et al., 1981) and the reduction of metMb to deoxyMb (Lee et al., 1999; Schaefer et al., 1995). Finally, ascorbic acid can also regenerate endogenous a-tocopherol after its oxidation to the a-tocopheryl radical (Schaefer et al., 1995). The post-mortem application of blending ascorbic acid into ground beef to retard lipid oxidation has been reported. A decrease in TBAR value between 38 to 58% by 0.05 to 0.1% ascorbic acid (v/w) over control was observed by Lee et al. (1999) while a decrease in TBAR 20 value by 73% was observed by blending ascorbic acid to a level of 0.5% (Mitsumoto et al., 1991a). However, topical applying or dipping of beef steaks with higher concentrations of ascorbic acid (1,5 and 10%) reduced its efficiency to lower TBAR value, producing a decrease between 24 to 56% (Harbers et al., 1981; Mitsumoto et al., 1991a; Mistumoto et al., 1991b). The decrease in efficiency in lowering TBAR value may be caused by inadequate penetration of ascorbic acid into the site of oxidation by the dipping treatment. Endogenous addition of ascorbic acid can improve beef colour stability. Hood (1975) demonstrated an increase in colour stability by 2 d, with only a 20% pigment conversion from oxyMb/deoxyMb to metMb (limit of commercial acceptability) by injection of ascorbic acid. Extended storage stability of meat redness was reported to be approximately 5 to 7 d longer than control when injected intramuscularly (Wheeler et al., 1996). Surface redness, as measured by Hunterlab a value for redness, was also stabilized after 3 d of storage by mixing ascorbic acid into ground meat (Lee et al., 1999). Moreover, metMb concentration was maintained below 20% of the total surface pigment for up to 5 d (Shivas et al, 1984), and up to 7 d for total extracted pigment (Mitsumoto et al., 1991a) using the same method of application. The efficiency of ascorbic acid in stabilizing surface colour has led to the USDA approval for addition of ascorbic acid to fresh beef and lamb to delay discoloration (USDA, 1994). The combined effect of ascorbic acid and a-tocopherol is to offer better protection against lipid oxidation and Mb autoxidation than when either compound is applied alone. Okayama et al. (1987) dipped beef loin steaks into an ethanolic solution containing ascorbic acid, a-tocopherol, or both, and found that the combination treatment had the least amount of lipid and Mb oxidation after 13 d storage. Similar results were obtained by Mitsumoto et al. (1991a and b) with beef loin steaks. Thus, it would be worthwhile to investigate the interaction of dietary a-tocopherol and ascorbic acid administered to muscle by ante-mortem infusion versus post-mortem addition to meat. 2 1 2.4.3 Antioxidants and Microbial Growth Lipid oxidation products, peroxides and free radicals may have antimicrobial properties. During irradiation, the free radicals produced from radiolysis of water and lipid oxidation can contribute to the overall reduction of the microbial population. Therefore, the addition of antioxidants (e.g. ascorbic acid or a-tocopherol) to beef may protect against the destruction of bacteria during irradiation or during refrigerated storage. The effects of antioxidants on microbial growth were studied by Cabedo et al. (1998). Aerobic bacteria, coliforms, E. coli 0157:H7 and Listeria monocytogenes were monitored in ground beef patties made from beef derived from cattle supplemented with a-tocopherol at 1000 and 2000 IU/head/day for 100 d. Throughout the 10 d storage study, conducted at 4 and 12°C, bacteria growth of aerobic organisms, coliforms and E. coli 0157:H7 was not affected by either a-tocopherol treatment or the two different temperatures (Cabedo et al., 1998). Colonies of Listeria monocytogenes were also not affected by a-tocopherol treatment, but proliferated more extensively at 12°C. Similar findings were observed with the endogenous addition of a-tocopherol (Arnold et al., 1993; Chan et al., 1995; Garber et al., 1992) and ascorbic acid to ground beef (Shivas et al., 1984). In the study by Cabedo et al. (1998), the visual discoloration of beef occurred before the microbial population reached an unacceptable level of 107CFU/g. 2.5 Experimental Objectives In the following three chapters, the mechanism of antioxidant activity of LAA and the effects of irradiation on bacterial count and physiochemical quality changes in beef will be examined. The objectives of the thesis experiments were to: 22 • In Experiment 1, the objectives are to determine the »OH generation in the presence of Hb, measure the L A A oxidation after irradiation and evaluate the effects of L A A and irradiation on lipid oxidation in various aqueous and emulsion model systems (Chapeter 3); • In Experiment 2, the objectives are to the examine the effects of irradiation, L A A and fat level on aerobic plate count, total coliform count, E. coli count and psychrotroph count of ground beef patties (Chapter 4); and • In Experiment 3, the objectives are to the examine the effects of irradiation, L A A and fat level on physiochemical quality changes of lipid oxidation, surface redness and hardness attribute of ground beef patties (Chapter 5). 23 3. Experiment 1: Model Studies 3.1 Materials and Methods All chemicals used were of reagent grade or better. Sodium dihydrogen orthophosphate, ferrous chloride, ferric chloride, metaphosphoric acid and L-ascorbic acid (LAA) were obtained from BDH Chemicals Co. (Toronto, ON). Tween 20 was obtained from Difco Laboratories (Detroit, MI). Linoleic acid (LA), BHA, Na 2 -EDTA, thiobarbituric acid (TBA), 2,6-dichloroindophenol, Tris-HCl, Tris-NaOH, hemoglobin (Hb) and Mb were purchased from Sigma Chem. Co (St. Louis, MO). Deoxyribose was acquired from Applied Science Lab Inc (PA). Sodium phosphate, sodium hydroxide, sodium bicarbonate, ethanol, hydrogen peroxide, hydrochloric acid, trichloroacetic acid (TCA), acetic acid and ammonium thiocyanate were purchased from Fisher Scientific Co. (Fair Lawn, NJ). Only distilled deionized water was used during the experimentation. 3.1.1 Hydroxyl Radical Generation Hydroxyl radical generation, in vitro, was measured according to the modified method of Halliwell et al. (1987). Stock solutions of Na 2 -EDTA (1 mM), FeCl 3 (10 mM) or Hb (0.01 g/mL), L A A (1 mM), H2O2 (10 mM) and deoxyribose (10 mM) were prepared in distilled deionized water. The assay was performed by adding 0.1 mL Na2-EDTA, 0.01 mL FeCl3 and/or 0.1 mL Hb, 0.1 mL H 2 0 2 , 0.36 mL deoxyribose, 1.23 to 1.33 mL Tris-buffer (5 mM, pH 7.4) and 0.1 mL L A A , in sequence, to a total volume of 2 mL. The mixture was then incubated at 37°C in a water bath for 1 h in the dark. Following incubation, a 1 mL aliquot of the incubated mixture was mixed with 1 mL of 10% T C A and 1 mL of 0.5% T B A (in 0.025 M NaOH containing 0.02% BHA) and heated in a boiling water bath for 15 min to develop colour. The pink chromogen of thiobarbituric acid reactive substance formed was measured at 532 nm, against a 24 heated reagent blank containing TCA, T B A and distilled deionized water, with a Shimadzu UV160 UV-visible spectrophotometer. 3.1.2 Ascorbic Acid Depletion Study Two simplified model systems were used to assess the depletion of L A A after irradiation. An aqueous model system in a total volume of 16 mL was prepared by mixing 15.5 mL of 10, 100 or 500 mM L A A (in 0.2 M sodium phosphate buffer at pH 7.0) with 0.5 mL of Mb (0.01 g/mL). An emulsion model system, of the same total volume, was also prepared by modifying the above model to contain 14.5 mL of either 10, 100 or 500 mM L A A (in sodium phosphate buffer), 0.5 mL of Mb, 0.5 mL L A and 0.5 mL Tween 20. All samples from both model systems were transferred to 30 mL tissue culture flasks and kept at 4°C during transportation to and from the irradiation facility. The samples were irradiated at 0 (control), 2.5, 5 or 10 kGy using an electron beam (Iotron Technologies Inc., Port Coquitlam) at room temperature. After irradiation, all samples were stored in 50 mL Erlenmeyer flasks at 4°C under normal lighting. Ascorbic acid in model systems was measured by modifications of the vitamin C:2,6-dichloroindophenol method (AOAC, 1980). Prior to analysis, a metaphosphoric-acetic acid solution (15 g H P 0 3 dissolved in 200 mL HO Ac and diluted to 500 mL) was prepared and stored in an amber bottle at 4°C. The colour dye 2,6-dichloroindophenol (50 mg dissolved in 50 mL distilled deionized water containing 42 mg sodium bicarbonate and diluted to 200 mL with distilled deionized water) was prepared immediately before use. Ascorbic acid was determined by titrating a 7 mL mixture containing an aliquot (0.01, 0.1 or lmL; depending on ascorbic acid concentration) of sample in metaphosphoric-acetic acid against an indophenol solution until the end-point, as indicated by a stable pink colour, occurred. Ascorbic acid content was calculated from three standard curves relating specific ranges of ascorbic acid concentration to the volume of indophenol required for titration (Appendix 1). 25 3.1.3 Ultraviolet Light-Induced Lipid Oxidation The effects of ultraviolet (UV) light and L A A on lipid oxidation was determined using a. model emulsion system. This model system was prepared by mixing L A (1 mL), Tween 20 (1 mL), 0.2 M sodium phosphate buffer at pH 7.0 (20 mL), L-ascorbic acid (1 mL) from stock solutions of either 10, 100 or 500 mM (prepared in distilled deionized water) together, and adjusting the total volume to 25 mL with distilled deionized water. The mixture was vortexed until an emulsion is formed and then transferred to a 30 mL Falcon Tissue Culture Flask (Fisher Scientific, NJ). All samples in tissue culture flasks were exposed to 15 min of U V light (A,=254 nm; intensity of 1250uW/cm2) (Fisher Scientific, N.J.) at a distance of 4 cm at 4°C. Afterwards, the emulsion was stored in 50 mL Erlenmeyer flasks at room temperature under normal lighting for 14 d. Samples were periodically removed to measure the degree of lipid oxidation by the ferric-thiocyanate method (Wong and Kitts, 2001). The assay was performed by adding 5 mL ethanol (75%), 0.1 mL ammonium thiocyanate (30%), 0.1 mL emulsion and 0.1 mL ferrous chloride (20 mM in 3.5% HC1) in sequence. The mixture was vortexed and allowed to stand for 5 min at room temperature. Colour development in the reaction mixture was then measured at an absorbance of 500 nm against ethanol (75%). 3.1.4 Electron Beam Irradiation-Induced Lipid Oxidation The model emulsion system was prepared according to Section 3.1.3. All samples, stored in tissue culture flasks, were kept at 4°C during transportation to and from the irradiation facility. The samples were irradiated at 0 (control), 5, 10 or 20 kGy by electron beam at room temperature. After irradiation, all samples were transferred to 50 mL Erlenmeyer flasks and stored at room temperature under normal lighting. An aliquot of the emulsion was periodically removed to measure the degree of lipid oxidation by the ferric-thiocyanate method mentioned in the previous section (Section 3.1.3). 26 3.1.5 Statistical Analyses All treatments in all experiments were done in triplicate. Each experiment was conducted twice to assess reproducibility. Data were all analyzed by either an one-way or a two-way A N O V A and Tukey's Test for significance at a probability of p<0.05, using the MiniTab Statistical Software version 12.0 (MiniTab Inc., PA). Linear trend analyses, linear trend equations and correlation coefficients were determined by Microsoft Excel program (Microsoft Corp., USA). 27 3.2 Results 3.2.1 Hydroxyl Radical Generation The degree of hydroxyl radical (*OH) generation, represented by the quantity of oxidized deoxyribose-TBA adduct detectable at 532 nm, is presented in Figure 3. Regardless of the effects of heating and exposure to UV, the catalyst ferrous ion (Fe2 +) (generated from the reduction of ferric ion by LAA) generated twice the quantity of «OH in comparison to that of Hb. The combination of Fe 2 + and Hb increased the total concentration of iron in the sample mixtures; however, *OH production was not found to be statistically different from that of Fe 2 + alone. Incubating both sample mixtures of Fe 2 + and Fe 2 + plus Hb at 37°C (i.e. heat treatment) produced a significantly greater (p<0.05) «OH response than with the U V exposure. An opposite trend was noted when Hb was used alone in the place of Fe 2 + . The combination of Hb with U V exposure generated a significantly greater (p<0.05) •OH response than the combination of Hb with heat incubation. 3.2.2 Ascorbic Acid Depletion Study L-ascorbic acid (10 mM) oxidation, induced by electron beam irradiation in the presence of Mb and linoleic acid, during 4°C storage is illustrated in Figure 4. In general, the L A A content of all treatments decreased over time. The L A A oxidation was affected by irradiation dose, in the order of 10 kGy>5 kGy=2.5 kGy>0 kGy. At 10 kGy, complete oxidation of L A A in the linoleic acid emulsion occurred by day 3. In general, greater oxidation of L A A occurred in samples containing linoleic acid (approximately 15 to 20% more oxidation) throughout the storage period. No significant differences between L A A concentration values measured from day 1 to day 14 were found for all treatments. Rather a rapid loss of 10 mM L A A (i.e. between 31.8 to 96.5+0.5% of the original amount) occurred within a period of 24 h post-irradiation. By day 14, the final concentrations of the 10 mM L A A treatments fell within a range of 0.8 to 3.8 mM. 28 CO CO o u 0.40 0.35 0.30 - 0.25 cn 0.20 0.15 0.10 0.05 0.00 ax by * bx Fe(II) Hb Catalyst ax aby Fe(II) + Hb Figure 3 - Deoxyribose degradation by hydroxy radical generated from Fenton reaction initiated by Fe(II) and/or hemoglobin (Hb) under the influence of heat incubation (open columns) or UV light illumination (checkerboard columns). All values are mean+SEM; n=6. abDenotes the statistically significant (p<0.05) effects of various catalysts. xyDenotes the statistically significant (p<0.05) effects of heat and UV light. A Day Figure 4 - Quantitative analysis of 10 mM L-ascorbic acid during storage as influenced by (A) the absence of linoleic acid and (B) the presence of 3.13% (v/v) linoleic acid with an initial exposure to irradiation at 0 kGy (diamond), 2.5 kGy (triangle), 5 kGy (circle) and 10 kGy (square). All values are mean+SEM; n=6. abcDenotes the statistically significant (p<0.05) concentrations of ascorbic acid over time. xyzDenotes the statistically significant (p<0.05) effect of irradiation at a particular interval of time. The oxidation of 100 mM L A A by electron beam irradiation is shown in Table 1. No statistical difference in L A A concentration was observed at dosages of 0, 2.5 and 5 kGy. Only the 10 kGy irradiated samples produced a significant (p<0.05) loss of L A A over time. A significant (p<0.05) decrease in L A A concentration (i.e. between 18.6 to 31.2+0.5% of initial concentration) was observed for all treatments on day 3. By day 14, however, only an average of 17.4+0.5% of the original L A A concentration was found to be oxidized in all irradiated samples. The oxidation of L A A was not significantly affected by the presence of linoleic acid in any treatment. A minimal oxidation of 500 mM L A A was observed during storage (Table 1), with a general decreased in concentration ranging from 7.0 to 26.7+0.5% between day 0 and day 3. At the end of the storage period, the L A A content of all samples ranged from 80.0 to 96.2 + 0.5% of the original concentration. Fat and irradiation were found to have no significant individual or interactive effect on L A A oxidation at 500 mM concentration. 3.2.3 Ultraviolet Light-Induced Lipid Oxidation Antioxidant activity of L A A against U V light-induced linoleic acid oxidation was evaluated based on the ability of L A A to suppress the formation of lipid peroxides. The presence of peroxide was detected by the extent of Fe 2 + oxidation to Fe 3 + , which when coupled to thiocyanate produces a red pigment with a maximum absorbance at 500 nm. Figure 5 illustrates the U V light-induced oxidation of linoleic acid. Immediately after the UV light exposure (i.e. Time 0), both the control and the 10 mM L A A sample exhibited a significantly greater (p<0.05) degree of lipid oxidation compared to both 100 and 500 mM L A A treatments. After the 46th h post U V light exposure, only the 10 mM L A A treatment was found to have a greater degree of lipid oxidation than the control. 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All values are mean+SEM; n=6. abcDenotes the statistically significant (p<0.05) levels of peroxide within a particular treatment over time. wxyzDenotes the statistically significant (p<0.05) effect of L-ascorbic acid at a particular time interval. peroxide were achieved by the 70l h in the 10 mM L A A samples. It was not until the end of the storage period that the control treatment reached the highest concentrations of lipid peroxides. In contrast, the 500 mM L A A treatment was effective at preventing the UV-induced production of lipid peroxides, throughout the entire storage period. 3.2.4 Electron Beam Irradiation-Induced Lipid Oxidation The same linoleic acid system used in the above experiment was also used to assess the antioxidant activity of 10, 100 and 500 mM L A A following electron beam (e" beam) irradiation at 0, 5, 10 and 20 kGy. The effectiveness of L A A treatments to retard irradiation-induced lipid oxidation is presented in Figure 6. All irradiated samples attained the highest production of lipid peroxides after 72 h of storage, which in general was 24 h earlier than with samples exposed to U V light. Notwithstanding this, the presence of 10 mM L A A once again demonstrated a prooxidant activity in all samples, regardless of the irradiation dosage. The 100 mM L A A sample significantly (p<0.05) inhibited irradiation-induced peroxide production, in comparison to the control, with an increase in antioxidant activity occurring as the irradiation dosages increased. The 500 mM L A A treatment prevented irradiation-induced production of lipid peroxide throughout the entire storage period. A significant interaction (p<0.05) was noted between L A A and the presence of the irradiation dosage for the peroxide content in the emulsion. To investigate the relationship between electron beam irradiation dosage and lipid peroxide production, the maximum absorbance values, obtained from control samples (free of the influences of L A A on lipid oxidation) during storage, was plotted against the respective electron beam irradiation dosage (Fig. 7). A trend analysis was conducted on the plotted data and a significant (p<0.01) correlation coefficient (r2 = 0.9564) was determined, thus demonstrating a linear relationship between the increase in electron beam dosage (up to 20 kGy) and the increase in lipid oxidation products. 34 1 8 -, 1 6 -1 4 -at 500 nn 2 -0 -l o 8 -in OL 6 -0 4 -0 2 -0 0 * 0 0 kGy bw | | 24 48 Time (h) 1.8 1.6 1.4 | 1.2 o •e o 1/1 3 0.6 0.4 0.2 0.0 -- bw 11 / / / -# / - aw / bx£ aw „ -1 >^bVI ^ . . . b z . 