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The effect of microwave and convection reheating on the vitamin B₁₂ content of roast beef produced in… Gellman, Catherine Elizabeth 1986

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THE EFFECT OF MICROWAVE AND CONVECTION REHEATING ON THE VITAMIN B 1 2 CONTENT OF ROAST BEEF PRODUCED IN A COOK/FREEZE FOODSERVICE SYSTEM by CATHERINE E1IZABETH GELLMAN B. Comm., University of Guelph, 1982 A/THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Food Science) We accept this thesis as conforming to thp required standard THE UNIVERSITY OF BRITISH COLUMBIA April, 1986 © Catherine Elizabeth Gellman, 1986 I n p r e s e n t i n g 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 o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e 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 r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 ABSTRACT The purpose of this study was to determine the effect of microwave and convection reheating on the vitamin B^2 content of roast beef produced in a cook/freeze foodservice catering system. Two concentrations of vitamin B-^ in a phosphate buffer, as well as vitamin B-^2 found in roast beef, were used throughout this study to detemine the effects of domestic and commercial microwave reheating and convection reheating on vitamin B-^. All the vitamin analysis was done using a radioisotope dilution (RID) assay (Bio-Rad Laboratories). In order to study the effects of microwave radiation without heat on vitamin B-^, two solutions containing 200 pg/mL and 1500 pg/mL (nominal) were exposed to microwave radiation for time periods of up to one hour in a 625 watt and a 1300 watt microwave oven. The temperature was maintained at 0*C by resting the vials in an ice bath. Retentions ranged from 86.57 + 3.79 % to 110.29 + 4.94 %. A fair amount of variability was present in the data, and as a result, the differences in retention were found to be non-significant. The differences between retentions in the two oven types as well as between the two concentrations of the vitamin were also found to be non-significant. •=»iia-Buffer samples were exposed to temperatures of 50 "C - 70"C in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180* C, to detemine the effect of heat on vitamin B^2. The destruction of the vitamin was not significant. Retentions ranged from 82.45 +_ 3.57 % to 103.3 + 4.6 %. Non-significant differences were found between the oven types and the two concentrations of the vitamin. Precooked, frozen and thawed roast beef samples were reheated to 70"C in the 2 microwaves and for 30 minutes in the convection oven set at 180"C in order to simulate a hospital cook/freeze catering system. Analysis of the samples indicated that all of the original B-^ was present after the samples were reheated. When the meat and drip portions of the samples were analyzed separately, 2.9 - 9.2 % of the vitamin was found in the drip, however, this amount was not significant. The amount of vitamin B-^ 2 present in the drip was found to be related to the amount of drip released from the sample. Samples reheated in the 625 watt microwave released significantly less (p< 0.05) drip than samples reheated in either the 1300 watt microwave or the convection oven. The results of these studies suggest that reheating does not appear to have a significant effect on vitamin B 1 2 retention. - • i i i -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i i i LIST OF TABLES v LIST OF FIGURES vii LIST OF APPENDICES v i i i ACKNOWLEDGEMENTS ix I. INTRODUCTION - 1 II. LITERATURE REVIEW A 1. Cook/Freeze Catering Systems A 2. The Effect of Convection Ovens on the Nutrient Composition of Foods 7 3. The Effect of Microwave Ovens on the Nutrient Composition of Foods 7 3.1 Moisture 7 3.2 Fat Soluble Vitamins 10 3.3 Water Soluble Vitamins 10 3.3.1 Thiamin 10 3.3.2 Riboflavin and Niacin 15 3.3.3 Pyridoxine and Folacin 17 3.3.A Ascorbic Acid 18 A. Effects of Heating Methods on the Vitamin Content of Frozen Foods. 20 5. Nomenclature and Function of Vitamin Bj_2 2A 6. Destruction of Vitamin B]_2 2 9 7. The Vitamin B 1 2 Content of Beef 30 III. MATERIALS AND METHODS 32 1. Extraction of Vitamin B].2 32 1.1 Extracting Solution 32 1.2 Phosphate Buffer 32 1.3 Glassware 32 l.A Procedure 33 2. Analysis of Vitamin B]_2 33 2.1 Standard Curve Preparation 35 2.2 Calculations 36 3. Determination of the Effect of Microwave Radiation on Vitamin Bi 2 at t=0-C A3 3.1 Sample Preparation A3 3.2 Sample Treatment AA 3.3 Expression of Data AA - i v-4. Determination of the Effect of Microwave and Convection Heating on Vitamin B12 in a Buffer System... 44 4.1 Sample Preparation 44 4.2 Sample Treatment 45 4.3 Expression of Data 45 5. Determination of the Effect of Reheating on Vitamin B12 i n R oa s t Beef 45 5.1 Sample Preparation 45 5.2 Sample Treatment 46 5.3 Crude Nitrogen Analysis 47 5.4 Expression of Data 48 6. Statistical Analysis 48 IV. RESULTS AND DISCUSSION 50 1. Preliminary Experiments 50 2. Determination of the Effect of Microwave Radiation on Vitamin Bj_2 in a Buffer Solution at t=0-C 59 3. Determination of the Effect of Microwave and Convection Heating on Vitamin B12 in a Buffer System... 70 4. Determination of the Effect of Reheating on Vitamin Bi_2 in Roast Beef 79 5. General Discussion 94 V. CONCLUSIONS 98 REFERENCES CITED 100 APPENDIX A 106 B 113 -V-LIST OF TABLES Page Table I. Moisture losses in microwave and conventionally cooked foods 9 Table II. Thiamin losses in microwave and conventionally cooked foods 12 Table III. Riboflavin and niacin losses in microwave and conventionally cooked foods 16 Table IV. Ascorbic acid losses i n microwave and conventionally cooked vegetables 19 Table V. • The effect of various reheating methods on the nutrient content of precooked frozen foods 21 Table VI. Typical data derived from a vitamin B12 RID assay of meat samples 37 Table VII. Recovery study of cyanocobalamin in an aqueous solution 52 Table VIII. Recovery study of different concentrations of cyanocobalamin added to meat extracts 53 Table IX. Repeatability of the RID assay using subsamples of A meat samples (Bj_2 expressed as ug/100 g sample). 57 Table X. Interassay variability 58 Table XI. Retention of Vitamin Bj.2 i n buffer solution A after exposure to varying periods of radiation in a 625 watt domestic microwave oven 60 Table XII. Retention of Vitamin Bj.2 i n buffer solution B after exposure to varying periods of radiation in a 625 watt domestic microwave oven 61 Table XIII. Retention of Vitamin B 1 2 i n buffer solution A after exposure to varying periods of radiation in a 1300 watt commercial microwave oven 62 Table XIV. Retention of Vitamin Bj_2 i n buffer solution B after exposure to varying periods of radiation in a 1300 watt commercial microwave oven 63 -vi -Table XV. Vitamin B]_2 retention in buffer solution A after exposure to heat in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180* C . . . 72 Table XVI. Vitamin B]_2 retention in buffer solution B after exposure to heat in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180* C . . . 73 Table XVII. Time in seconds for 250 mL buffer samples to come to temperature in three oven types 78 Table XVIII. Vitamin B12 retention (ug/lOOg moist basis) of meat plus drip samples reheated to 70 -C in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180-C 82 Table XIX. Nitrogen content (%) of meat and drip from meat samples heated in three oven types 85 Table XX. Retention of Vitamin B12 i n meat plus drip samples heated to 70-C in a 625 watt microwave, a 1300 watt microwave, and a convection oven set at 180«C. ug/100 g N 86 Table XXI. Contribution of drip from heated meat samples to the total B_2 content and total sample weight 91 Table XXII. Number of treatment replications needed to detect a significant difference of 28 % (p<0.05) 97 0 -vi i -LIST OF FIGURES Page Figure 1. Conventional and cook/freeze catering operations 5 Figure 2. The structure of a cobalamin molecule 25 Figure 3. Meat and buffer standard curves expressed as CPM vs. B12 pg/mL 38 Figure 4. Meat and buffer standard curves expressed as logit % B/Bo vs. log B 1 2 39 Figure 5. Retention of vitamin B 1 2 in buffer solution A after exposure to 625 and 1300 watts of microwave radiation for 15 minute time periods 64 Figure 6. Retention of vitamin B12 i- n buffer solution B after exposure to 625 and 1300 watts of microwave radiation for 15 minute time periods 65 Figure 7. Retention of vitamin B12 i- n buffer solution A after heating to 50-70«C in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180*C... 74 Figure 8. Retention of vitamin B12 in buffer solution B after heating to 50-70-C in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180- C . . . 