24 48 Time (h) 72 10 kGy 20 kGy 24 48 Time (h) 72 24 48 Time (h) 72 Figure 6 - Effects of 0 mM (diamond), 10 mM (square), 100 mM (triangle), and 500 mM (circle) L-ascorbic acid and electron beam irradiation at 0 kGy, 5 kGy, 10 kGy and 20 kGy on the oxidation of linoleic acid in an emulsion system. All values are mean+SEM; n=6. abcDenotes the statistically significant (p<0.05) levels of peroxide within a particular treatment over time. wxyzDenotes the statistically significant (p<0.05) effect of ascorbic acid at a particular time interval. 35 10 Dose (kGy) 15 20 Figure 7 - A regression analysis of the relationship between irradiation dosages and oxidation of linoleic acid emulsion system without the influence of L-ascorbic acid. AH values are mean+SEM; n=6. 36 3.3 Discussion In biological systems, ' O H is commonly produced from the oxidation of H2O2 and soluble Fe 2 + (Aruoma et al., 1987; Puppo and Halliwell, 1988). As expected, the addition of Fe 2 + (from the reduction of Fe 3 + in FeCl3), solubilized by EDTA, directly generated the highest concentration of ' O H (Fig. 3). The ' O H was also generated in the presence of Hb. Puppo and Halliwell (1988) have reported similar findings but concluded that intact Hb does not generate •OH by direct interaction of H 2 0 2 with the heme iron, but rather by the released Fe 2 + from the H 2 0 2 oxidized metHb. In this study, the exposure of the Hb samples to UV light generated more »OH compared to conventional heat incubation. Andley and Clark (1989) have reported the oxidation of protein and the formation of H 2 0 2 and 02* by exposure to U V light under aerobic conditions. Specifically, Whitburn et al. (1982) observed the generation of H 2 0 2 , by y-radiolysis of an aqueous ferrimyoglobin solution that resulted from the union of two * 0 H produced from oxidized proteins. It is possible, therefore, that the observed effect of U V light on Hb catalyzed •OH generation in the present study was indeed due to the oxidation of Hb, resulting from the direct generation of * 0 H . The rapid oxidation of 10 mM L A A samples after 24 h of post-irradiation storage appeared to be more pronounced when the dosage of irradiation was increased, even thought no statistical difference (p>0.05) was found between L A A concentration of all irradiated samples. A moderate loss of L A A in both 100 and 500 mM samples was also observed by day 3 of storage, even thought no statistical difference (p>0.05) was found in 500mM L A A samples. These findings agree with the previous results of Tobback (1977), who also showed that the sensitivity of L A A increases as the dilution of L A A increases. Since L A A oxidation can also be induced by soluble iron, Mb (a chelated form of iron) was added to the L A A to enhance the electron beam induced L A A oxidation. However, the iron in Mb was probably unavailable for 37 L A A oxidation since Hb generated less *OH than EDTA-solubilized iron (Fig. 3). Therefore, oxidation of L A A in the present study is likely due to the presence of «OH generated from radiolysis of water or the oxidation of Hb or Mb during irradiation (Fig. 3). Furthermore, our results showed that the lipid peroxyl radical scavenging activity of L A A could also lead to the oxidation of L A A in the lipid emulsion model. This suggestion was confirmed by the greater depletion of L A A observed in samples containing linoleic acid. Thus the combination between irradiation-induced oxidation and free radical scavenging activity of ascorbic acid collectively contributed to the rapid lost of L A A in our study. Surprisingly, the destruction of L A A (i.e. decrease in concentration) detected after 24 h. storage was slower in our study than that observed by other workers. Lee et al. (1999) observed a complete oxidation of L A A in buffer catalyzed by C u 2 + within 25 min of reaction at room temperature. Perhaps the storage temperature used in our study (4°C) delayed the oxidation of L A A relative to that of Lee et al. (1999). This observation has also been observed by Allan (1950), where the time for L A A oxidation in butter stored at -13°C was about 12 h, compared to 2 h when the temperature was raised to 20°C. After an initial reduction in L A A concentration, a subsequent increase in L A A concentration in both the 100 and 500 mM concentration samples occurred after 3 d storage. This temporal change of L A A content suggests an accumulation of the reversible dehydroascorbic acid (DAA), which can be regenerated back to L A A in the presence of a reducing agent. This observation did not occur in the 10 mM L A A samples, indicating a likely rapid oxidation of D A A to diketogulonic acid at low concentrations before D A A was able to be reduced back to L A A . Tobback (1977) has also observed similar findings and proposed that a disproportion mechanism occurring between partly oxidized and partly reduced L A A was the explanation. Taken together, these results suggest a potential self-regeneration process between two L A A molecules, which could occur only in an oxygen-free aqueous solution. Therefore, our findings may be explained by the possibility that the dissolved oxygen in the buffer, or in the headspace of the flask, was 38 consumed by day 3 of storage due to oxidation consumption during the aerobic oxidation pathway. This would provide a limited oxygen environment in the aqueous solution for the self-regeneration of L A A . High intensity U V light (k=254 nm) induces photooxidation of PUFA. Melo and Mahmoud (1988) have shown, by electron pair resonance (EPR), that U V light (X<310 nm) produced both superoxide radical (CV) and peroxyl radical (LOO*) by homolytic cleavage on PUFA under aerobic conditions. In the presence of 100 and 500 mM L A A , the scavenging of 02* and/or LOO* by excess L A A could have prevented lipid peroxide formation immediately after U V light exposure. It is possible that 10 mM L A A was unable to scavenge all the generated 02* and/or L O O , which in turn resulted in a lipid peroxide content not statistically different from that of the control. Once the L A A donated two hydrogen (FT) atoms to free radials, an ascorbate radical anion would be formed, before further conversion to D A A (Tannenbaum et al., 1985). Moreover, accumulation of an ascorbate radical anion initiates lipid oxidation (Mahoney and Graf, 1986), therefore, eliciting a prooxidant effect. This observation occurred for the 10 mM L A A concentration starting at approximately 25 h of incubation, since 40% of the initial L A A content degraded at this time was coupled to a marked increase in peroxide level. By the 40th h of incubation, the peroxide content present in the 100 mM L A A treatment was equal to that measured in the control. This result paralleled the finding that 25 to 30% of the initial L A A content had been oxidized after 40 h. The results suggest that as the amount of peroxide formed exceeded the capacity of the remaining 100 mM L A A to scavenge free radical, an ineffective concentration for antioxidant activity against U V light photooxidation occurred. This theory is supported by the observation that 500 mM L A A treatment retained a significant amount of L A A to retard the formation of lipid peroxides during the entire storage period. 39 The same linoleic acid emulsion model was also subjected to e" beam irradiation. Similar to U V exposure, irradiating the model system by e" beam produces free radicals that can initiate lipid oxidation reactions. Since water was the main component of our model, radiolysis of water by e" beam irradiation likely occurred to produce e"aq, *OH and H2O2, with all three by-products having the potential to initiate oxidative damage of macromolecules (Thakur & Singh, 1994). In general, oxidation reactions in the control and the 10 mM L A A treatment at all irradiation dosages began to increase after 48 h of storage. This change was expected due to the loss of 40% L A A by 24th h of storage. Similar to the U V light-induced oxidation experiment, all 10 mM L A A treatments accumulated lipid peroxides to an extent greater than the control. In contrast, both the 100 and 500 mM L A A concentrations exhibited antioxidant activity; with 100 mM L A A retarding the degree of oxidation during propagation, while 500 mM L A A prevented propagation from occurring during 72 h storage. Former studies have shown that a low dose of irradiation (as low as 5 Gy) of unsaturated fatty acids (e.g. methyl oleate or linoleic acid) produces lipid peroxides and that the rate of the autoxidation reaction varies markedly with the irradiation dose (Nawar, 1977). The peroxide content observed in the irradiated model system used herein was also dose-dependent, depicted by the peroxide content of irradiated control samples seen after 72 h of storage. This was best illustrated by the fact that an increase in the irradiation dose exhibited a linear increase in the peroxide content. However, a similar trend was not observed when L A A was added to the mixture. 40 3.4 Conclusion Under the current experimental conditions, both EDTA-solubilized Fe 2 + and Hb generated *OH via the Fenton reaction. However, exposure of Hb to U V light increased the production of *OH. Therefore, the heme iron in Hb was concluded to be inactive in the Fenton reaction when in its native form, but with possible denaturation or degradation of Hb by U V light, the release of iron from heme can participate in the Fenton reaction, the oxidation of ascorbic acid and the peroxidation of unsaturated fatty acids. L-ascorbic acid, at 10 mM, was rapidly degraded by 60 to 80% of its initial concentration after an electron beam irradiation dose of less than 5 kGy, following 1 d of storage at 4°C. Increasing the dose of irradiation to 10 kGy completely degraded all of the added L A A after 1 d of storage. Both 100 and 500 mM L A A also exhibited a moderate (20%) loss of L A A content by day 3 of storage. However, the regeneration of L A A was thought to have occurred when an increase in L A A concentration, comparable to the original amount added, was observed after 3 d of storage. U V (X=254 nm) photooxidation produced maximum peroxide after 96 h of storage while e' beam irradiation produced even higher levels of peroxide after only 72 h of storage. Regardless of the forms of irradiation, a 10 mM L A A solution exhibited potential prooxidant activity while a 100 mM L A A solution demonstrated marginal antioxidant activity. Only the 500 mM L A A solution was observed to exert superior antioxidant activity in both situations. These results indicated a biphasic concentration dependent prooxidant/antioxidant activity of L A A in the linoleic acid emulsion under the influence of both U V and e- beam irradiation. 41 4. Experiment 2: Irradiation Effects on the Intrinsic Factors and Microbial Population in Ground Beef Patties 4.1 Materials and Methods 4.1.1 Cattle Raising and Ascorbic Acid Infusion Three healthy non-pregnant beef cows (Hereford x Angus), between 5 to 8 y of age and weighing between 500 to 650 kg, were raised in the Agriculture and Agri-Food Canada Research Station in Lacombe, Alberta. All cows were raised on pasture and finished on a diet consisting of 90% barley silage and 10% rolled barley for 2 m prior to experimentation, which resulted in an approximate weight gain of 1.3 kg per head per day. At time of slaughter, the cows were stunned, shackled by both hind legs, raised and exsanguinated. Afterwards, the abdominal cavity was opened, contents removed, and the carcass was arbitrarily divided into a control half (left side) and an infused (i.e. treatment) half (right side). Before infusion of ascorbic acid solution started, the femoral vein on the left half (control) of the carcass was severed to limit circulation of the infused ascorbic acid solution to the left hindquarter. Approximately 17.5 L of sodium ascorbate solutions of 10 mM, 100 mM or 500 mM, were infused, respectively, into the right half at 37°C and 15 psi. An equal volume of saline solution was infused into the control, left half of the carcass at the same temperature and pressure. The carcass was then chilled at 2°C for 24 h. Six major muscles groups (i.e. semimembranosus, semitendinosus, biceps femoris, gluteus medius, adductor and tip muscle) were removed from both halves of the carcass and vacuum sealed, frozen (at -18°C) and transported (within 6 h) to the Department of Food Science at The University of British Columbia for further analyses. 4.1.2 Production of Ground Beef Patties Production of ground beef patties, from the frozen muscles stored at -18°C for 4 wk., was carried out at 4°C. All beef muscles were first trimmed of visible fat and connective tissue. 42 Muscles receiving 10, 100 and 500 mM of L A A infusion were pooled together and analyzed as one treatment (i.e. L A A infused). All muscles (i.e. from both control and L A A infused treatment) were cubed (approximately 3 x 3 x 3 cm) and ground (3/16 in plate) in a Hobart Grinder Model 84142 (Don Mills, ON), without added fat to obtain extra lean ground beef (with a crude fat content determined to be approximately 4% (w/w) by extracting the lipid in the patties with a 2 parts chloroform to 1 part methanol solvent followed by analysis of the lipid concentration by the gravimetric method described by Folch et al., 1957). Lean and regular ground beef were obtained by the addition of an appropriate amount of tallow to achieve a final patty crude fat content of 17 or 30% (w/w), based on a lean beef lipid content of 4%. The resulting ground beef were manually shaped, with a hand-held patty maker, into patties of 150.0+1.0 g weight, and dimensions of 10.5 cm (diameter) x 1.5 cm (thickness). Each patty was stored in a ZipLoc 1 M polyethylene bag and frozen at -30°C for 5 d until irradiated. 4.1.3 Electron Beam Irradiation Procedure Individually packaged frozen patties were placed in a single layer to be irradiated at room temperature on one side using a 60 kW high capacity high energy (10 MeV) electron beam accelerator (Iotron Technologies Inc., Port Coquitlam) at 5, 10 and 20 kGy. Control samples (i.e. 0 kGy) were not irradiated but were also kept at room temperature during the duration of the irradiation treatment. After this physical handling, the frozen ground beef patties were stored at 4°C in their original ZipLoc™ bags for a period of 14 d. As a result of the combination between the infusion of ascorbic acid, adding three different levels of fat exposed to four irradiation dosages, a total of 24 different treatments were generated (Figure 8). All 24 different treatments, performed in triplicate, were subjected to analysis described herein at pre-determined intervals during storage. Ground Beef Saline-Infused F at 4' F at 17 % (irradiation kGy 0 5 10 20 F at 30 % Irradiation kGy 0 5 10 20 Ascorbic acid-Infused F at 4' y„ F at 17 % Irradiation kGy 0 5 10 20 Irradiation kGy 0 5 10 20 F at 30 % Irradiation kGy rradiation kGy 0 5 10 20 0 5 10 20 Figure 8 - Flow diagram of experimental design. 44 4.1.4 pH of Ground Beef The pH values of the ground beef patties were periodically measured by removing two (2.0+0.5 g) portions from ground beef patty and homogenizing in a laboratory blender with distilled deionized water to a total of 20+0.5 mL. The homogeneous mixture was filtered through Whatman #540 filter paper and the pH of the filtrate was measured, at room temperature, using an Accumet pH Meter (Fisher Scientific, NJ). 4.1.5 Microbial Population 4.1.5.1 Aerobic Plate Count Enumeration of aerobic bacteria was determined according to the MFHPB-34 method (Health Protection Branch, 1997). Two 25.0+0.5 g portions were aseptically removed from each ground beef patty and added to 225.0+1.0 mL of 0.1% peptone water (1:10 dilution) in a stomacher bag (Seward, UK). All samples were then homogenized in a Stomacher Lab-Blender 400 (Seward, UK) for 1 min, and serial dilutions (i.e. 1:10 to 1:100,000) were made in 0.1% peptone water. A 1.0+0.1 mL aliquot of the diluted homogenate was plated onto a 3M Petrifilm Aerobic Count Plate (St. Paul, MN) and incubated for 48±2 h at 35±1°C. 4.1.5.2 Escherichia coli and Total Coliform Enumeration of Escherichia coli (E. coli) and total coliform was conducted according to the procedures described in Section 4.1.5.1; with the modification of plating a 1.0+0.1 mL aliquot of the diluted homogenate onto a 3M Petrifilm E. coZ/VColiform Count Plate (St.Paul, MN), prior to incubation at 48±2 h at 35±1°C. 4.1.5.3 Psychrotroph Count Enumeration of psychrotroph bacteria was determined according to the procedures described in Section 4.1.5.1. A modification involved plating a 1.0+0.1 mL aliquot of the diluted homogenate onto a 3M Petrifilm Aerobic Count Plate (St.Paul, MN) prior to incubation at 20±2°C for 5 d. 45 4.1.6 Statistical Analyses All treatments, in triplicate, were sampled twice periodically in all experiments. The effect of the levels of L A A infusion on pH and microbial data recorded in the ground beef patties was first assessed with a /-test and the probability of making a Type II error was computed. Analysis of variance (ANOVA) was performed on data using the General Linear Model (ascorbic acid, fat, dose, time, and all combinations) using Minitab Statistical Software version 12.0 (MiniTab Inc., PA). Significant difference between means were determined by Tukey's Test (p<0.05). Correlation (Pearson's product moment) were also determined using the Minitab Statistical Software. 46 4.2 Results 4.2.1 pH of Ground Beef The p H values of all ground beef patties are given in Table 2a and 2b. In general, initial pH values ranging from 5.05+0.16 to 5.56+0.03 were obtained for all patties. During storage up to 14 at 4°C d, a gradual increase in p H values was observed for all patties regardless of the experimental treatment. Therefore, time was determined to be significant (p<0.05) towards the change in p H of ground beef patties during storage. In particular, the increase in p H towards neutrality was more pronounced at the later stages of storage (i.e. day 7 to day 14). A n A N O V A revealed that L-ascorbic acid ( L A A ) infusion significantly (p<0.05) affected the meat patty pH (Table 2a and 2b). A comparison of means by Tukey's test showed that L A A infusion decreased the meat pH observed. Adjusting the ground beef patties to various fat contents had a significant (p<0.05) impact on the change in p H . In particular, 4% fat patties (in both control and L A A infusion) had higher p H values than corresponding 17% and 30% fat patties. The interaction between L A A and fat content was significant (p<0.05), which resulted in a marked decrease in p H values for all patties made from L A A infused carcasses at both 17% and 30% fat, in comparison to the control patties of the same fat content throughout storage. Only the 4% fat patties made from carcasses infused with L A A had higher p H values than the control patties, at the same fat content. Irradiation of patties at different dosages also significantly (p<0.05) affected the pH. Non-irradiated patties (i.e. 0 kGy) were found to have a higher pH value than all irradiated patties (i.e. 5, 10 and 20 kGy). The interaction of L A A and irradiation did not affect the p H of ground beef patties, while the interaction of fat and irradiation was significant (p<0.05). The combination of increasing fat (from 4% to both 17% and 30%) and increasing irradiation dosage (from 0 to 5, 10 and 20 kGy) resulted in a decrease the p H values of patties observed at day 14. 