75 Figure 9. Vitamin B12 retention (ug/100 g sample) of samples from Animal 1 reheated to 70*C in a 625 watt microwave oven, a 1300 watt microwave oven, and a convection oven set at 180* C for 30 minutes 83 Figure 10. Vitamin B 1 2 retention (ug/100 g sample) of samples from Animal 2 reheated to 70«C in a 625 watt microwave oven, a 1300 watt microwave oven, and a convection oven set at 180 *C for 30 minutes 84 Figure 11. Vitamin B12 retention (ug/100 g N) of samples from Animal 1 reheated to 70*C in a 625 watt microwave oven, a 1300 watt microwave oven, and a convection oven set at 180 «C for 30 minutes 87 Figure 12. Vitamin B^2 retention (ug/100 g N) of samples from Animal 2 reheated to 70-C in a 625 watt microwave oven, a 1300 watt microwave oven, and a convection oven set at 180-C for 30 minutes 88 -vi i i -LIST OF APPENDICES Page Appendix A. S t a t i s t i c a l Analysis 106 1. Time experiments 106 2. Temperature - Solution A 107 - Solution B 108 3. Meat (ug/100 g sample) - Animal 1 109 - Animal 2 110 A. Meat (ug/100 g N) - Animal 1 I l l - Animal 2 112 Appendix B. Example of calculation to convert vitamin concentrations from ug/100 g sample to ug/100 g N 113 -ix-ACKNOWLEDGEMENTS I would like to express my appreciation to my Supervisor, Dr. J. Vanderstoep, for his support throughout this study. I also wish to thank the members of my committee, Dr. S. Innis, Dr. D. Kitts, and Dr. J. Richards, for their help and suggestions. I wish to thank Sherman Yee and Lynn Robinson for their help in technical matters and the rest of the members of the Food Science Department for their help at various times. Finally, I wish to thank my family for their moral support and criticisms. This study was made possible through a grant to Dr. J. Vanderstoep from the Natural Sciences and Engineering Research Council of Canada. -1-I. INTRODUCTION Recent trends in foodservice catering have shown a move away from the 'conventional' catering methods and towards cook/freeze and cook/chill operations. The production of frozen foods was first introduced into hospital kitchens in 1967 and into school kitchens in 1970. Since this time, a number of cook/freeze operations have been developed for use in the industrial catering sector for feeding factory workers, as well as for use in hotels, restaurants, and airline catering. Economic, organoleptic and nutritional advantages have been cited as reasons for the change in catering methods (Glew, 1970). It is generally agreed upon in the literature that the cook/freeze catering system offers an overall nutritional advantage over traditional catering methods. However, since the method involves cooking, freezing, and subsequent reheating of foods, it is necessary to know how the essential nutrients are affected by each process. A large amount of experimental data has been published on the effects of various conventional cooking and freezing methods on the nutrient content of foods. Microwave and convection ovens are the two most commonly used reheating methods. Very little data exist on their effect on vitamins when used for reheating foods. Conflicting data exist on the effect of microwaves on nutrients when used for cooking foods (Cross and Fung, 1982). -2-The vitamins studied in relation to microwave and convection reheating have been limited to the least stable ones; thiamine, riboflavin, and ascorbic acid. To the knowledge of this author, there is no literature available on the effects of reheating methods on the other water soluble vitamins or the fat soluble vitamins. This study looked at the effect of microwave and convection reheating on vitamin B 1 2 . Vitamin B^2 * s a n essential component of the diet, necessary for the normal production of red blood cells (erythrocytes). Although the incidence of Vitamin B-^ deficiency is low largely due to the low requirements for the vitamin and the fact that the liver can store as much as 2 mg, an increased frequency of the deficiency disease, 'pernicious anemia', has been noted among strict vegetarians and the elderly. Beef was chosen as the food source for this study as it is one of the highest contributors of vitamin B 1 2 to the diet. Based on an average daily consumption of 46 g of beef per person in the United States and an average B^ 2 content in beef of 2 ug/100 g, the daily consumption of vitamin B^2 from beef is 0.9 ug, or 30 % of the recommended dietary allowance (Bennink and Ono, 1982). This study was carried out to determine the effect of reheating on Vitamin B 1 2 in beef. Specifically, the objectives of this research were: 1) to determine whether microwave radiation (t=0*C) has any effect on vitamin B 1 7 in a buffer system. 2) to determine whether microwave heating has any effect on vitamin B 1 9 in a buffer system. -3-3) to determine the effect of microwave and convection reheating on the vitamin B^2 content of precooked, frozen and thawed roast beef. 4) to consider the effect of different power levels in a domestic microwave oven and an institutional microwave oven on the vitamin B-^ -, content of precooked, frozen and thawed roast beef. -4-II. LITERATURE REVIEW 1. COOK/FREEZE CATERING SYSTEMS The cook/freeze food service system was developed in the late 1960's in response to an investigation by Piatt et al . (1963). This study, of the foodservices in 152 hospitals in England and Wales, demonstrated that the best quality food was produced in small hospitals of less than 60 beds. In larger hospitals, 55-60 % of the food served was returned uneaten on trays due to its poor palatability. At the time this investigation took place, long delays occurred between the cooking and serving of food, resulting in large nutrient losses. One third of the hospitals studied cooked green leafy vegetables for longer than 55 minutes. A 75 % loss of ascorbic acid occurred. One half of the hospitals cooked potatoes for one and one half hours, resulting in a complete loss of Vitamin C. Two of the hospitals cooked vegetables for as long as 115 minutes. Glew (1970), demonstrated that cooking cabbage for 80 minutes resulted in a 77 % loss of ascorbic acid, while cabbage cooked for 140 minutes lost 90 % of its original ascorbic acid content. Not only does the conventional catering service result in large nutrient losses, but it is also very inefficient with respect to utilization of labor and equipment. A typical hospital food service system must operate 12 to 14 hours a day, 7 days a week, in order to provide three meals a day for patients and staff. There are usually peaks of great activity around the meal period followed by lulls between meals. -5-With the above problems in mind, the Catering Research Unit at the University of Leeds set out to develop new methods of hospital catering. The most flexible system developed is known as the "cook/freeze" catering system (Glew, 1970). As illustrated in Figure 1, the steps of purchasing, preparation, cooking and portioning are the same in the cook/freeze and traditional catering systems. They differ at the stage of transport. Instead of being transported hot to the wards for service, the food is rapidly frozen, stored, transported in the frozen state and reheated at the point of service usually by microwave or convection heating. Purchasing Raw food storage 1 . Preparation 1 Cooking Portioning Transport HOT Food I Freezing I Cold Storage X Transport FROZEN Food 1 Reconstitution Service Conventional Catering Cook/Freeze Catering Figure 1: Conventional and Cook/Freeze Catering Operations (Glew, 1970). -6-Frozen storage allows for the building of an inventory of cooked products which can be kept for 6 months, removing the need for daily production. The meals needed for the week are produced in a normal AO hour work week with resultant efficient utilization of labour and equipment. A more important advantage of the cook/freeze system is removal of the hot transport step. Transportation of hot food has been shown by various researchers to have a detrimental effect on its nutritive value. The extent of this effect varies depending on the vitamin, the food, the method of hot transport, the temperature and the length of holding time. Westerman (19A8), and Erickson and Boyden (19A7), found no significant losses of thiamine in sliced roast pork and turkey held hot on a steam table for 30 minutes. Kahn and Livingston (1970), observed a 21.8 % loss of thiamine from four products held at 180'C for 1 hour on a steam table. These same products lost 26.1 % of original thiamine when held hot for 2 hours and 32.6 % when held for 3 hours. Lachance et al. (1973), noted a 27 % decrease in the thiamine content of chicken pies held at 180*C on a steam table for 100 minutes. Thomas (19A9), reported losses of the original ascorbic acid in the range of 7A % to 100 % from nine vegetables held hot for 1 hour on a steam table. Similarly, Harris (1960), reported losses of ascorbic acid ranging from 0 % to 9A % from nine vegetables held hot for 3 hours. Branion et al . (19A7), reported losses of ascorbic acid of 51 %-98 % from potatoes cooked by various methods and held hot for 2 hours. Wagner (1971), reported 72.8 % to 89.1 % losses of ascorbic acid from nine vegetables held hot for 3 hours in an insulated container. -7-In a well operated cook/freeze system, in order to minimize nutrient losses, the freezing process should achieve an ice front penetration rate of 0.5 inches per hour (Glew, 1970). Storage at -18'C allows food to remain wholesome and palatable for many months (Harris, 1977). When required for use, the food is removed from storage and transported frozen to the ward kitchens where it will eventually be reheated. 