47 es > S3 a > •a s cs Vi ii WO cs Vi O -a a o •a cs CS a; s o •_ u a; "3 es CN ju es H W) es •-o +^ Vi U o WO _c es a. eu •a s 3 o •~ W) "3 o u 0) e^  o o O s cj e« ON i n CN F -1 — 1 CN CN CN o, O O O + 1 + 1 + 1 + 00 o t> t--VO 00 CN VO v d v d v d v d < < < < Z rt ^ o 1-1 c o O « 2 8 o CN vd CN O o +1 ON i n °. © VO + 1 CN VO i—i cn CN CN o o o o o o o o +1 +1 +1 +1 v~> o i n m vo vo c o O i n i-i CN O O O o o o +1 +1 +1 i—i CN 0 0 h VO vd i n i n i n I CN O o +1 o VO o ?'l oo i n m vo cn O ? l o o + 1 ov VO i n i n i n i n i n i n m X X X X X X X X X X <n X) XI JS XI ctj CN CN CN CN cn cn cn cn VO VO VO O O O O o o o o o o o o o o o o o o o © ' , o, o +1 +1 +1 +1 + 1 + 1 + 1 + 1 +1 +1 + 00 00 00 00 VO VO VO VO 1—1 r—1 1—1 cn cn cn cn i n m m m cn cn cn i n i n i n i n i n m m m i n m m o o vo vo o o +1 cn m o m O CN O cn a o U W w G CD B "S u O i—< CD + 1 cd a fe T3 "a 3 - ' o M o o SC Q c 0) cs cn >—i wo ON q o o o + 1 +1 +1 +| 00 00 1/1 p i 00 00 T f NO NO NO NO NO < ^ < <! ^ z ^ CN o i n o ca cj - O u, O o oo < CN O CN o CN o 0 , 0 0 0 +1 +1 +1 +1 i n i n i n i n T t T - t T t T t i n to i n O CN O CN r - H O o o +1 +1 O NO O m o o +1 00 m cn O O + 1 o NO NO i n i n i n —i o O r - H o +1 CN to o +1 i n CN O © +1 NO IT) o © +1 00 i n m i n m in o o +1 o T t o o +1 o T t r--o o +1 o T t o o +1 o T t i n i n m - 2 S o ca o o o oo < o ?'l NO cn NO' CN O o +1 oo i n CN o o +1 NO i n CN o CN o o + i n o © o o o +1 +1 +1 + 00 r - H r - H CN T t NO NO NO i n m m i n NO NO NO NO O + 1 i n o m o, o o + 1 +1 + m m m o o o i n m i n o CN O cn o o o oo w oo ¥ CD o u. O IH IH CD T3 IH ca ID fl ca +-» 00 +1 u B <D IH CS CCJ > o o CD I oo CU -fl oo CD fl ca > c CD CU o IH CD s ca 00 OJ -a oo OJ 3 ca > a CD CD CD - Q CD C J c CD IH (D X) CD cj fl CD IH CD & OH o IH o 00 IH CD OH oo ta CD IH r«J 3 * £ o Q H CD OH 3 oo CD IH 3 -4.2.2 Microbial Population 4.2.2.1 Aerobic Plate Count The aerobic bacteria population (CFU/g beef) of all non-irradiated ground beef patties increased during storage at 4°C (Tables 3a and 3b). During the storage period, the aerobic bacteria count increased from a minimum of 84+21 CFU per gram of beef, to a final count of greater than 2.5 x 107 CFU per gram of beef in all non-irradiated patties. The only exception was for the 4% fat control patties, where a maximum count of 2.5 x 106 CFU per gram of beef was obtained. No viable aerobic bacteria in irradiated patties were detected (at 1:10 dilution) throughout the entire storage period; indicating the destruction of bacteria by e" beam irradiation. The L A A infusion had no significant (p>0.05) effect on the aerobic plate count, as compared to the control patties. The amount of fat and irradiation dosage both significantly (p<0.05) affected the aerobic bacteria population (Table 3a and 3b). An increase in fat (from 4% to either 17 or 30%) resulted in an increase in the aerobic bacteria count while an increase in irradiation dosage resulted in an overall decrease in aerobic bacteria. Interaction of fat content and irradiation dosage also significantly affected the aerobic bacteria population. There was an increase in the aerobic bacteria population in both the 17% and 30% fat patties, irradiated at 0 kGy (control) in comparison to 4% fat patties irradiated at 0 kGy. The infusion of L A A , in combination with fat and irradiation, resulted in a significant (p<0.05) interaction, as observed with the statistical difference noted between the aerobic bacteria population of non-irradiated 4% control patties and non-irradiated 4% patties made from L A A infused carcasses on day 14. 4.2.2.2 Total Coliforms and Escherichia Coli The bacteria counts for total coliforms and Escherichia coli (E. coli) are shown in Tables 4a and 4b and 5a and 5b, respectively. Similar to the aerobic bacteria count, the time variable had a significant (p<0.05) effect on bacteria growth, resulting in an increase in both total 50 M es o o o o +1 ON o +1 o NO f-ON CN CN <N CN + 1 +1 +1 +1 Tf TT 00 00 0 0 T J -00 o IQ o o - H CN c CU E re cu T3 'o o o o oo o NO* ? l Tt NO o +1 oo ? l cn i n m m o o o +1 +1 +1 cn cn cn CN m o + 1 cn CN CN CN O CN o cd O o o on < O O H ? l ON ? l o o o X m CN A X m CN A X i n CN A ^ 2 o m o o o o o o o o o" o w ¥ CD a o S H o fa !D c3 ID C cd + 1 c cd CD a <D CO td a .9 ~ -3 if I -c 2 *o o o i n CN a cd -a H-> l-H CD t3 <D IH 00 3 O o cd •c CD H - i U cd -D oo 0) o Cl a> Q O H cd oo •a 3 <2 o "o o m CN a o od oo oo <D Cl 3 O o 2 'EH CD -t-> o Cd -D 00 tD •!-> o c CD Q Cl o m o irT (p<0 o o o (p<0 o V •4—* CH "—' CD IH H—» Ci f-H CD IH td -3 1 nits J>N H3 nits j > N *4H 3 . i f ific can a J-H .2P •a 'oo ilony s are are s o o o m CN u 43 .-tt CD O 3 C ° IH o "5 o o m CN u * p IH M 2 u IH CD CD S3 © cd CD U 3 O o ' C CD H-> o cd HO oo CD O Cl CD T 3 a o o cd Cl 3 O o O Cl CD IH o IH cd Cl a 3 o o - H *H ' -H O 51 3 .2 <J cu DC es es Q U o + 1 o \ i CO O O + 1 o eu co es fe S es eu I * CN CN + 1 +1 OO 00 CN •sr oo 00 o o <—' CN o cd O o CO < U v o o +1 v d o + 1 00 > x > r s o» CN CN o O O o i — f r-*H I D I T ) in m o o o o +1 +1 +1 + 1 cn cn cn cn CN CN v ' O m o T—H O CN o cd o o o o U H E—1 o + 1 O v cn o + 1 o cn X! in CN A o o o r H i—i X X X in in in CN CN CN A A A o CN o cn o cd o o co < A o o o o o O fl — i o oo ¥ CD a O 1-1 o fa CD - O U i cd -a +1 e cd CD a CD fe cd t d -a cd | 2 a .-2 o a fl fl o -g ° ^ in gj> fl I J ! 8 CD fl o T3 O cd cd CD t -bO •a o o cd O cd Xi to CD -t-> O fl CD Q U H fl o o o o cd CO -t-» 'c 3 fl° *o o o m CN C cd . f l fl fl° o *o o cd m X> CN CO £3 § ^ ^ 9> cd CD 3 O o in o o V fl CD 1— & >^  cd o ' f l op ' c O CD u . cd CD CD CD u fl CD +-» O cd Xi CO CD O fl CD Q U H in o o V OH fl CD '-3 >> 1 • 1—I fl .SP ' t o CD fe CO l-l CD -i-> CD fl CD 1-TJ .f l •s 9 cd C fl 3 O o "cd • t—I o cd fl fl 3 O o o o 52 CU OD CJ •-o o A ro o + 1 NO v i O 'x ? l NO CN + 1 c-» oo o o o O p o NO NO NO NO + 1 +1 +1 +1 co co m ro O O - H CN to e cu u IH fl o O O o ro o +1 NO ON o A ?'l NO CN O CO o +1 ON O A CO o A CO o 1—I A CO o CO o o o o +1 +1 +1 +1 o o —i CN fl o O O O o CN" o +1 TT o o +1 NO X V o O O r - H r - H r - H X X X V V ) V r -H ,—' ,—i A A A ^ 2 o ro O O O c ' • i n • 3 o w CU s <+H O T3 C a +J + 1 <U S I H KS cd H-» cd T3 oo -4-» •a fl° fl^ O o -fl +-> oo co CU •a o o cd ' C • cu t i CO - O « <U fl _o , 3 - 3 o o o o" o a oo •»-> •a 3 0 0 fl o fl u r N S H 8 £ fl o - 3 o o o etj fl 3 OX) c > N fl o "3 o o V c o • —H - 3 o -C cu td cu I-H 0 0 fl-3 o o 2 CU H-» o cd - O oo (U -*-» o fl cu Q fl cd - f l cd <D 6 V o o V OH fl CU I-H > N CO o •a (DO c S >> c O o o V cd oo +-> 'fl 3 0 0 fl -fl *o o o V ) 2 cU ts cd - O C*0 <u +-> O fl cu Q U H CD V -cd oo I H CU - ctt I-H LT-J cu od - f l 60 i> 3 rt 3 o o o o C3 fl fl 3 O o o V o o V OH fl CD I-H '-3 cd O H3 -a 0 0 CD fe OO i H CD (D c CD VH - 3 - A > o I H cd c fl 3 O o "2 - 3 o I H o 53 0) eu X> -o c s o u cm CJ CJ X p u c o s a. o a. 5 .S o CJ o c o "3 > CJ •o e cs CS E CS CJ X c o •_ +J u "eg i— o X CS u o •a eg es u CS CJ -a CJ CO c c j CS Xl u o c j co es • CJ T3 CS = ed O O a CJ E es c j u H S O 0 0 o + 1 o\ o +1 m CN + 1 ON vo + 1 CN CN in in m L O + 1 +1 +1 +1 o o CN O cd O IS u o o co CN o + vd o + 1 CN o +1 00 p o p i _ ^ CN, CN CN CN, + 1 +1 +1 +1 Os Os Os Os sO VO VO vo o i n o o • - i CN o cd o o co O H o + 1 CN CN o +1 VO m in X in X in A A A A o © •-i CN O c n o cd o x> U i O o co o o o o 00 co CO CD CI O *a xi o o o cd co -t-» "a 3 00 a <2 E? o "o o o i n xl m o o V OH « CD U i -a o m o o V a CD I -I "3 x> •t-> a cd o • i-H C bO CD fe co t i CD I « £ ,tS CD cd *2 £ § S 00 3 o o cd 'Ci CD +-> O cd X) CO CD +-> O a <D Q O H CD fe CO U i CD a CD u X i o U i cd C 3 O CJ *2 x> o u o 54 cU « S-O re Q o i—i o +1 co C li-re CU IH + 1 ON +1 NO + 1 ^ TT CN o o o o ro ro ro ro + 1 +1 +1 +1 no i n m i n o o r — f » c o O O H H s CO O i—i X o + 1 o +1 CN jo m m m CN, CN, CN CN, +1 +1 +1 +1 CN CN CN CN 0\ Os Os 0\ c o U O O o T f ? l o CN CN O + 1 X^ CN~ X^ CN" CN" o o o o +1 +1 +1 +1 Os Os Os Os t> r-' t> t> o o r - H CN o CO fl o o fl ' - r H o CrO in o CU T3 T3 fl od <D fe cd -+-> cd T3 S-H "3 o c cd r d CU fl fl O o cd + 1 s o X ) 00 m +-> o fl CD Q o .a _ 3 M cd 00 H—> ' f l fl fl° o o o cd c fl 00 fl o o o " o cd o o o i n fl cd r f l Q £ m —^^  O m d ° . V o O H V W O H <D I-H a * £ 3 § oo ,2 I-H o a -y © C 1 fl^ O O o o m fl 00 cu fe I H CD Ji CD cd cu I H oo ta fl o o cd •c CD cd . O o fl cu Q O H cd <D I H 00 t i fl o o I H <D ts ccd X> oo CU H-> o a cu Q a o o cd fl fl 3 O o IH X ) o UH o fl CU u •a op ' c« CU fe r H « tt fl CD 8 cd £ ?-> X3 .tt X3 cd fl fl 3 O o _o o I H o 55 o O CJ es fe C CD es eu o + 1 cs C<1 VO 00 +1 oo o o o o CN CN CN CN + 1 +1 +1 +1 c n c n c n c n O O •-i CN O a o o o CO O H O cs O A 00 o + 1 i n i n m i n CN CN CN CN + 1 +1 +1 +1 VO vo vo VO 00 00 00 00 o m o O CN a cd a X> u, O o CO o o + 1 Ov . + 1 Ov CN CN CN CN X X X X CN* CN" CN CN o d. d. d + Ov c--' r-' r--' r-' + 1 +1 Ov Ov O CN O c n o cd o •e o o CO T J O cd CO II 00 CD a -CD fe cd td T J cd X J C+H o 1-o H UH CD JD T J " ~ ' fe T J + 1 A cd CD flO CD cd °C CD o cd X ) CO CD •»-> o o CD Q fl o fl fl o O \fl '•^ XJ 3 [ f l o fl o O x5 "° X ! o o o r f l -a cd ^ CO •a fl* 1 § 2 o o o m fl cd X I H-» l -CD td CD I i 00 fl o o cd •cl CD -t-» CD cd X ) CO CD O fl CD Q CJ H o CD c cd X H-» u, CD fl fl O CD cd •c CD -t-> CD cd X ) O fl CD Q i n o d V OH O o o <d -a 2 § " UH cd -o fl fl £ =5 3 fe 5P 2P rrt fl c ^ a © 5 <2 O c2 O o CD o o in m CD cd CD GO 60 cd CD C fl o O <D H-> CD cd X> CD CD O fl CD Q i n o d V O -fl CD UH to T J >-> fll cd O cfl ' f l op 'co CD u. cd C O u. CD •B tS u. .SP 'co CD fe CO WH CD -*-> H-> CD •H CD C CD fl CD l-H •S T J fl a o CD O i— cd .9 - s CO « fl fl O O fl fl o o 13 3 X o l i o O CJ X i O 56 coliform and E. coli counts during storage for non-irradiated patties. Similar to the aerobic plate count, 4% fat patties were observed to have a significantly (p<0.05) lower total coliform and E .coli count compared to the 17% fat patties, which were both lower than the 30% fat patties. Irradiation was found to have a significant impact on total coliform and E. coli growth. No coliform or E. coli was ever detected (at 1:10 dilution) in irradiated patties throughout 14 d storage. No significant differences between the mean values of coliform populations, of 4% fat non-irradiated control beef patties and 4% fat non-irradiated patties from L A A infused carcasses were observed. However, mean values of E.coli count from the 17% fat non-irradiated patties derived from the control were significantly higher than the corresponding non-irradiated patties derived from the L A A treated carcasses. The interaction between L A A infusion and fat level of the patties was also significant (p<0.05); an increase in fat content of ground beef patties made from control carcasses lead to a greater increase in E.coli population in comparison to similar patties made infused carcasses from the L A A . 4.2.2.3 Psychrotroph Count The psychrotrophic bacteria counts up to 14 d of storage are presented in Tables 6a and 6b. As expected, time was a significant (p<0.05) factor affecting the psychotropic bacteria growth, leading to an increase in population during storage. The infusion of L A A had no significant (p>0.05) effect on the number of psychrotrophic bacteria counted. After irradiation, no viable psychrotophic bacteria were detected (at 1:10 dilution) in irradiated patties containing 4 and 17% fat. Therefore, irradiation dosages had a significant effect on psychrotoph count. However, the effect of irradiation was less effective for the 30% fat patties stored for 7 and 14 d at 4°C, when psychrotrophic bacteria was detected. On day 7, the 5 kGy irradiated 30% fat patties were starting to show the presence of psychrotrophic bacteria and on day 14, all irradiated 30% fat beef patties had psychrotrophic bacteria. 57 CU CU U. 3 O . O n. u ea X X a, p o u X u >> c o "53 > •a c ea c © -a ea e ? - O ea X c o -a a u o u cj cj ea NO JU X ea H ea OJ <<-cu cu X T3 C 3 O Ml ea o O c CU ea CU H NO O *X + 1 TT o A TP ? i A CN" o +1 TT A C ON" g A A t>r <N" o o +1 +1 CN CN O A o +1 CN o A CN" d +1 CN O H A ON" ? l ON CN CN O + 1 c--s CD o A CN" ? l CN o o o X X CN" CS" A CN" A o o o o +1 +1 +1 +1 CN CN CN CN o m ^ o CN - 2 8 o t-H c o O a o O A A T f " rZ" +1 +1 oo oo O A CN" o + 1 NO ? l ON A CN" o +1 cn CN ;_, jr, ;_, i—i o i n CN A O m O tn O r—1 r—1 T—H X X X i n i n m CN CN CN A A A - 2 S o co o U 58 CO xs • ~ es o «« £ -2 5? .5 »• no .y > x <-> -1 s o x) cs CS a X -a c 3 o CJ CJ X OA P fe U ^ r V B CJ CS CJ I-H d + 1 CN d + 1 m CN" d + 1 cn O H d + 1 cn CN" d + 1 cn o o CO o a CO o o cn <—> d d + 1 +1 cn oo CN ^ o ? l oo ? l CN ? l VO CN CN O A CN O CN o CN O A CN o A CN o A 0 s CN o A CN o A CO o 1—1 X i n CO o X i n CO O i n CO o m d + 1 o_ d + 1 r-_ d + 1 d + 1 t> +1 00 + 1 00 +1 00 + 1 00 CN A •<r CN A CN A CN A i n m m i n o i n o o CN o m o o CN O m o o CN "=* I> O cn *2 XJ 'o cd 'o cd 'o CCJ o • 1—1 o o co < c _ o '•3 o cd w </} fl Cd CD a <+* O 0) XJ S -4-> CO +1 C cd CD a CD I i cd cd td XJ § .2 XJ ^ 1 1 2 § cS &? " o o i n CN fl cd X CO CD fl fl O o cd •c CD CD cd X CO CD <*-> o fl CD Q cd C fl OC fl CO r l •A a a 00 fl f-1 *T< o * o CD O m CN s X -*-» I i <D td CD u, 00 § 3 3 1 O fl CD Q fl * - f l XJ o fl o '-fl j g XJ o o cd CO H-> ' f l 3 00 fl c2 O * o o o i n CN C cd X +-> i i CD td CD I i 00 fl cd X H-> i i CD fl fl O CD CD cd X CD cd X O fl <L> Q o c CD Q H H z z H H CN CO m o d V OH c CD >-H - ~ & +2 -fl -fl co ccj CO CO ^ H-> H-> x? fl fl « 3 3 § 00 00 fl fl 'fl e * o o o o i n m CN CN a u O o fl cd X +-> i i CD cd CD CD H-> CD cd X co Si CD CD CD fl 3 O CD cd •a CD HHJ CD cd X O fl CD Q H H c 00 CD d cd i i CD C a. i i • 1—1 XI X cd CD I i U J> bO 00 !> O o cd fl fl fl O CD X o i n o d V OH •s CD I i XJ >> CD cfl •a 'co CD I i cd CO UH CD CD C CD XJ X o I i cd G C 3 O CD 1? X o u o 59 The level of fat was a significant (p<0.05) factor for the psychrotroph count. The difference was noted from day 4 onwards where 4% fat patties had a lower bacterial count than both the 17 and 30% fat patties. This trend was more pronounce as the storage period continued to days 7 through to 14. The interaction between fat content of patties and irradiation dose was also significant. Bacterial growth was only noted in those patties that had the highest level of fat (i.e. 30%) and the lowest dosage of irradiation (i.e. 5 kGy) on day 7, as well as in the 30% patties irradiated with 5, 10 and 20 kGy on day 14. However, the application of 10 and 20 kGy dosages of irradiation were sufficient to retard psychrotroph growth noted also in 5 kGy irradiated patties on both days 7 and 14. The combination of LAA-infusion with different levels of fat and irradiation dosages did not significantly affect the psychrotroph count. 4.2.3 Correlations Pearson's product moment coefficient of correlations, calculated for the correlation between pH, aerobic bacteria, total coliform, E. coli, and psychrotrophic bacteria, are given in Table 7. The correlation between pH and aerobic bacteria, total coliform and E. coli, and psychrotrophic bacteria were significantly positively correlated; thus, an increase in pH correlated well with an increase in all bacteria growth. A positive correlation existed between the four types of bacteria. Higher values (i.e. > 0.700) of coefficient of the correlation were also noted, suggesting a strong relationship between the growth of various types of bacteria. In particular, aerobic bacteria growth was strongly correlated with coliform (r=0.925) growth, but surprisingly, it was weakly correlated wifh.fi'. coli (r=0.790) growth. 60 CU O. c*-eu ii -Q -a a 3 o OX) O +J cs w ^ cu U "55 o. C on •c s -S-8 cu CS -a M o o •_ u s cu CU 5 s 4- . CU C C/) S O JS "3 s- o U cs i > r~- cu J U "5 H cs f ' 43 O > N t / J OH O N j -4 U o t-H CU < ft o o o o CN p—i o o CN o o o 1 ' d p, o NO o c n <ET o T t o T t o o 00 o 00 o d d d d 1 — 1 ,_, o IT) o o o T t o o CN o ON o r- o <p ON o o 00 o , ; d p i d P l d p, o 00 <*-} ET T t o1 o ET o o ON o CN o ON o o CN p o CN o o , ' d d d d d d d 1 — 1 1 — ' l__J ft 43 O u <U < "o U t*5 43 O. O 43 O > N CD 43 $3 cu cd CH a =5 o 43 co CU IH cd CO <D 43 O 61 4.3 Discussion Initially, three cattle were each infused with either 10 mM, 100 mM and 500 mM sodium ascorbate solution. Since the concentration of L A A deposited in the muscles were unknown (due to a lack of assessment of L A A in the muscles after infusion), the muscles from all infusion treatments were pooled together and would serve as triplicates for the infusion of L A A . This decision was supported by the findings of an analysis of the pH and bacterial data by the T-test, which revealed no significant difference (p<0.05) between mean values from all three levels of L A A infusion. Moreover, there is a high probability of accepting a dose effect of L A A when there is none after the computations of Type II error (i.e. failure to reject a false hypothesis) resulted in a probable error between 25 to 37% . The ultimate pH of muscle is determined by the amount of lactic acid produced from glycogen during anaerobic glycolysis of rigor mortis. For mammalian tissue, such as beef, the ultimate pH is approximately 5.4-5.5 (Lawrie, 1985). The pH values of the ground beef patties on day 0 were approximately in the range of the reported ultimate pH. Only the 30% fat patties were lower than the reported ultimate pH. One explanation for this observation is that adding more fat to the patties, increased the hydrogen ion content through contribution of fatty acid hydroxyl groups from the free fatty acids, which in turn decreased the pH of meat. Surprisingly, the infusion of L A A to the carcass did not affect the initial pH of the ground beef patties. This finding is different from that reported by Lee et al. (1999), who examined the keeping quality of ascorbic acid and carnosine treated ground beef patties. These authors reported that a decrease in pH of the ascorbic acid treated patties when blended with a 1% ascorbic acid solution that had an initial pH of 2.5. Harbers et al. (1981), also observed a decrease in the ultimate pH of meat to 4.99, when a 5% ascorbic acid solution was applied to beef steaks. This was also observed in ascorbic acid treated ground beef patties (Lee et al., 1999) and ground buffalo meat (Sahoo et al., 1998). However, proteins and phosphates present in meat are good buffering agents (Hultin, 62 1985) and so, the infusion of L A A was not likely to have drastically decreased the pH of the ground beef patties used in this study. During storage, the pH of all patties increased in the present study. In particular, pH values of the 4% fat patties at all irradiation dosages as well as the 17 and 30% fat patties at 0 kGy, were especially high (pH of 5.997 to 6.883). A similar observation was observed in ground beef (Lefebvre et al., 1994; Cabedo et al., 1998), in steaks and ground beef (An-Hung et al., 1995) and in ground buffalo meat (Sahoo et al., 1998). This increase in pH in non-irradiated patties over time was probably due to the growth of microorganism in the beef that produced alkaline ammonia and amines from substrate urea and amino acids (Lefebvre et al., 1994; An-Hung et al., 1995). However, the bacterial population in the 4% fat beef patties was much lower than in either the 17% and 30% fat beef patties and therefore should produce the least amount of ammonia and amine. Thus, the increase in pH specific to the 4% fat patties, cannot be fully explained by the presence of microbial growth alone Irradiation of beef has been widely accepted as an effective measure in controlling microbial growth in beef. In particular, Murano (1995) reported that a 3 kGy irradiation dose would eliminate 99% of pathogenic bacteria, such as E. coli 0157:H7, Salmonella spp., Campylobacter jejuni, and Listeria spp. in meats and ground beef. Furthermore, spoilage organisms such as Pseudomonads and Enterobacteriaceae, typically found in ground beef, are also destroyed by low-dose irradiation (Roberts and Weese, 1998). In general, the aerobic, total coliform, E.coli and psychrotroph bacteria count for all non-irradiated beef patties increased during storage. An increase, ranging from 104 to 10b CFU/g beef, was observed in all viable bacterial counts. Similar increases in bacterial count were also reported by Lefebvre et al. (1992) and Ju-Woon et al. (1999) in ground beef and by Okayama et al. (1987) on beef steaks. On day 14, all 17% and 30% fat patties had a population greater than 2.5 x 107 CFU/g beef. This has reached the unacceptable microbial level of 107CFU/g tissue for raw ground beef (Lefebvre et 63 al., 1992; Roberts and Weese, 1998); suggesting that the raw ground beef stored under the current experimental condition may not be considered safe to consume after 14 d. The low oxygen tension in the Z ipLoc™ bag might have retarded the microbial growth in the raw ground beef (Labadie, 1999; Lopez-Gonzalez et al., 1999), in addition to antimicrobial effect of the irradiation treatment. Viable bacterial counts were not detected (at 1:10 dilution) in all irradiated (5, 10 and 20 kGy) beef patties, except for psychrotroph growth in the 30% fat patties on days 7 and 14. A maximum difference of 10 6 C F U / g between the irradiated and non-irradiated ground beef patties was observed. This significant (p<0.05) effect of irradiation on microbial growth was also observed by Roberts and Weese (1998) with irradiation dosages reaching 7 kGy, by Lefebvre et al. (1992) with irradiation dosages up to 5 kGy and by Ju-Woon et al. (1999) with dosages up to 3 kGy. However, a low viable bacterial count obtained by the traditional total plate count method does not necessarily suggest the presence of viable pathogenic or spoilage organisms in minute amounts in the ground beef patties. Ray (1979 and 1986) has reported that sub-lethal treatment of food with irradiation wi l l only injure microorganisms and cellular repair is often observed during post-irradiation storage. These injured microorganisms often exhibit more exacting nutritional and physical requirements, thereby requiring a longer lag phase for growth and may exhibit different physiological and taxonomical characteristics, which exclude them from being detected by traditional growth medium and enumeration techniques (Lucht et a l , 1998). Nonetheless, the 10 and 20 kGy are relatively high doses sufficient to destroy microorganism in beef. Ascorbyl-2-polyphosphate (a source of dietary ascorbic acid) applied to dairy cattle has previously been shown to be deposited in various muscle groups such as biceps femoris (Macleod et al., 1999). Unfortunately in the present study, quantification of L A A in muscles after infusion was not successful and therefore these data are not available. However, pre-64 slaughter injection of cattle with sodium ascorbate has also been reported to be a satisfactory method of delivering ascorbic acid to meat tissues (Hood 1975; Wheeler et al., 1996). The primary application of ascorbic acid to cattle is to improve overall quality (e.g. colour, lipid oxidation and texture) of the resulting meats; which was demonstrated by both Hood (1975) and Wheeler et al. (1996) by means of L A A injection studies. However, a secondary benefit of applying ascorbic acid to cattle is to improve their health status. Macleod et al. (1999) demonstrated this by feeding ascrobyl-2-polyphosphate to dairy cattle and observing an increase in neutrophil content, the primary leukocytes responsible for destroying invading pathogens in animals. L-ascorbic acid infusion did not significantly affect the aerobic plate count and psychrotrophic bacteria count. Since L A A infusion did not markedly lower muscle p H to less than 4.0, no antimicrobial effect of L A A would be expected. Earlier investigators examining the effects of ascorbic acid on microbial growth also did not find a significant effect of L A A on total viable bacteria count in treated ground beef (Shivas et al., 1984; Ju-Woon, 1999) and beef steaks (Okayama, 1987) in comparison to control beef. The level of fat in the ground beef patties was a significant factor in determining the size of the population of aerobic, total coliform, E. coli and psychrotroph bacteria found in beef patties. A n increase in fat led to an increase in both the initial bacteria count as well as the final psychrotrophic bacteria count after 14 d storage. Since ground beef patties of all fat levels were made from the same pool of beef tissue, the difference in the initial viable bacteria count observed was due mainly to the introduction of the fat. Therefore, the 4% fat patties had the lowest viable bacteria count since no fat was added to formulate the patties while the 17% and 30% fat patties had a respective higher initial viable bacteria count. Nevertheless, the initial viable count (aerobic plate count between 84 to greater than 2.5 x 103 CFU/g) obtained from ground beef patties of all fat levels in this study was far less than that reported by Berry et al. 65 (1980), with an aerobic plate count between 2.0 x 105 to 1.0 x 107 CFU/g were detected in ground beef patties formulated with fat trimmings from the brisket and the kidney area. The increase in the viable bacteria count following the addition of fat is contrary to the findings of Shoup and Oblinger (1976) who observed a lower aerobic plate count in higher fat beef products. However, Berry et al. (1980) have found that fat trimmings used in ground beef can have a high bacteria count, and that the level of the bacterial population differs with different anatomical sources of fat trimmings. Thus, the sources of our fat trimming could have been the reason for the high level of bacteria. In this study, no microbial analysis was conducted on the fat nor was the anatomical source of the fat known. Bacteria growth in foods is highly dependent on the intrinsic factors of the sample, such as temperature, pH, moisture content, water activity (aw), nutrient content, competing microorganisms and antimicrobial constituents, and extrinsic factors in the immediate surrounding such as temperature of storage and oxygen availability (Jay, 1996). Low temperature preservation, less than 4°C, has generally been used to control coliform and E. coli growth in foods (Jay, 1996) and thus this effect could be responsible for the 1000 times low viable total coliform and E. coli count, in comparison to aerobic and psychrotophic bacteria count, observed in this study. A pH value between 4.0 to 7.0 is the optimum range for bacteria growth in foods. Since the ground beef sample fell within this range, viable bacteria would multiply during storage period. In particular, as the pH increased towards neutrality in the later stages of storage, a higher rate of bacteria replication was observed. However, a weak, but significant positive correlation between pH and aerobic plate count (r=0.258), total coliform count (r=0.195), E. coli count (r=0.224) and psychrotroph count (r=0.190) (p<0.05) was determined by Pearson's coefficient of correlation. Therefore, the increase in the ground beef pH was only partially explained by the amine and ammonia content produced during proliferation of the total viable bacteria. A highly significant correlation was found between all 66 total viable counts. In particular, aerobic plate count and total coliform count was highly correlated with a Pearson's coefficient of correlation of 0.925 (p=0.000). A l l bacteria were shown to proliferate together during storage, but at a different rate and to a different extent. Although no positive identification of any specific strain of bacteria was conducted on the patties used in this study, the high total viable bacteria count in non-irradiated samples probably consisted of mostly non-pathogenic spoilage organism due to sanitary production procedures. O f the spoilage organisms identified in foods, Pseudmonads spp., Enterobacteriace and lactic acid bacteria are the primary microorganisms found in fresh meats (Labadie, 1999; Roberts and Weese, 1998). Depending on the health status of the cattle and the sanitary conditions of the slaughtering process, pathogenic bacteria such as E. coil OJ57:H7, Salmonella spp., Camplyobacter jejuni and Listeria spp. can sometimes be isolated from ground beef and meat products (Roberts and Weese, 1998). Due to low initial aerobic, total coliform, E. coli and psychrotroph plate counts, the microflora of the ground beef in the present study would consist mostly of spoilage bacteria. Since a low dose of 3 kGy is capable of destroying pathogenic organisms (Roberts and Weese, 1998), it stands to reason that a minimum dose of 5 kGy employed in this study would effectively eliminate all pathogenic bacteria and some spoilage organisms (due to proliferation of psychrotroph at day 7 and 14 with 30% fat patties). With irradiation, only patties with a low initial total viable bacteria count remained below the detection level after 14 d storage. Thus, the bacterial load in the meats and fat trimmings used for ground beef is likely the primary factor determining the overall quality and shelf-life of irradiated product. Intervention technologies such as modified atmosphere-packaging, low temperature storage, irradiation, or the application of antioxidants can only minimize or delay the quality changes that occur during storage. 67 4.4 Conclusion The increase in p H of all ground beef patties during 14 d of 4°C storage may be partially explained by the production of bacterial ammonia and urea produced during bacteria growth in the patties. The increase in fat and irradiation both decreased the p H of ground beef. Ascorbic acid infusion was shown to have no effect on ultimate p H of ground beef patties. The initial counts of aerobic, total coliform, E. coli and psychrotroph bacteria were approximately 2.5 x 103 C F U / g beef or less, indicating good quality meat and sanitary processing techniques. The addition of fat increased the initial bacteria count of ground beef patties, and this trend was observed throughout storage. This effect was likely due to the fat trimmings that are often contaminated with bacteria. A final count of greater than 2.5 x 10 7 C F U / g beef on day 14 is beyond the acceptable level of bacteria count of 10 7 C F U / g beef. Irradiation of patties, at all dosages, was shown to be effective at destroying bacteria since no colony forming unit was detected, at the lowest dilution of 1:10, throughout storage. Only the 30% fat patties were found to have psychrotophic bacteria after irradiation by 5 kGy on day 7. Similar results were obtained at 5, 10 and 20 kGy on day 14. The low oxygen environment, combined with low temperature storage was the primary reason for the low total coliform and E. coli counts observed during storage. Ascorbic acid infusion had no effect on bacteria count. Therefore, the microbial quality of the irradiated ground beef patties in this study depended mainly on the quality of the raw material, in particular the source and quality of the fat. 68 5. Experiment 3: Irradiation Effects on the Physiochemical Attributes of Ground Beef Patties 5.1 Materials and Methods 5.1.1 Materials All chemicals used were of reagent grade. Trichloroacetic acid, o-phosphoric acid, hydrochloric acid, sodium hydroxide, acetone, methanol, sodium phosphate and sodium dihydrogen orthophosphate were purchased from Fisher Scientific Co. (Fair Lawn, NJ). Butylated hydroxytoluene and 1,1,3,3 tetraethoxypropane were obtained from Sigma Chemical Co. (St. Louis, MO). Only distilled deionized water was used for experimentation. Ground beef used in this experiment was obtained from ascorbic acid infused carcasses described in section 4.1.1 Cattle raising and ascorbic acid Infusion and the corresponding patties were made according to section 4.1.2. Irradiation of ground beef patties by electron beam irradiation was the same as section 4.1.3. All patties were individually stored in Zip-loc™ bags at 4°C under normal lighting for 14 d. 5.1.2 Heme Iron Concentration Heme iron concentration in ground beef patty was measured according to Lee et al. (1998). In short, duplicate 2.0+0.5 g samples from each ground beef patty were weighed into separate centrifuges tube and 9.0 mL of acid acetone (90 acetone: 8 water: 2 hydrochloric acid, v/v) were added. The ground beef was then thoroughly macerated with a glass rod and allowed to stand in the dark for 1 h at room temperature. Afterwards, the mixture was filtered through a Whatman #42 filter paper and the absorbance at 640 nm was read against an acid acetone blank with an UV-visible recording spectrophotometer (Model UV-160, Shimadzu Co., Kyoto, Japan). Heme iron concentration (ppm) was calculated by first converting the absorbance reading to total 69 hematin using a conversion factor of 680 and the heme iron concentration was derived based on the 8.82% of iron concentration in hematin, as follows: Total Pigments (ppm) = A 5 4 0 x 680 Equation 1 Heme Iron (ppm) = Total Pigments (ppm) x 8.82/100 Equation 2 5.1.3 Lipid Oxidation The degree of lipid oxidation was assessed by the production of malondialdehyde ( M D A ) during storage. M D A was measured with a modified aqueous extraction procedure of the thiobarbitric acid ( T B A ) method of Buege and Aust (1978). Duplicate 2.5+0.5 g samples from each ground beef patty were homogenized with 25 m L of 20% trichloroacetic acid containing 1.6% o-phosphoric acid, and diluted to 45 mL with distilled deionized water. A l l cloudy homogenates were then clarified with a C-18 Sep-Pak cartridge preconditioned with methanol. A 2.0+0.1 mL aliquot of the clarified homogenate was mixed with 1.0 mL of 0.5% T B A solution in 0.025 m M N a O H containing 0.02% B H T and heated in a boiling water bath for 15 min. Absorbance readings of the resulting pink chromogen were measured at 532 nm against a reagent blank with a spectrophotometer. A l l absorbance readings were converted into mg M D A equivalent/g beef according to the standard curve derived from 1,1,3,3 -TEPP solution (Appendix 2). 5.1.4 Colourimetric Determination Surface colour of ground beef patties was determined using the Hunterlab L, a and b colour system. Prior to analysis, the Hunterlab spectrocolorimeter was first standardized by scanning standard colour plates in Zip- loc™ bags. A l l ground beef patties were allowed to "bloom" for approximate 1 h in their individual Zip-loc™ bag at 4°C and Hunterlab colour scores were then determined by scanning patties with a Hunterlab Scan 6000 model spectrocolorimeter (Hunterlab Associated Laboratories Inc., Reston, V A ) under standard illuminant D 6 5 (simulating daylight, correlated colour temperature ~6500K). Each patty was 70 scanned twice at two different locations with a 5.0 cm aperture, which, in total, covered 45% of the entire surface area of the patty. Colour difference between the patties made from the saline infused and L-ascorbic acid infused carcasses was calculated as delta E (AE) according to the following formula (Faustman et al., 1989): AE = (AL2 + Aa2 + Ab2)05 Equation 3 5.1.5 Metmyoglobin Concentration Metmyoglobin concentration in ground beef patties was quantified according to Lee et al. (1998). Approximately 5.0+0.5 g of ground beef was weighed into a centrifuge tube and mixed with 25.0 mL ice-cold 40 mM phosphate buffer (pH 6.8). The mixture was homogenized for 15 s at high speed with a laboratory blender and was then allowed to stand for 1 h at 4°C. Afterwards, the mixture was centrifuged at 2450 x g for 30 min at 4°C and the supernatant was filtered through Whatman #1 filter paper. The collected filtrate was read at 700, 572 and 525 nm against the phosphate buffer with a UV-Visible recording spectrophotometer. Absorbance readings were converted to percentage metMb using the formula of Krzywicki (1982) listed below: % metMb = {1.395-[(A572-A7oo)/(A525-A7oo)]} x 100 Equation 4 5.1.6 Texture Analysis The hardness of ground beef patties was analyzed by punching through the patties with a single blade using a Texture Analyzer TA-XT2 (Texture Technologies Corp., NY). Each patty was subjected to three parallel punching with a rectangular blade (with a contact area of 2.2 cm2) at room temperature, to a distance of 1.5 cm, at a speed of 1 mm/s with a minimum contact force of 5 g. Maximum force (i.e. the highest peak force) recorded in a force deformation curve during the analysis is interpreted as the hardness of the ground beef patty. 71 5.1.7 Statistical Analyses A l l treatments, i n triplicate, were sampled at least twice i n a l l experiments. A n a l y s i s o f variance ( A N O V A ) was performed on data us ing the General L i n e a r M o d e l i n M i n i t a b Statistical Software vers ion 12.0 ( M i n i T a b Inc., P A ) . Signif icant differences between means were determined by Tukey ' s Test (p<0.05). Correla t ion (Pearson's product moment at p<0.01) were also determined us ing the M i n i t a b Statistical Software. Regress ion analysis ( r 2 value) were determined us ing the 97' Mic roso f t E x c e l P rogram (Microsof t C o . , U S A ) . 72 5.2 Results 5.2.1 Heme Iron Concentration and Lipid Oxidation The heme iron concentration of ground beef patties is shown in Table 8. On day 14, the beef patties contained heme iron concentrations in the range of 7.6 ppm to 22.4 ppm (Table 8A). An increase in fat level was shown to significantly (p<0.05) decrease the heme iron concentration of patties (especially from 4% to 17% fat), with a decrease of approximately 13 ppm. Irradiation was also a significant (p<0.05) factor affecting the heme iron concentration of patties, where an increase of irradiation dose from 5 kGy to either 10 kGy or 20kGy corresponded to a general decrease in the heme iron concentration by about 1 ppm. The post-slaughter infusion of L A A did not affect the heme iron concentration. No interactions between L A A infusion, irradiation dosages and fat level were present for the heme iron concentration of ground beef patties on day 14. The effect of time on heme iron concentration was only tested with the 30% fat ground beef patties due to a shortage of ground beef patties for the 4% and 17% fat treatments on days 0 and 7 (Table 8B). During storage, a significant decrease in heme iron concentration for all patties was observed, and this was not affected by infusion of ascorbic acid. Analysis of variance revealed that the interaction of irradiation dosage with time had no significant effect on the heme iron concentration. However, mean values of patties irradiated at 0 kGy and 5 kGy were significantly different (p<0.05) from patties irradiated with 10 kGy and 20 kGy, according to Tukey's test. Irradiation dosages had a significant effect on heme iron concentration during storage, as determined by Tukey's test. A two-way A N O V A performed on the data by the General Linear Model did not demonstrate any significant interactions between time, L A A infusion or irradiation dosages and heme iron concentration. 73 Table 8 - Effects of electron beam irradiation, L-ascorbic acid and fat level onheme iron concentration (ppm) in ground beef patties on day 14 of storage (A) and in 30% ground beef pattties on days 0, 7,14 of storage (B) at 4°C . A . • Treatment Fat Content Dose (kGv) (%) 0 5 10 20 Control 4 22 .45±1 .56x N/A N/A N/A 17 8 .46±0 .24 a y 9 .65±0 .40a x 8 .67±0 .19 a x 8 .75±0 .18 a x 30 8.93+0.30az 8 .74±0 .25a y 7 .80±0 .35 b y 7 .62±0 .29 b y L A A 4 21 .38±0 .38x N / A N/A N/A 17 9 . 9 6 ± 0 . 8 1 a y 9 .62±0 .25a x 9 .09±0 .28 a x 8 .34±0 .27 a x 30 8 .63±0 .21 a z 8 .88±0 .38a y 8 .27±0 .44 b y 7 .58±0 .27 b y B Treatment Day Dose (kGy) 0 5 10 20 Control 0 19 .48±0 .85a x 19 .48±0 .85 a x 19 .48±0 .85 a x 19 .48±0 .85a x 7 17 .79±0 .64a y 18 .37±0 .43 a y 18 .38±0 .43 a y 19 .18±0 .38a y 14 8.93+0.30az 8 .74±0 .25a z 7 .80±0 .35 b z 7 .62±0 .30 b z L A A 0 2 0 . 2 9 ± 0 . 5 9a x 2 0 . 2 9 ± 0 . 5 9 a x 2 0 . 2 9 ± 0 . 5 9 a x 2 0 . 2 9 ± 0 . 5 9a x 7 17 .79±0 .40a y 17 .84±0 .60 a y 18 .61±0 .40 a y 17 .12±0 .42a y 14 8 .63±0 .20 a z 8 .88±0 .38a z 8 .27±0 .44 b z 7 .58±0 .27 b z All values are mean + SEM; n-18 a b cData in a row with different letters are significantly different (p<0.05) x y zData within column with different letters are significantly different (p<0.05) N/A Not available Lip id oxidation of all ground beef patties was monitored during the 14 d storage at 4°C and in general, a significant (p<0.05) increase in M D A was observed after 14 d storage (Figs 9a and 9b). The M D A concentration in the irradiated patties reached an initial maximum on day 4 of storage, followed by a rapid decline in concentration by day 7. A subsequent increase of M D A to a final maximum occurred on either day 10 or 14 of storage were detected. The addition of fat (to 17% and 30%) had a significant (p<0.05) effect on the susceptibility to lipid oxidation, as evidenced by the fact that an increase in M D A concentration was detected in ground beef patties with increasing fat content. In general, the infusion o f L A A produced a minimal but significant (p<0.05) increase in the M D A concentration compared to the saline-infusion. Applying electron beam irradiation to the patties had a significant (p<0.05) effect on the M D A concentration. Non-irradiated patties (i.e. OkGy) were found to have a significantly lower (p<0.05) M D A concentrations than all irradiated patties. Similarly, 5 kGy irradiation resulted in significantly lower M D A concentrations compared to patties receiving the 20 kGy dosage. However, the use of 10 kGy and 20 kGy dosage of irradiation generated similar levels of M D A in the ground beef patties. In order to predict the extent of lipid oxidation in ground beef patties during 4°C storage, several regression analyses were performed on the data and the corresponding quadratic equations, with the coefficient of determinations are given in Appendix 3. Because of the potential of having two maximum stages of M D A accumulation occurring in the patties during the study period, a quadratic equation of the third order was found to provide the best model for describing lipid oxidation over time. Only non-irradiated control 4% fat patties had a r 2 > 0.9281, indicating a good prediction of the M D A content over time. Furthermore, data from most treatments had positive quadratic equations in the model, which suggests the possibility of detecting further increases in M D A concentration beyond day 14 of storage. The treatments with negative quadratic equations in the models (i.e. 4% control at 0 kGy and 5 kGy, 30% control and 75 F i g u r e 9 a - E f f e c t s o f f a t l e v e l a n d e l e c t r o n b e a m i r r a d i a t i o n d o s a g e s a t 0 k G y , 5 k G y , 1 0 k G y a n d 2 0 k G y o n m a l o n d i a l d e h y d e c o n t e n t ( M D A ) i n c o n t r o l g r o u n d b e e f p a t t i e s d u r i n g s t o r a g e a t 4 ° C . A l l v a l u e s a r e m e a n + S E M ; n = 1 8 . • 17%-Fat 30%-Fat OkGy Day 10 12 14 2.0 n •4%-Fat • 17%-Fat 30%-Fat 10 Day 10 kGy 12 14 •4%-Fat •30%-Fat 17%-Fat Day 10 20 kGy 12 14 Figure 9b - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on malondialdehyde content (MDA) in ground beef patties made from L-ascorbic acid carcasses during storage at 4°C. All values are mean+SEM; n=18. L A A - i n f u s e d at 5 k G y , 3 0 % control and L A A - i n f u s e d at 10 k G y , and 4 % L A A - i n f u s e d at 20 k G y ) w o u l d therefore suggest the opposite trend where the M D A concentration w i l l most ly l i ke ly continue to decline beyond day 14 o f storage. 5.2.2 Colourimetric Determination and Metmyoglobin Concentration Hunterlab L, a, b colour values were used to assess the surface colour o f ground beef patties. In this study, only a and L were o f concern, and this is reported in F i g s 10a and 10b and 11a and l i b , respectively. A n a l y s i s o f variance showed that t ime was a significant (p<0.05) factor affecting the redness o f the ground beef patties and the redness, i n general, decreased w i t h increasing storage t ime (Figs 11a and l i b ) . Th is gradual decrease i n a value was significant (p<0.05) up t i l l day 11 o f storage and the a values recorded between days 11 and 14 o f storage were not s ignif icant ly different. Patties that appeared to have a pale red or p ink hue under normal laboratory l igh t ing had a m i n i m u m a value o f 8.00. Therefore, under the conditions o f this study, ground beef patties w o u l d presumably lose acceptable surface redness between days 7 and 11 o f storage. The infusion o f antioxidant L A A into the carcass did not protect the redness o f the beef patties during storage, but rather significantly decreased the recorded a value for redness. Irradiation dosage had a significant effect on a value and the degree o f impact was observed to f o l l o w i n descending order o f 20 k G y > 1 0 kGy>5 k G y > 0 k G y dosages for the 17% and 3 0 % fat patties. However , a reverse order o f i rradiat ion dosage effect was noted for the 4 % fat patties. There was no interaction between irradiat ion dosages and storage t ime on a value. Regress ion analyses conducted on the data revealed many first and second order models, w i t h fairly h igh r 2 values, for predict ing the changes i n a values for redness over t ime (Append ix 4). L o w fat patties (i.e. 4%) tended to exhibit either first or second power models regardless o f the i r radiat ion dosages applied or the infusion o f L A A or saline. This result impl ies a linear (first power) or gradual (second power) decrease i n the redness o f ground beef patties dur ing storage, 78 0 2 4 6 - 8 10 12 14 0 2 4 6 ^ 8 10 12 14 Day 0 . 2 4 6 8 10 12 14 Day 0 2 4 6 ^ 8 10 12 14 Figure 10a- Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on Hunterlab a value for redness in control ground beef patties during storage at 4°C. All values are mean+SEM; n=18. •4%-Fat 17%-Fat- •30%-Fat O k G y Day 10 12 14 15 -, -4%-Fat- • 17%-Fat • •30%-Fat 5 k G y Day 10 12 14 Day 10 12 14 Day 10 12 14 Figure 10b - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on Hunterlab a value for redness in ground beef patties made from L-ascrobic acid infused carcasses during storage at 4°C. AH values are mean+SEM; n=18. which are not severely affected by irradiation dosages and antioxidant addition. Changes in a values for higher fat patties (i.e. 17% and 30%) were more complex and required third power/order models to predict those changes during storage. Interestingly, almost all regression models for irradiated patties exhibited negative signs in the regression equations. Such a finding suggests the possibility of further decrease in a values beyond 14 d of storage after exposure to irradiation. Non-irradiated patties, with positive signs to most of the regression equations, would suggest that minimum a values were obtained before or by day 14 of storage. Only non-irradiated 4% fat patties showed the potential for further decreases in a value after 14 d of storage. During storage, a gradual lost in surface redness of the ground beef patties would theoretically be accompanied by a development in brown pigmentation. This change in colour might have an effect on the perception of lightness of the ground beef patties and so the Hunterlab L value was monitored (Fig. 11a and l i b ) . During 14 d of storage, a significant but minor increase in L values in 17% and 30% fat patties were observed on day 3. Depending on the fat content of the patties, Hunterlab L values recorded during storage ranged from 25 to 45. Patties with higher fat content would naturally be lighter in colour and this was confirmed by the significantly higher L values observed in the order of 30% fat>17% fat>4% fat. Irradiation dosages have a significant (p<0.05) effect on L values and the mean L values for 20 kGy irradiated patties were significantly lower than from 0 kGy, 5 kGy and 10 k G y irradiated patties. This significant difference is small in terms of changes in the absolute L values though. L -ascorbic acid infusion did not affect Hunterlab L values. Since there were only marginal changes in L values (i.e. essentially similar) over time, no regression analyses were conducted on these data. Further investigation into the influences of L A A infusion on the overall colour of ground beef patties was carried out by computing A E - an arbitrary unit that compares the overall colour 81 Day 10 12 14 Day 10 12 14 Day 10 12 14 Day 10 12 14 Figure 11a - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on Hunterlab L value for lightness in control ground beef patties during storage at 4°C. All values are mean+SEM; n=18. 50 -I value 45 -value 40 -A x> CS 35 -CO 30 | t X 25 -20 --•4%-Fat •17%-Fat •30%-Fat O k G y Day 10 12 14 •4%-Fat 17%-Fat -A - 3 0 % - F a t 5 k G y Day 10 12 14 50 -i u 45 -13 > 40 & 35 -a +-} 30 it S3 3 25 -70 - -•4%-Fat •17%-Fat - A - 3 0 % - F a t 1 0 k G ? -fc-Day 10 12 14 Day 10 12 14 Figure l ib- Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on Hunterlab L value for redness in ground beef patties made from L-ascrobic acid infused carcasses during storage at 4°C. AH values are mean+SEM; n=18. difference (accounting for all three Hunterlab colour components) between an untreated and a treated sample - and shown in Figure 12. Delta E values for all patties were not consistent during the storage period and so, the effects of L A A infusion on overall colour may be dependent on time of storage. In fact, higher A E values were typically observed from day 3 to day 11 of storage in most patties. The colour difference between control patties and patties made from L A A infused carcasses was significantly affected by fat content where a greater colour difference was observed in higher fat patties, especially patties containing 17% fat. Similarly, increasing dosages of irradiation resulted in an increase of A E . However, an exception was noted for the 4% fat patties where non-irradiated patties were determined to have a significantly higher A E than irradiated patties. On the whole, A E values for all patties receiving different treatments were all within a range of 3.0 to 8.0. Colour pigmentation in ground beef is due mainly to the presence of oxyMb (red), deoxyMb (dark red) and metMb (brown), and the ratios between all the M b derivatives wi l l determine the overall surface colour of the beef. Hunterlab colourimetry was helpful in monitoring the overall colour resulting from the presence of all three M b derivatives on the surface of the beef patties but no indication is given as to the amount of each M b derivative present. Therefore, metMb concentration was independently measured as a percentage of all the derivatives of M b present, in all patties to aid in the explanation of the observed decrease in redness during storage. As is the case with measuring heme iron, there were sufficient patties from the 4% and 17% fat treatment groups for metMb determinations on day 14 only. Results relating to the development of metMb over time would be limited exclusively to the 30% fat patties. Analysis of metMb concentration (as a percentage of the total M b derivatives present) in ground beef patties on day 14 showed that increasing patty fat content (from 4% to either 17% or 30%) resulted in a significant decrease of metMb concentration (Table 9A). The exposure to 84 O k G y 10 12 14 D a y 5 k G y 10 12 14 Day 30% 1 0 k G y 10 12 14 Day <D 4 Q 3 _| 2 1 0 0 3 0 % 2 0 k G ? 4 10 12 14 Day Figure 12 - Colour difference (delta E) between ground beef patties made from control and L-ascorbic acid infused caracasses during 14 days at 4°C storage as affected by fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy. All values are mean+SEM; n=18. Table 9 - Effects of electron beam irradiation, L-ascorbic acid and fat level on metmyoglobin content (%) in ground beef patties on day 14 of storage (A) and in 30% ground beef pattties on day 0, 7,14 of storage (B) at 4°C . A Treatment Fat Content Dose (kGy) (%) 0 5 10 20 Control 4 65.87+3.04x N/A N/A N/A 17 57.30+3.54ay 85.98+4.45bx 79.26±2.24 b x 84.88+2.74bx 30 56.17±2.07 a z 57.88±6.11 a y 80.56±1.36 b x 77.70+3.85by LAA 4 68.92±2.57 x N/A N/A N/A 17 59.33+2.86ay 87.16±0.60 b x 86.13±0.85 b x 88.38±0.79 b x 30 58.91±1.28 a z 57.87+4.20ay 83.81+1.92bx 76.59±2.33 b y B Treatment Day Dose (kGy) 0 5 10 20 Control 0 54.69±7.19 a x 66.55±4.71 b x 56.17±2.07 a x 54.69±7.19 a x 7 70.25+5.24ay 85.98±0.71 b y 54.69±7.29 c x 66.11±3.67 a y 14 80.54±1.36 a z 76.20±4.29 a z 64.36+4.85by 77.71±3.85 a z LAA 0 66.94±2.38 a x 76.29±0.93 b z 71.67±2.57 a z 66.94±2.38 a y 7 79.17±0.78 a y 87.16±0.60 b y 66.94±2.37 o y 76.57±0.73 a z 14 83.81±1.92 a z 66.93±2.38 b x 72.32±1.27 c z 76.59+1.86az All values are mean+SEM; n=18 abcDatain a row with different letters are significantly different (p<0.05) xyzData within column with different letters are significantly different (p<0.05) N/A Not available irradiation significantly increased the metMb concentrations, from concentrations of 57.3% to 84.8% in 17% fat patties and 56.2% to 77.7% in 30% fat patties. The infusion of L A A also significantly (p<0.05) increased the metMb concentration, however, the increase was minimal. Increasing storage time was observed to increase metMb concentration in 30% fat patties (Table 9B). Infusion of L A A into these patties further increased the metMb concentration over time. Even though the exposure to irradiation significantly affected the metMb concentration, its effect was dependent on time. The interaction between irradiation and time resulted in a marginal increase in metMb concentration of patties in day 0 whereas a general decrease in metMb concentrations of patties was detected on days 7 and 14. Patties irradiated with a 5 kGy dosage exhibited the highest metMb concentration observed on days 7 and 14, regardless of the carcass infusion by L A A or saline. 5.2.3 Textural Analysis A punch test conducted with a rectangular blade was used to assess the textural property (i.e. hardness) of all ground beef patties. Maximum force (in grams) required to punch through the patty was interpreted as the hardness. As illustrated in Figs 13a and 13b, maximum force or hardness of the ground beef patties was affected by the fat content. In particular, 17% fat patties had significantly higher punch forces than 30% and 4% fat patties. Regardless of the initial hardness, all patties were detected to have comparable hardness values after 3 d of storage at 4°C. Continuing storage of the patties led to a gradual increase in hardness, until a maximum hardness in all patties was detected on day 11. Infusion of L A A decreased the punch forces detected in all patties and therefore had a significant impact in lowering the hardness of ground beef patties. Irradiation was also a significant factor affecting the hardness of beef patties, but dosages of 5 and 10 kGy had opposite effects on hardness. This was found to be dependent on the individual fat content of the patties. Regardless, a significant increase in hardness was detected in 4% fat patties while a significant decrease in hardness was detected in 17% and 87 J 3000 ' 2000 1000 8000 ^ 7 0 0 0 130 ^ 6 0 0 0 £ 5000 -£ 4000 -| 3000 2000 ] 1000 8000 ^ 7 0 0 0 -| @6000 | 5000 s 4 0 0 0 ^ I 3000 2000 1000 Figure 13a - Effects of fat level and electron beam irradiation dosages at 0 kGy, 5 kGy, 10 kGy and 20 kGy on maximum single blade shear force in control ground beef patties during storage at 4°C. AH values are mean+SEM; n=18. Figure 13b - Effects of fat level and electron beam irradiation dosages at 0 k G y , 5 k G y , 10 k G y and 20 k G y on maximum single blade shear force in ground beef patties made from L-ascorbic acid infused carcasses during storage at 4 ° C . A l l values are mean+SEM; n=18. 30% fat patties with the exposure to these two irradiation dosages. This interaction between fat and dosages was also evident when comparing the hardness of 17% and 30% fat patties between 0 kGy and 20 kGy dosages. Maximum punch forces required for 17% fat patties were higher than those required for 30% fat patties when irradiated at 20 kGy. Irradiation of 4% fat patties was also shown to increase hardness, with patties treated with 20 kGy dosage requiring the highest punch force observed, followed by 10 kGy, 5 kGy and 0 kGy. However, the increase in hardness between 5 kGy and 10 kGy irradiated 4% fat patties was not significant (p>0.05). 5.2.4 C o r r e l a t i o n s Correlations between the various physiochemical qualities (i.e. lipid oxidation, colour and texture) were determined using the Pearson's product moments coefficient of correlation (ocO.01). Table 10A provides the coefficients of correlation between the various attributes of all ground beef patties tested. Since there were insufficient data for heme iron and metMb concentration (data only available for day 14) for 4 % and 17% fat patties, no correlation coefficient between these two attributes and the other qualities could be computed. However, since a complete set of data (i.e. data for days 0, 7 and 14) was available for 30% fat patties, a second correlation analysis was performed on all attributes of 30% fat patties only (Table 10B). According to Table 10A, there were several attributes that were not correlated; namely between L value and a value, b value and punch force, and M D A and punch force. A negative correlation was observed for a value and punch force, as well as for heme iron and metMb concentration. Even though most of the attributes were significantly correlated to one another, the correlations were relatively weak. Therefore, the changes observed in one physiochemical attribute would not necessarily correlate to a change in another attribute of the ground beef patties under the conditions used in this study. Table 10 - Correlations between maxmium shear forces, Hunterlab colourvalues, malondialdehyde concentration, heme iron content and percentage metmyoglobin for electron beam irradiated ground beef patties of 4%, 17% fat content (A) and 30% fat content (B) containing L-ascorbic acid. Shear L a MDA HemeFe metMb Shear 1.000 L 0.079 1.000 a [0.005] -0.168 0.013 1.000 MDA [0.000] -0.015 [0.652] 0.284 0.092 1.000 HemeFe [0.599] N/A [0.000] N/A [0.001] N/A N/A 1.000 metMb N/A N/A N/A N/A -0.274 1.000 [0.000] B Shear L a MDA HemeFe metMb Shear 1.000 L 0.001 [0.981]1 1.000 a 0.072 [0.223] -0.158 [0.007] 1.000 MDA 0.286 [0.000] -0.294 [0.000] -0.105 [0.074] 1.000 HemeFe -0.124 -0.101 0.019 0.166 1.000 [0.035] [0.087] [0.754] [0.005] metMb 0.368 0.112 -0.145 0.116 -0.269 [0.000] [0.058] [0.014] [0.050] [0.000] Probabilities are shown in parentheses N/A Not available The relationships between various attributes of all the ground beef patties are less correlated when examining 30% fat patties only (Table 1 OB). Only a few attributes were related, which includes the positively related M D A and heme iron, M D A and punch force, metMb and punch force, and a and b value. Negatively correlated attributes included L value and a value, L value and M D A , b value and M D A , b value and heme iron, and heme iron and metMb. Therefore, fat content might be an important factor affecting the relationship of various physiochemical qualities of ground beef patties. 92 5.3 Discussion Heme iron in beef has two distinctive biological properties of importance. Firstly, heme iron in beef is the more bioavailable form of iron (in comparison to non-heme iron) in foods, with a typical uptake of 15 to 35% by the body (Monsen and Balintfy, 1982). Secondly, heme iron in beef has long been known to catalyze lipid oxidation (Monahan et al., 1993; Kristensen and Andersen, 1997; Harel and Kanner, 1985). Few researchers have measured the heme iron concentration in irradiated ground beef and none have evaluated the relationship between heme iron concentration and oxidative damages observed after irradiation. Therefore, heme iron in the ground beef patties was quantified and the association with oxidative damage was examined. Using the Hornsey (1956) method for heme iron determination, the ground beef patties employed in this study contained between 19.5 to 22.5 ppm of heme iron on day 0. This is similar to the concentration (20+4 ppm) of heme iron reported by Carpenter and Clark (1995) in raw ground beef. The final heme iron concentration detected in the irradiated ground beef patties on day 14, ranged from 7.58 to 9.96 ppm, is also similar to the heme iron reported in cooked ground beef (approximately 9.0 ppm) on day 9 of storage (Lee et a l , 1998). The decrease in heme iron concentration observed with increasing storage time at 4°C was attributed to the destruction of porphyrin rings of heme pigment by cooking, resulting in the release of non-heme iron (Lee et al., 1998). There has been no study to show the spontaneous destruction of heme iron in untreated raw ground beef during storage. However, the observed decrease in the heme iron concentration during storage in the present study suggests that the interaction of both time and irradiation had an effect on heme iron destruction. This irradiation-induced destruction of heme iron is probably a free radical mediated oxidation of the M b heme ring arising from the nucleophilic attack by a hydroxyl radical, produced from radiolysis of water. Such oxidative destruction of heme iron can also be enhanced by ascorbic acid (Schricker and Mil ler , 1982), possibly due to the oxidation by ascorbate radical anion. However, this was less likely to have 93 occurred since L A A didn't significantly decrease the detected heme iron concentration in all patties. In the absence of oxygen, the free radical oxidized M b molecule, in theory, can form aggregates with other oxidized M b to form dimers, trimers and even tetramers without the release of iron (Diehl, 1990; Delincee, 1983). In addition, without an analysis of non-heme iron concentration, it is not possible to say for certain that the destruction of heme pigments in the patties was accompanied by an increase in non-heme iron due to the possible formation of heme aggregrates. Nevertheless, the presence of oxygen in the Z ipLoc™ bags during irradiation and subsequent storage should have prevented the formation of aggregates and thus enhance the denaturation and fragmentation of proteins into low molecular weight peptide fragments (Giroux and Lacroix, 1998). This could have led to the release of non-heme iron, or the increase in reactivity of heme iron (still bound to peptide fragment), due to a decrease in stearic hindrances found in the original intact M b molecule (Thakur and Singh, 1994) A n increase in lipid oxidation in model and meat systems has been observed in the presence of both heme iron (Monahan et al., 1993; Kristensen and Andersen, 1997; Harel and Kanner, 1985; Verma et al., 1985) and non-heme iron (Monahan et al., 1993; Apte and Morrissey, 1987; Chen et al., 1984). Heme iron, mainly in the form of metMb, was thought to enhance phospholipid oxidation by homolytic scission of preformed phospholipid hydroperoxides to form free radicals, which in turn initiate nucleophilic attack on non-oxidized unsaturated fatty acids (Harel and Kanner, 1985b). Furthermore, the initiation of lipid oxidation by metMb can also be explained by the proposed mechanism of Harel and Kanner (1985b), involving the following reactions: (a) autooxidation of oxyMb and deoxyMb to metMb accompanied by oxygen activation to hydrogen peroxide; (b) catalyst activation of metMb by hydrogen peroxide to porphyrin cation radical (P+-Fe4+=0); (c) initiation of lipid oxidation by P +-Fe 4 +=0, via a two-electron reduction of the catalyst to yield P-Fe 3 + , a lipid peroxyl radical and a hydroxyl ion; and (d) activation of metMb by hydroperoxides generated during lipid oxidation. 94 In the present study, metMb-induced lipid oxidation was less likely to have occurred as suggested by the weak and insignificant correlation (r=0.116, p=0.050) found between M D A content and metMb concentrations in the beef patties. However, non-heme iron is generally accepted to initiate lipid oxidation by abstracting a hydrogen atom from an unsaturated fatty acid. This hypothesis was rejected by a weak but significant association (r=0.166, p=0.005) established between the increase in heme iron and the increase in M D A . Therefore, the presence of heme iron, in the F e 2 + form of either oxyMb or deoxyMb, was likely to have enhanced lipid oxidation in the early periods of storage by generating a superoxide anion during oxidation to metMb. This superoxide anion wi l l in turn be converted to H2O2 and its breakdown product, • O H can initiate lipid oxidation reactions. The reason for proposing this explanation was that the negative correlation between heme iron and metMb concentrations indicated that the heme iron present in the patties was not metMb and thus mostly likely to be either deoxyMb or oxyMb. In the later stages of storage, a significant accumulation of both metMb and non-heme iron can catalyze lipid oxidation reactions via direct metal catalyst or the activated metMb pathway, respectively. Aside from the potential effects of heme and non-heme iron on lipid oxidation, other factors such as the irradiation dosages and the addition of antioxidant can also affect the severity of lipid oxidation in ground beef. In particular, oxidative damage attributed to irradiation has been shown to be dose dependent and the rate of oxidation is dependent on the dissolved oxygen content in the meat or the immediate surroundings (Lefebvre et al., 1994; Katusin-Razem et al., 1992). This conclusion agrees with the finding in this thesis, as patties irradiated with 20 kGy contained a higher M D A content than those irradiated with 10 kGy, 5 kGy or non-irradiated control patties. It is of interest that the concentrations of M D A detected in 5 kGy and 10 kGy irradiated patties were not significantly different. Hampson et al. (1996) also observed a similar trend in irradiated muscles from beef, pork, lamb and turkey legs xand suggested that lipid 95 oxidation in these meat muscles was independent of irradiation dose effect in the range of 0 kGy to 10 kGy. The dose-dependent oxidative damage of lipid in ground beef patties therefore seems to be detectable only if a threshold irradiation dosages (e.g. 10 kGy) is achieved. The source of initiation of lipid oxidation in irradiated meat was thought mainly to be the hydroxyl radicals generated from the radiolysis of water molecules present in meat tissues (Thakur and Singh, 1994). This theory is highly plausible, since beef muscles contains as much as 75% water (Labdie, 1999). Other contributors, or secondary initiators of lipid oxidation, in irradiated meats include reactive oxygen species (ROS) derived from the irradiation of dissolved intra- or extracellular oxygen (Diehl, 1995), and the direct ionization of the electron deficient areas in the hydrocarbon backbone (e.g. regions with an oxygen atom or a double bond) of the fatty acid (Thakur and Singh, 1994). In the present study, the initiation of lipid oxidation in ground beef patties was probably not entirely the result of irradiation since low levels of M D A were detected also on day 0 before irradiation of the patties. Cellular disintegration arising from handling, cutting and grinding during the conversion of muscle to ground beef has been cited as a contributor to lipid oxidation of raw meats (Chen et al., 1999; Morrissey et al., 1998). Because the patties were immediately frozen after processing, the preformed lipid peroxides in the non-irradiated patties would be expected to localize in the region where the peroxides were formed since diffusion of molecules in a dry or frozen material is restricted (Thakur and Singh, 1994). A spontaneous recombination of two lipid peroxides would then be highly favoured, producing a stable non-radical dimer. Upon thawing, the reduced pool of non-reacted lipid peroxides continues to propagate lipid oxidation in meats, but to a lesser extent than if the meats were not previously frozen. Thus, non-irradiated patties were observed to obtain a maximum accumulation of M D A either by day 3 or day 7 (4% fat patties only). With the exposure of meat patties to irradiation, larger amounts of oxidized catalysts would be expected to be formed in addition to the preformed lipid peroxides. Even though recombination of lipid peroxides occurs 96 during freezing, the increase in cellular oxidizing catalysts (e.g. hydrogen peroxides, superoxide anions, ionized unsaturated fatty acids) contribute further to a greater source of oxidized products that would remain upon thawing, thus allowing initiation and propagation of lipid oxidation to occur. This increase in cellular oxidizing agents, coupled with a relatively slow disappearance (in comparison to lipid peroxides), is responsible for the delay or "after effects" of irradiation on oxidation of lipid, proteins and water-soluble vitamins (Thakur and Singh, 1994; Diehl, 1983 and 1975; Thakur and Axya, 1993). Hence a maximum accumulation of M D A in irradiated, 17% and 30% fat patties was detected only on either day 10 or day 14 of storage. The basis for the catalytic role of free radicals or free transition metals on lipid oxidation reactions is due to the nucleophilic attack on the electron deficient site of an unsaturated fatty acid. Therefore, saturated fatty acids are not as susceptible to lipid oxidation. Typically, the lipids in ground beef contain 46% saturated, 51% monounsaturated and 3% polyunsaturated (e.g. phospholipids of membranes) fatty acids. The addition of tallow to ground beef to adjust for a final fat content would further increases the saturated fatty acid content and reduce the relative proportion of polyunsaturated fatty acid in the product. The practice of adding tallow to ground beef to increase the overall fat content of ground beef patties would, in theory, lower or not affect the severity of lipid oxidation. This theory is supported by the findings of Shivas et al. (1984) who reported no increase in M D A content of ground beef patties containing 20% and 25% fat. However, in the present study, the increase in fat content of ground beef patties was found to be related to a greater degree of lipid oxidation. Other studies have demonstrated that lipid oxidation can be detected in an emulsion made up of lard or tallow, with either metMb or inorganic iron salts (Verma, 1985). Furthermore, irradiation can directly ionize fatty acids at location of electron deficiency, which further react with oxygen to form peroxides (Thakur and Singh, 1994). Taking these facts into consideration, it can be concluded that the addition of 97 saturated fats to the ground beef patties did not improve oxidative stability, as previously thought, when exposed to irradiation, to highly reactive transition metals, or to both. Enrichment of muscle meats with ascorbic acid is not often practiced, since orally administered ascorbic acid would largely be degraded by ruminant microorganisms prior to absorption. Furthermore, injection of cattle with ascorbic acid is labour intensive with little assurance of adequate deposition in muscles. However, earlier studies have successfully administrated ascorbic acid to cattle by injecting a solution at a concentration of 0.25% to 50% (w/v) (Hood, 1975; Wheeler et al., 1996), or by oral administration of ascorbyl-2-polyphosphate at a dose of 20 g/day/head (Macleod et al., 1999). The resulting residual concentrations of ascorbic acid (between 100 to 200 mg/kg) detected in muscles after injection was noted after 10 d of storage at 0°C (Hood, 1975). Since analysis of ascorbic acid in plasma or muscle after injection, or in ground beef patties before and after storage was not possible in the present study, it can only be speculated that the deposition of sodium ascorbate (concentrations between 10 m M to 500 mM) into muscles by infusion resulted in residual levels of ascorbic acid even after 14 d of storage. This assumption is supported by the findings or other authors (Hood, 1975; Wheeler et al., 1996) and the observation that a significant difference in M D A content, as well as a value, metMb concentration and punch force, was observed in all patties made from carcassess infused with sodium ascorbate. Ascorbic acid is a reducing agent and functions as an antioxidant by scavenging lipid free radicals, scavenging dissolved oxygen and chelating transition metals (Borenstein, 1987). The addition of ascorbic acid to meats has been associated with an overall increase in oxidative stability by decreasing lipid oxidation (Lee et al., 1999), decreasing metMb formation (Shivas et al., 1984; Hood, 1975), and regenerating endogenous a-tocopherol (Schaefer et al., 1995). However, an opposite opinion towards the effectiveness of ascorbic acid to stabilize oxidative reactions in meat has also been documented (Morrissey et al., 1998; Schaefer et al., 1995; 98 Mahoney and Graf, 1986; Shivas et al., 1984). Lipid oxidation, measured as M D A content, was not suppressed in the presence of L A A in the present study. In fact, a greater concentration of M D A was detected, indicating a prooxidant effect of ascorbic acid under the conditions studied here. This suggests that the ascorbic acid infused was rapidly oxidized to the ascorbyl radical anion through the loss of two hydrogen atoms. Thakur and Singh (1994) have postulated that the radiosensitivity and destruction of vitamins in foods is due mainly to an indirect effect through the scavenging of secondary free radicals or carbonyls generated from the oxidation of fatty acid, protein and carbohydrate induced by irradiation. It was further stated that direct destruction of ascorbic acid by irradiation was less probable because the low concentration of ascorbic acid present in meats reduced the probability of interaction with primary hydroxyl radicals produced. Because of the infusion of a high level of ascorbic acid into the carcass, a prooxidant effect observed on day 1 of storage in irradiated patties may be attributed to the ascorbyl radical anions produced from the direct irradiation-induced destruction of ascorbic acid. Furthermore, applying higher dosages of irradiation (up to 20 kGy) to beef patties could increase the production of lipid free radicals immediately after irradiation, which also could rapidly deplete the infused ascorbic acid. It is concluded therefore that an antioxidant activity of infused ascorbic acid might have been noted with lower dosage of irradiation, resulting in a lower degree of lipid oxidation. This observation was also reported by Kanatt et al. (1998), where the antioxidant activity in chicken meat irradiated at 2.5 kGy was established to the 0.01% ascorbic acid applied. In addition to the direct prooxidant effect of ascorbate radical anion, ascorbic acid can also increase lipid oxidation through the generation of hydroxyl radicals. Mahoney and Graf (1986) demonstrated that the presence of ascorbate with F e 3 + increased lipid oxidation as indicated by the increased production of M D A derived primarily by the generation of increased amounts of hydroxyl radicals. The production of hydroxyl radicals occurred from the Haber-Weiss cycle (i.e. superoxide anion reduces F e 3 + to F e 2 + and two superoxide anions combine with 99 two hydrogen atoms to form hydrogen peroxide) and the Fenton reaction (i.e. F e 2 + oxidizes hydrogen peroxide to produce hydroxyl radical, hydroxyl ion and Fe 3 + ) . In meats, the oxidation of deoxyMb to metMb is accompanied by the formation of superoxide anion from oxygen, which in turn can be converted to hydrogen peroxide by superoxide dismutase (Schaefer et al., 1995). Alternatively, hydrogen peroxide can be produced by mitochondrial microsomes, peroxisomes, cytosolic enzymes and phagocytic leukocytes (Harel and Kanner, 1985a). The reduction of F e 3 + to F e 2 + is achieved by ascorbic acid, thereby allowing the Fenton reaction to proceed (Brown et al., 1963). Therefore, the observed increase in lipid oxidation in patties made from L A A infused carcasses may also be attributed to the production of hydroxyl radicals via the Fenton reaction. Myoglobin is responsible for the colour of fresh meat. Various forms of M b can impart different beef colours. The oxygenated form of M b , oxyMb, is responsible for the bright red colour, whereas the oxidized form of M b , metMb, is responsible for the brown discolouration of fresh beef. Ground beef is known to be more susceptible to oxidative deterioration (i.e. lipid and M b oxidation) during prolonged storage because grinding exposes more surface areas to air and microbial contamination, as well as, reduces intracellular reductants (O'Grady et al., 1998; Mitsumoto et al., 1993; Renerre, 1990). Hence the study of beef discolouration in this study is an important criteria for assessment of overall meat quality. The lightness of ground beef patties was measured as the Hunterlab L value. Surface lightness of beef patties were minimally affected by irradiation, ascorbic acid infusion and prolonged storage. Only fat content was shown to affect the L value, with increasing L value noted for increasing fat content in beef patties. This result was attributed to the lack of colour offered by fat and/or the reduction in the proportion of beef tissue in the patties. Murano et al. (1998) and Nanke et al. (1998) also found no change in L values of irradiated ground beef patties and beef steaks, respectively. The L values reported by Murano et al. (1998) and Nanke et al. 100 (1998) were comparable to those obtained in this study, which ranged from 25 to 45, depending on the fat content of the beef patties. The oxidation of the F e 2 + moiety in the deoxyMb/oxyMb complex to F e 3 + moiety in metMb is the main cause for discolouration of beef (Renerre, 1990). Previous studies have found that autooxidation of F e 2 + in beef can be enhanced by exposure to U V light (Bertelsen and Skibsted, 1987) and irradiation (Murano et a l , 1998; Nanke et a l , 1998). A significant decrease in Hunterlab a value for redness of beef exposed to electron beam irradiation was observed by Murano et al. (1998) in ground beef patties stored at 4°C for 45 d and by Nanke et al. (1998) in beef steaks stored at 0°C for 84 d. Similar decreases in redness of electron beam irradiated beef were observed in the present study with patties containing 17% and 30% fat, respectively, after 7 d of storage, but a higher a value was obtained in this study in comparison to that of Murano et al. (1998). The generation of hydroxyl radicals from radiolysis of water was probably the main oxidizing species responsible for the oxidation of deoxyMb to metMb. Moreover, lipid free radicals generated from electron beam irradiation can also induce deoxyMb oxidation (Chan et al., 1997; Lanari et al., 1996; Mitsumoto et al., 1993; Renerre, 1990). The latter explanation for the oxidation of deoxyMb in the present study can be rejected since irradiated patties containing higher fat content contained higher a values throughout storage. On the contrary, the presence of increased fat content in patties may exerted an inhibitory effect on deoxyMb oxidation by absorbing irradiation or reducing the oxygen tension below that required to initiate oxidation of deoxyMb in the patties. Even though electron beam irradiation was found to decrease the a value in beef patties, the dosages applied had a negligible effect on the redness values. Careful examination revealed that a values for redness of 4% fat beef patties that had undergone irradiation in this study followed, in descending order of magnitude from 20 kGy>10 kGy>5 kGy. Such an increase in redness of 4% fat meat patties at higher irradiation dosages has also been observed by Nanke et 101 al. (1998) who applied 1.5 kGy to 10.5 kGy dosage of electron beam irradiation. The increase in redness at higher dosages of irradiation could be the result of an increased generation of aqueous electrons that reduce electron deficient atoms, such as a ferric ion in the metMb, to a ferrous ion in the deoxyMb and oxyMb. Ascorbic acid has been demonstrated to prevent the discolouration of ground beef (Lee et al., 1999; Shivas et al., 1984; Hood, 1975; Wheeler et al., 1996; Mitsumoto et a l , 1991b; Harbers et al., 1981). A proposed model of an oxidation-reduction relationship in beef relating to the phospholipid and M b oxidation to vitamin E and C has been proposed by Schaefer et al. (1995) to involve ascorbic acid, which can protect redness of beef (i.e. maintain oxyMb) by inhibiting the autooxidation of deoxyMb to metMb. Ascorbic acid can also regenerate vitamin E , which in turn prevents the mediation of deoxyMb oxidation by lipid peroxyl radicals. Other roles of ascorbic acid, such as a reducing agent, a metal sequestering agent and an oxygen scavenger, cannot be overlooked in regards to preventing discolouration (Borenstein, 1987). However, in this study, the infusion of ascorbic acid was observed to induce a greater lost in the a value for redness in the ground beef patties. One possible explanation for this observed prooxidant effect is that the ascorbic acid can be oxidized by deoxyMb (containing Fe 2 + ) to produce a reactive free radical, ascorbate radical anion, and a metMb. Also, it is possible that the reduction of inorganic salts of F e 3 + to F e 2 + by ascorbic acid increased the generation of hydroxyl radicals, which in turn induce lipid oxidation, thereby producing peroxyl radicals capable of oxidizing deoxyMb. Alternatively, ascorbic acid can donate two hydrogen atoms to a superoxide anion (generated during oxidation of deoxyMb to metMb) to form hydrogen peroxide, which subsequently can oxidize F e 2 + in deoxyMb to produce a hydroxyl radical capable of inducing lipid peroxyl radical production. Further research is necessary to elucidate the process mechanism of this observed effect. 1 0 2 Another way of monitoring discolouration of beef is to measure the percentage of metMb present in relation to the deoxyMb and oxyMb concentrations. A decrease in the a value, caused by the oxidation of deoxyMb, is related to an increase in the metMb concentration. Surprisingly, the loss of redness (i.e. oxyMb) of beef patties in the present study was not significantly associated with the formation of metMb (r=-0.145, p=0.014). Renerre (1990) suggested that Mb in beef could yield a more stable red/pink chromophore with an absorption spectrum similar to that of oxyMb, in addition to the formation of metMb after irradiation. This explanation might explain the higher a value observed in irradiated samples and the lack of an association between a decreased a value (i.e. lost of oxyMb) and an increased metMb concentration. Nevertheless, the noted decrease in redness of the 30% fat patties over time followed a parallel increase in the metMb concentration. This increase in metMb concentration detected in the higher fat patties was probably due, in part, to the oxidation by lipid peroxides since 17% and 30% fat patties yielded similar percentages of metMb and a values by day 14 of storage. Moreover, increasing the dosages of electron beam irradiation did not necessarily increase the metMb concentration, which suggests that the increased production of hydroxyl radicals at higher dosages does not directly oxidize beef patty deoxyMb, but rather mediates the production of other oxidizing species (e.g. lipid peroxyl radical). This explanation of lipid peroxyl radical mediated formation of metMb is weak, as induced by the lack of any association between M D A and metMb concentration (r=0.116, p=0.050). The infusion of L A A has no antioxidant effect against deoxyMb oxidation in ground beef patties. In fact, higher concentration of metMb was formed in patties made from L A A infused carcasses. The percentage of metMb obtained from patties made from L A A infused carcasses (between 57.87% to 88.38%) in this study was much higher than that reported by Shivas et al. (1984) at a range of 48.03% to 62.11%, and by Lee et al., (1991) and Mitsumoto et al. (1991a) at 28% and 32%, respectively, in ground beef and by Okayama et al. (1987) at 15% in steaks. 103 Taken together, the finding of this study agrees with others and indicates that the formation of metMb is related to the ascorbate radical anion. To better illustrate the effect of ascorbic acid on colour retention of ground beef patties, a numerical total colour difference (AE), based on all three Hunterlab colour scores, was calculated and is'graphically represented in Fig 15. On the whole, the colour difference between control patties and patties made from L A A infused carcasses fluctuated during storage. Irradiation of patties led to a greater change in A E in ground beef patties immediately after exposure, possibility resulting from the rapid oxidation of deoxyMb to metMb as confirmed earlier by the decrease in a value. This rapid oxidation was probably a result of the hydroxyl radical formation occurring during irradiation. A drastic difference in A E , induced by ascorbic acid, for irradiated 17% and 30% fat patties was noted between days 3 to 11 of storage, which corresponded to a major difference in a value in those same patties. This result seems to suggest that lipid oxidation products, which are related to the oxidation of deoxyMb, was the cause of the significant change in A E at this particular time since the hydroxyl radical should have dissipated due to its relatively short half-life. However, a weak positive correlation between M D A and a value rejects the idea of lipid oxidation products being the cause of the colour difference. Since the higher content of M D A detected in the 30% fat patties was not associated with a greater A E than the 17% fat patties, it is more likely that the formation of ascorbate radical anion during this time period of increased lipid oxidation was the primary factor in oxidizing deoxyMb. The result of this reaction is a decrease in redness, and therefore a change in A E . Also, L A A in the ground beef made from infused carcasses is located in the sarcoplasm, where it is in direct contact with Mb, thus allowing for a prooxidant effect before lipid peroxides would otherwise come into contact with the Mb. It is noteworthy that the 4% fat patties maintained the most uniform colour change almost independently of the effect of ascorbic acid. 104 Protein oxidation in meats by irradiation has been well reviewed by Giroux and Lacroix (1998), Thakur and Singh (1994) and Delincee and Paul (1981). According to Giroux and Lacroix (1998), protein oxidation is initiated by hydroxyl radicals that denature the protein through breakage of hydrogen bonds, disulfide linkages and other intermolecular linkages involving both tertiary and quaternary protein structure. In the presence of oxygen, the denatured protein is fragmented at the a-carbon rather than at the peptide bond, producing low molecular weight polypeptide chains of various lengths. In the absence of oxygen, the denatured protein aggregates to form high molecular weight dimers, trimers and even tetramers which are stabilized by covalent, rather than disulfide linkages (Giroux and Lacroix, 1998). In general, the toughness of all ground beef patties was shown to be greater after prolonged storage at 4°C. During storage, a gradual release of beef patty moisture, in the form of a drip-loss, was visually detected in all storage bags. This loss in intracellular water may account for the increased cross-linking of muscle fibers in beef, leading to the increase in toughness in later stages of storage (Currie and Wolfe, 1980). Addition of fat did not lower the observed maximum punch force, as predicted by Trout et al. (1992) and Berry and Leddy (1984) in cooked ground beef patties. Perhaps the cooking of beef patties liquefied the saturated fats and fat patties containing more fat were detected to be less tough. The tallow present in beef is solid at 4°C and therefore is harder in texture when compared to the finely ground beef tissue. In addition, the high punch force observed in more concentrated fat patties might also be due to the induction of protein oxidation by lipid oxidation products and formation of protein-lipid oxidation product (e.g. MDA-protein adducts) (Delincee and Paul, 1981). Thus, the increased lipid oxidation during the later stages of storage could have had a role in the rapid increase in toughness observed in this study. However, no significant association was found between M D A content and maximum punch force under the conditions studied herein. Furthermore, 30% fat patties (with the highest M D A content) had a lower maximum punch force than the 17% fat 105 patties, which further disproves the hypothesis that the induction of protein oxidation was linearly related to lipid oxidation product. A n equally puzzling phenomenon observed was the finding that increasing dosages of irradiation from 0 kGy to 10 kGy decreased the punch force of the ground beef patties. Only 20 kGy irradiated patties had punch forces comparable to those of non-irradiated patties. Hence dehydration of beef patties was as effective as high irradiation dosages in toughening the meat product. Increasing irradiation dosage was thought to increase oxidation by hydroxyl radicals, followed by subsequent cross-linking of denatured protein, leading to an increase in toughness (Giroux and Lacroix, 1998; Delincee and Paul, 1981). Therefore, the decrease in toughness observed in this thesis is hypothesized to be due, in part, to the partial fragmentation of protein at the early stages of storage. The availability of oxygen prior to aggregation in the later stages becomes a limiting factor, as a result of the increased demand from lipid and M b oxidation. Aggregation of meat proteins can therefore lead to a decrease in protein solubility and this has been reported by Joo et al. (1999) to mask the red colour of meat by affecting light reflectance or light scattering. This explanation could therefore account for the significant negative association between punch force and a value (r=-0.168, p<0.000) found in this study. The negative effects of irradiation and dehydration on texture were counteracted by the infusion of ascorbic acid. Scavenging of hydroxyl radical by ascorbic acid was probably the main cause for the observed effect in lowering punch forces in ground beef patties made from L A A infused carcasses. 106 5.4 Conclusion Heme iron in the ground beef patty was oxidized by exposure to electron beam irradiation. Heme iron concentration in the 30% fat patties was shown to decrease during storage, possibly releasing non-heme iron. This decrease in heme iron concentration occurred parallel to an increase in lipid, oxidation of ground beef patties. However, the oxidation of deoxyMb to metMb, which generates superoxide anion as a by-product, was most likely the explanation for the increased lipid oxidation in the presence of M b derivatives. Increasing the dosage of irradiation was also a significant factor in the increased lipid oxidation observed. The production of hydroxyl radicals from radiolysis of water was probably a major oxidizing agent inducing lipid oxidation. However, exposure to 5 kGy and 10 kGy dosages of irradiation resulted in a similar degree of lipid oxidation, which was greater than that in non-irradiated patties and less than that observed in 20 kGy irradiated patties. Addition of tallow to the ground beef patties was found to decrease the oxidative stability of the patty. Ground beef patties with higher fat content had higher lipid oxidation during storage. The antioxidant activity of L A A was rapidly lost likely due to hydroxyl radical and lipid peroxyl radical scavenging activities. Therefore, infusion of L A A enhanced lipid oxidation in beef patties. In the presence of iron, in the form of oxyMb or deoxyMb, the L A A likely oxidized to a prooxidant form of ascorbyl radical anion. This explanation could be the reason for the decease in redness of ground beef patties infused with L A A . The lose of redness observed during storage was lower in low-dosage irradiated patties. In general, higher dosages of irradiation were responsible for the lower redness values observed at the end of 1 4 d storage. The previous findings that lipid oxidation products mediate the oxidation of deoxyMb in beef, or the loss of redness, was not observed here. Furthermore, the loss of redness in the ground beef patties was not necessarily associated with an increase in metMb concentration. Irradiation and infusion of ascorbic acid were key factors responsible for the increased metMb concentration noted over time. Therefore, an overall 107 colour change (delta E) due to the infusion of ascorbic acid was found to be affected by hydroxyl radicals likely produced during irradiation and not the lipid peroxyl radical. Hardness of ground beef patties was affected by addition of tallow. Ground beef patties containing 17% fat had higher hardness value than patties containing 4% or 30% fat. Irradiation below 10 kGy decreased maximum punch force in 17% fat patties in comparison to non-irradiated patties. Ascorbic acid was only effective at lowering the hardness of ground beef patties. 108 6. General Discussion The objectives of this thesis were to assess the effectiveness of electron beam irradiation in reducing the microbial load in fresh ground beef and the capacity of infused L A A in minimizing the oxidative damages induced by electron beam irradiation. Like most food systems, ground beef is a complex medium consisting of numerous food components that are interrelated to each other. Therefore, simplified model systems were used in Chapter 3 to isolate and study the key components in ground beef that were mostly likely involved in oxidative damage. Conclusions drawn from those model systems can be helpful in explaining the oxidative changes in a real food systems such as ground beef patties. Hydroxyl radicals, generated from radiolysis of water, are widely accepted as the free radicals responsible for irradiation-induced damage to both microorganisms (Diehl, 1990; Lopez-Gonzalez et al., 1999) and food components (Thakur and Singh, 1994; Giroux and Lacroix, 1998). In addition, »OH can also be generated in beef by the oxidation of H 2 0 2 (produced during oxidation of deoxyMb to metMb) by F e 2 + in the Fenton reaction (Arouma et al., 1987). Since the majority of iron in beef is in the bound form of heme iron, the conflicting opinions toward the capacity of heme iron (either as Hb or Mb) to act as a catalytic iron source in the Fenton reaction still exist. According to Figure 3, substitution of FeCb by Hb in the Fenton reaction, exposed to either U V radiation or thermal incubation, led to an approximate one half decrease in the «OH produced. Therefore, intact Hb or Mb would not be expected to significantly increase the production of ' O H . The indirect destruction of bacteria by free radicals in the ground beef patties was not due to the production of «OH from the Fenton reaction but rather from the radiolysis of water. Moreover, the presence of transition metals such as copper and iron should directly catalyze lipid oxidation. However, no significant correlation was found between M D A and metMb concentration in the ground beef patties and therefore, the inactivity 109 of intact Hb/Mb towards oxidative reactions was further demonstrated. The level of «OH generated from radiolysis of water far exceeds that generated from the Fenton reaction, which increases its probability of interaction with cellular and/or food components in the patties. To minimize oxidative damages to food components by ' O H , an infusion of L A A into the carcass post-mortem was examined. Three carcasses were infused on the right side with 17.5 L of either 10 m M , 100 m M or 500 m M sodium ascorbate solution. Muscles from all three L A A treatments were pooled together to make ground beef patties for experimentation. Unfortunately, no assessment of L A A deposition into the muscles after infusion was performed and so the amount of L A A deposited is unknown. Moreover, a lack of ground beef patties prevented the measurement of L A A concentration in patties during storage. Therefore, L A A depletion in buffer solutions after exposure to irradiation was studied in Chapter 3 in order to estimate the concentration of L A A in ground beef patties during the storage. At a low concentration of 10 mM, the rapid oxidation or depletion of L A A in buffer by • O H was noted after 1 d of storage for all dosages of irradiation but at higher concentrations (i.e. 100 mM), only a marginal loss of L A A was detected (Fig. 4). At 500 m M of L A A , no net lost was detected after 14 d of storage at 4°C due to self-regeneration. Therefore, assuming adequate deposition of L A A into the beef by infusion, low levels of L A A would not be expected to provide protection against damages by oxidative reactions such as lipid oxidation and deoxyMb oxidation in the ground beef patties beyond 1 d of 4°C storage after irradiation. Instead, the oxidation of L A A to ascorbate radical anion, under aerobic conditions, would have exhibited prooxidant activity and enhanced oxidative reactions thereafter. This was clearly demonstrated in emulsion models containing all levels of L A A that were exposed to both U V radiation and electron beam irradiation at 5 kGy, 10 kGy and 20 kGy (Figs 5 and 6). Thus, these findings can be generalized to a more complex food system such as irradiated ground beef patties, but with far less prooxidant activity expected since other food components might have insulated the L A A no from the absorbed dosage of irradiation and decreased its oxidation or depletion. This was the case with the infused patties having greater lipid and deoxyMb oxidation than non-infused patties (Figs 9, 10 and 11, 12), as suggested in Chapter 5. Moreover, the prooxidant activity of low levels of L A A (i.e. 10 m M and 100 mM) was probably more dominant than the antioxidant activity of high levels of L A A (i.e. 500 mM) in the ground beef patties examined under the conditions of this study, and this resulted in a net increase in lipid and deoxyMb oxidation when the pro/-anti-oxidant effects of all three levels of L A A were taken into account in the reported mean M D A and metMb values. Another factor enhancing lipid oxidation reactions in beef is ionizing irradiation. Increasing dosages of ionizing irradiation in the model system were shown to increase lipid oxidation, as reported by many investigators in the past. A similar increase in lipid oxidation was observed in the ground beef patties with increasing dosages of irradiation. However, the increase in lipid oxidation was not as linearly associated with the increase in dosages, as seen in the model systems. This is similar to the above mentioned decrease in activity of L A A in ground beef patties where direct ionization of fatty acids in a complex food system is reduced by the presence of other food components that are absorbing the applied dosage and indirectly insulating the fatty acid. Furthermore, the increased production of the radiolytic product, ' O H , is less likely to have interacted with unsaturated fatty acids in the ground beef due to its insolubility in the non-aqueous medium. This was not the case, as observed in Chapter 1, since forming an emulsion in the model systems increased the chance of contact between * O H and unsaturated fatty acid. Interestingly, a decline in M D A content was noted on day 7 of storage. Such disappearance of M D A was also observed in beef (Rhee et al., 1997) and in tuna (Quaranta et al, 1984). Quaranta et al. (1984) have attributed this decline in M D A to be due to the interaction with other tissue constituents, such as amino acids, proteins or vitamins while Rhee et al. (1997) 111 proposed the assimilation of M D A by microorganisms and/or reactions with the amine compound produced during bacterial metabolism. Since L A A was acting as a prooxidant in the ground beef patties, the antioxidant activity of vitamins (i.e radical scavenging activity) suggested by Quaranta et al. (1984) can be rejected. The proposed explanation by Rhee et al. (1997) was more possible in this case since a rapid growth of all bacteria types (i.e. aerobic bacteria, total coliform, E. coli and psychrotrophic bacteria) between days 4 and 7 was paralleled by a decline in M D A concentration. In addition, a negative association between all microbial growth and M D A concentrations in the beef further supported the proposed explanation (Appendix 5). However, low coefficients of correlation suggest that other reasons for the decline in M D A content (e.g. decomposition of M D A or removal of M D A by way of drip-lost) was also possible. In addition to its effect on M D A accumulation, microbial growth can also impart an effect on beef colour. Renerre (1990) had proposed that a large increase in metMb concentration during logarithmic growth of aerobic bacteria to be the result of bacterial reduction in oxygen tension on the surface of the meat. The author further proposed that by-products of bacteria metabolism (e.g. H2O2 or hydrogen sulfite (H2S)) could ionize the iron moiety to become sulfmyoglobin (greenish tint) or cause a degradation of M b to beyond porphyrins and to bile pigments. This proposed hypothesis was supported in this study by establishing a negative association between microbial growth and metMb concentration in the patties (Appendix 5), indicating that microbial growth led to an oxidation of deoxyMb/oxyMb to sulfmyoglobin or other oxidized M b derivative beside metMb. The latter reaction by metabolic by-products from bacteria, which can degrade deoxyMb/oxyMb to a colourless pigment, was also demonstrated in Chapter 5 with an increase in L value during microbial growth giving a positive correlation (Appendix 5). Together,* these two findings indicated that microbial growth could decrease the overall intensely bright red colour on the surface of ground beef. However, a weak positive 112 association between a value for redness and all microbial growth was found in all ground beef patties. Therefore, the role of microbial growth on oxidation of deoxyMb/oxyMb is not clear and the oxidation by free radicals was probably more dominant than microbial effects. The proliferation of microorganisms in beef would often lead to a degradation of food components such as carbohydrate, lipids, vitamins and proteins. Utilization of protein as a nitrogen source for bacteria growth is especially detrimental to meat quality since it can lead to a lost of texture in the beef. As predicted, an inverse association was found for all bacteria growth and maximum punch force. In particular, hardness of ground beef patties was especially affected by the growth of aerobic and psychrotrophic bacteria. According to Appendix 5, a positive association between heme iron concentration and bacteria growth was established, indicating that heme iron oxidation during storage was independent from bacteria proliferation. 113 7. General Conclusions Two mechanisms of irradiation-induced injury to microorganisms and food components have been identified, namely the direct ionization of cellular and food components and the indirect ionization of the aforementioned components by hydroxyl radicals generated from radiolysis of water. A third mechanism of injury to both microorganisms and food components was proposed, in this thesis, to be the hydroxyl radicals generated from the Fenton reaction. However, the inability of hemoglobin to generate hydroxyl radicals in the Fenton reaction was clearly demonstrated in Chapter 3. Moreover, the inactivity of Hb and M b towards oxidative damages was further demonstrated when an decrease in heme iron and metMb was not shown to be strongly associated with an increase in lipid oxidation. Contrary to the early findings, no statistically significant relationship was established between Mb oxidation and lipid oxidation and vice versa. Therefore, hydroxyl radicals generated from radiolysis of water remained the putative main free radical responsible for the majority of the oxidative damage observed in the ground beef patties used in this study. The formation of hydroxyl radicals was also proposed to be the main cause for the observed oxidation of L A A in a buffer solution after exposure to all irradiation dosages applied. In addition, the lipid peroxide scavenging activity of L A A can also lead to its oxidation, as observed in the aforementioned system containing oxidizing unsaturated fatty acids. As a result, ascorbate radical anion was assumed to be present in both model systems and ground beef patties after irradiation. Therefore, the free radical activity of ascorbate radical anion was believed to be the cause of the increased lipid oxidation observed in model systems exposed to both ultraviolet light and electron beam irradiation. This assumption could also be made for the enhanced lipid oxidation, greater loss of a value and greater accumulation of metMb concentration observed in the ground beef patties. 114 Increasing the fat content of the ground beef patties, from 4% to either 17% and 30% by addition of tallow, was shown to adversely affect the quality of the product. Higher microbial populations were detected in patties with added tallow. In addition, such an increase in saturated fat did not improve the overall oxidative stability as expected, but rather increased lipid oxidation and overall hardness of the ground beef patties. Nonetheless, a retention of redness and a decrease in the accumulation of metMb was observed in higher fat patties. The metabolic by-products of microbial growth appeared mostly to be amine, as shown by the increase in p H during storage. Microbial growth in ground beef patties was shown to be positively associated to the loss of colour due to the oxidation of deoxyMb to sulfmyoglobin or to other colourless M b derivatives. Similarly, a loss of texture was shown to be associated with microbial growth. This was probably the result of a demand for nitrogen that was supplied by proteins in the ground beef. However, assimilation of M D A by microorganisms could have been established based on the negative association between these two variables. This assimilation of M D A can be misinterpreted as a decrease in lipid oxidation reactions, by underestimating the actual M D A concentration, which may have a positive effect on quality perception of ground beef patty. In the present study, electron beam irradiation of ground beef patties was shown to drastically reduce the microbial population in the fresh product. However, adverse effects to lipid oxidation, M b oxidation and texture were observed. Increasing dosages of irradiation did increase the severity of oxidative damage in beef. Infusion of L-ascorbic acid into beef carcasses post-mortem was not beneficial in controlling the irradiation-induced oxidative reactions in ground beef patties. Instead, a prooxidant activity at the levels of L-ascorbic acid used in this study (10 m M to 500 mM) was the cause of the observed increase in oxidative damages in the patties. Oxidative stability was further decreased by the addition of tallow to ground beef patties 115 to increase the fat content to either 17% or 30%. As a result, an increase in the microbial population, lipid oxidation and hardness value of the fresh ground beef patties was reported. Because of the great concern of E. coli 0157:H7 present in ground beef, the food safety aspect of fresh ground beef has become the single most important criteria for assessing the quality of raw meats. Electron beam irradiation was demonstrated in the present study to be an effective tool in reducing the microbial load to an acceptable level in raw ground beef patties for a period of 7 to 14 d at 4°C storage. In doing so, the consumers are assured a safe ground beef supply from the local supermarket and this wi l l in turn result in an increase in profit for the meat processor. Many investigators in the past have cited adverse effects of irradiation on the colour stability and the off-flavour development in meats. Without a sensory evaluation of the irradiated ground beef patties in this study, it is unclear as to whether the changes induced by irradiation wi l l be noticeable by the consumers. Nevertheless, meat processors can reduce the side effects of irradiation to meats by packaging frozen lean beef under vacuum prior to irradiation. The addition of antioxidants to the beef prior to irradiation can be beneficial, only i f the antioxidant is not irradiation sensitive and not too costly to apply. In short, electron beam irradiation is a safe and effective measure in ensuring food safety of raw ground beef without drastically affecting the physicochemical attributes of the product. In this study, the infusion of L A A to beef carcasses only provided marginal protection against textural changes induced by electron beam irradiation in the ground beef patties. Therefore, it would be noteworthy to examine the antioxidant activity of other natural foodgrade antioxidants such as alpha-tocopherol or culinary spices in ground beef patties. Moreover, it would be interesting to examine the effects of electron beam irradiation on beef steaks. 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Journal of the Science of Food Agriculture, 35, 662-670. 126 Appendix 1 - Standard curves derived for determining ascorbic acid concentrations between the ranges of 0 mM to 25 mM (A), 25 mM to 100 mM (B) and 125 mM to 500 mM (C). 0 5 10 15 20 25 L-ascorbic acid concentration (mM) 100 200 300 400 500 L-ascorbic acid concentrtion (mM) Al l v a l u e s are m e a n s of a populat ion of n=3. A p p e n d i x 2 - T h e m a l o n d i a l d e h y d e content i n re la t ions to the a b s o r b a n c e r e a d i n g as m e a s u r e d us ing the t h i o b a r b i t u r i c a c i d test. 0 2 4 6 8 10 MDA concentration (uM) Al l v a l u e s are m e a n s of a populat ion of n=3. A p p e n d i x 3 - R e g r e s s i o n equa t i ons f o r best- f i t l ines d e p i c t i n g m a l o n d i a l d e h y d e content i n g r o u n d bee f pat t ies d u r i n g s to rage . Irradiation Fat L e v e l Infusion of Equa t ion R 2 (kGy) (%) asco rb i c ac id 0 4 N o y = 0 . 0 2 5 2 X 3 - 0 . 6 5 4 7 X 2 + 4 . 6 7 3 X + 1 0 . 0 6 0 0.9281 4 Y e s y = 0 . 0 4 9 2 X 3 - 0 . 7 3 9 0 X 2 + 2 . 4 1 9 9 X + 1 1 . 2 9 9 0.7697 17 No y = 0 . 0 1 2 2 X 3 - 0 . 1 9 8 4 X 2 + 0 . 5 1 6 1 X + 8 . 6 5 7 9 0.3482 17 Y e s y = 0 . 0 4 7 X 3 - 0 . 8 8 7 7 X 2 + 3 . 8 6 2 5 X + 1 2 . 0 0 9 0.5588 30 No y = 0 . 0 4 5 8 X 3 - 1 . 1 7 1 1 X 2 + 7 . 4 3 3 4 X + 7 . 7 4 0 8 0.8384 30 Y e s y = 0 . 0 0 9 7 X 3 - 0 . 3 8 5 6 X 2 + 3 . 2 2 3 4 X + 1 2 . 7 3 8 0 0.7176 5 4 No y= -0 .0025X 3 - 0 .0419X 2 +1 .2903X+12 .248 0 .3840 4 Y e s y = 0 . 0 8 8 5 X 3 - 1 . 2 9 2 0 X 2 + 4 . 6 4 0 2 X + 1 2 . 7 3 2 0 .5823 17 No y = 0 . 0 8 3 4 X 3 - 1 . 5 4 7 8 X 2 + 6 . 8 4 5 2 X + 8 . 8 8 2 0 0 .8500 17 Y e s y=0 .0886X 3 - 1 .7758X 2 +8 .8442X+13 .731 0.6471 30 N o y= -0 .0434X 3 +0 .5943X 2 +0 .4966X+9 .0848 0.6661 30 Y e s y= -0 .0427X 3 +0 .5231X 2 +0 .1701X+18 .725 0 .5345 10 4 No y = 0 . 0 7 7 7 X 3 - 1 . 3 9 4 3 X 2 + 6 . 8 6 2 8 X + 1 0 . 9 0 6 0 0 .7845 4 Y e s y = 0 . 0 8 3 1 X 3 - 1 . 4 2 2 6 X 2 + 6 . 2 4 0 5 X + 1 2 . 4 9 3 0 0 .5856 17 ' No y = 0 . 0 7 5 7 X 3 - 1 . 4 3 2 8 X 2 + 7 . 0 3 6 8 X + 8 . 3 9 2 8 0 .8219 17 Y e s y = 0 . 1 0 3 2 X 3 - 2 . 0 3 7 6 X 2 + 1 0 . 3 0 6 X + 1 2 . 5 3 8 0 .8677 30 N o y= -0 .0262X 3 +0 .2197X 2 +2 .4419X+9 .360 0 .5559 30 Y e s y= -0 .0376X 3 +0 .4029X 2 +1 .8002X+14 .188 0 .8533 20 4 No y = 0 . 0 7 1 X 3 - 1 . 2 1 8 4 X 2 + 5 . 3 4 7 3 X + 1 0 . 3 0 9 0.8078 4 Y e s y= -0 .0008X 3 - 0 . 2103X 2 +2 .1154X+13 .030 0 .4080 17 No y = 0 . 0 3 9 3 X 3 - 0 . 7 5 9 5 X 2 + 4 . 3 6 9 8 X + 9 . 6 9 3 7 0.6556 17 Y e s y = 0 . 0 6 6 6 X 3 - 1 . 2 3 1 8 X 2 + 6 . 0 8 7 7 X + 1 5 . 4 5 6 0 .6944 30 No y=0 .0269X 3 ' 0 . 9469X 2 +8 .4728X+9 .359 0.7844 30 Y e s y = 0 . 0 1 2 4 X 3 - 0 . 6 4 8 5 X 2 + 6 . 9 7 1 X + 1 3 . 6 7 9 0 .8516 129 A p p e n d i x 4 - R e g r e s s i o n equa t ions f o r best- f i t l ines d e p i c t i n g H u n t e r l a b " a " v a l u e f o r redness in g r o u n d beef pat t ies d u r i n g s to rage . Irradiation Fat L e v e l Infusion of Equa t ion FT (kGy) (%) asco rb i c ac id 0 4 No y=-0 .079X2 +0.570X+10.010 1.0000 4 Y e s y=-0 .7990X+15.259 0.9864 17 No y= 0 . 0 0 9 3 X3 - 0 . 1 7 8 X 2 + 0 . 1 8 6 7 X + 1 3 . 3 6 4 5 0.9960 17 Y e s y= 0 . 0 1 6 4 X3 - 0 . 3 2 6 3 X 2 + 1 . 1 5 6 1 X + 1 0 . 3 1 2 0.9697 30 No y= =0 .0149X3 ~0 .3041X 2 +1 .070X+11 .970 0 .8643 30 Y e s y= 0 . 0 1 2 5 X3 - 0 . 2 4 0 2 X 2 + 0 . 6 5 0 7 X + 1 1 . 3 0 3 0.9068 5 4 No y=-0 .0625X2 +0.4875X+8.500 1.0000 4 Y e s y= -0 .0508X2 +0.4992X+6.700 1.0000 17 No y=-0 .4920X+13.1030 0.9651 17 Y e s y= - 0 . 0 0 5 6 X3 - 0 . 1 5 3 9 X 2 - 1 . 3 7 4 5 X + 1 0 . 1 4 5 0.9868 30 No y= -0 .0021X3 +0 .0213X 2 +0 .4720X+11 .592 0.9624 30 Y e s y= - 0 . 0 1 3 0 X3 + 0 . 3 0 0 1 X 2 - 2 . 0 3 0 0 X + 1 1 . 2 6 9 0.9021 10 4 No y=-0 .1225X2 +1.6275X+2.820 1.0000 4 Y e s y= -0 .1125X2 +1.4375X+3.600 1.0000 17 No y= - 0 . 0 1 6 7 X3 + 0 . 4 0 1 6 X 2 - 2 . 7 8 7 9 X + 1 3 . 5 4 2 0.9410 17 Y e s y= - 0 . 0 0 3 2 X3 + 0 . 0 8 4 1 X 2 - 0 . 8 9 3 5 X + 1 0 . 1 5 0 0.9897 30 No y : =0 .0035X3 - 0 .0391X 2 - 0 .4815X+11 .832 0 .9542 30 Y e s y= - 0 . 0 0 5 6 X3 + 0 . 1 4 7 9 X 2 - 1 . 3 8 0 0 X + 1 1 . 0 9 0 0.9997 20 4 No y=-0 .1083X2 +1.2917X+4.500 1.0000 4 Y e s y= -0 .1075X2 +1.3425X+4.140 1.0000 17 No y=0 .0268X2 - 0 .7486X+13 .382 0.9550 17 Y e s y=0 .0148X2 +0 .5441X+10 .403 0.9834 30 No y= 0 .0166X2 - 0 . 1156X+11 .684 0.9589 30 Y e s y= -0 .0303X2 -0 .7396X+11 .019 0.9958 130 o If o , *H '•S o o IW o o o 03 o o © 2 * o o o CN r - l OS o o o n © © O O O ~ o © © © co C N o i—i in oo © O V O C N O £ s s § o o r~! o o o 9 o m ON" © o ^ PH CO fi CJ © © © © © © Tt-C N - 9 P . <i <; © © © © OS © © f—1 © ^H o © CS © T f © CN O CN © © © CN © © p , © p' ( ( ! © SO © CO © •n © © T f © T f © OS © © oo © oo © >—i © r—< o p , ©' © ' , © © © in © © © * T f oo © © CN © OS o r~ © in o © OS © r- © oo © CN © r—<' ©' p , ©' p^ ©' p , © ©^ CN © © ,_ ©^ r- SO o 1 OS © SO © T f © ro © Os © in © m © T f © Tt- © T f © © ©^ © © ©' © © © © © so © so •n OS in OS © i-H © ro © m © OS T f © •n © Tt- © -H o © © CO © m © ©' ©^ ©' ©^ © ©^ © ©^ ©' © Tt- cS1 Tt- ET © © * r- ro l > © CN © T f o ro o r- © CN © CN © © CN © o © ©^ © p , © o © o o cri , , I-H © © i-H © © © SO ro T f © r—l © T f © SO © ro T f r-H © <-H © © o © •n © ©^ ©' P , ©' © © ©' ©' *——1 , ( © CN © SO © ro © CN r-in o I-H © ro © OS © I-H >n © CN © CN © © ' 1 © © ©' © © © ©' ©' © ©' ©' ^— j 1 — ' ' — ' ' — 1 OS ©^ SO ©^ in T f ©^ 00 Tt- © OS © in © in © •n CO ro © CN © CN © ro © © CN O © ©^ © ©^ © ©^ ©' ©^ o In < U o CJ W Xi ft o f o .tS o 

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