2. EFFECT OF CONVECTION OVEN REHEATING ON THE NUTRIENT COMPOSITION OF  FOODS Very little information is available on the effects of convection oven heating on nutrients in foods. Dahl-Sawyer et al. (1982), found no difference in the thiamine retention of beef loaves, peas and potatoes reheated in a convection oven compared with those reheated in a conventional oven. To the knowledge of this author, no other studies have been done on vitamins or foods reheated in convection ovens. 3. EFFECT OF MICROWAVE OVEN HEATING ON THE NUTRIENT COMPOSITION OF  FOODS There are a large number of conflicting studies available in the literature comparing the effect of microwaves on nutrients in foods with that of conventional cooking methods. 3.1 Moisture There is general agreement that microwave cooking increases the loss of moisture from animal products compared with conventional cooking (Cross and Fung, 1982). -8-Table 1 summarizes the published reports which compare the effects of microwaves on moisture in various foods with those of conventional cooking methods. Marshall (1960), found a significantly greater weight loss, drip loss and evaporation loss in top rounds of beef cooked in a microwave oven than those cooked in a conventional oven. Apgar et al . (1959), found significantly greater drip losses but lower evaporation losses from microwave cooked pork patties and roasts. Pork chops were found to have significantly lower weight and evaporation losses and slightly lower drip losses when microwave cooked. Kylen et al. (1964), showed greater weight losses in microwave-cooked beef roasts, pork roasts, beef loaves and pork loaves. These losses were significant for all products except for pork roasts. Drip losses were significantly greater in microwave-cooked beef and pork roasts but were the same for beef and pork loaves cooked by either method. Evaporation losses were significantly greater in beef roasts, beef loaves and ham loaves after microwave cooking. Ziprin and Carlin (1976), found that cooking in microwave ovens, in comparison with conventional ovens, increased the total cooking losses from beef loaves, beef-soy flour loaves and beef-soy concentrate loaves. Moisture loss and evaporation loss were significantly greater in microwave-cooked loaves but drip loss was less than in those which were conventionally cooked. Headley and Jacobson (1960), found significantly greater weight loss and evaporation loss from microwave cooked legs of lamb but slightly lower drip loss. Wing and Alexander (1972), noted significantly greater weight and moisture losses in microwave-cooked chicken breasts but, as with Headley and Jacobson (1960), and Ziprin and Carlin (1976), lower drip loss was observed. Table I . Moisture losses in microwave and conventionally cooked foods. Reference Sample Treatment Internal % Loss  Temperature Moisture Weight Drip Evaporation (-C) Marshall Top round microwave 80 60.6 13.8 28.4 (1960) roasts of conventional 80 - 34.7 6.5 19.7 beef Apgar et a l . pork patties microwave 87. 8 37.2 32.6 4.6 (1959) conventional 87. 8 - 36.3 22.1 14.3 pork roasts microwave 87. 8 - 26.5 12.4 14.3 conventional 87. 8 - 25.0 9.1 15.7 pork chops microwave 87. 8 - 17.7 14.7 2.9 conventional 87. 8 - 26.4 16.8 17.7 Kylen et al. beef roasts microwave 57 26.9 38.8 18.8 20.0 (1964) conventional 58 14.8 17.5 7.4 10.2 pork roasts microwave 86 25.5 37.3 17.1 20.2 conventional 82 21.4 34.1 12.3 21.9 beef loaf microwave 85 5.8 26.9 8.9 18.0 conventional 85 5.8 24.3 9.1 15.2 pork loaf microwave - 12.1 28.3 5.6 22.8 conventional - 5.7 18.2 5.2 13.Q Ziprin and beef loaves microwave 74 14.0 27.0 5.0 22.0 arlin conventional 74 5.0 19.0 12.0 7.0 (1976) beef-soy flour microwave 74 14.0 24.0 2.0 22.0 loaves conventional 74 5.0 14.0 8.0 7.0 beef-soy microwave 74 14.0 24.0 2.0 22.0 concentrate conventional 74 5.0 14.0 6.0 7.0 Headley and lamb roasts microwave 65 _ 43.0 15.0 27.0 Jacobson(1960) conventional 82 - 35.0 17.0 19.0 Wing and chicken microwave 88 11.5 26.6 2.69 -Alexander(1972) conventional 88 8.8 22.7 3.95 --10-The reason for the observed increase i n moisture loss and weight loss i n microwave cooked animal products has not been determined. Apgar et a l . (1959), and Kylen (1964), suggested that the increase i n moisture loss may be a result of the higher internal post-cooking temperature r i s e observed after microwave cooking, causing dehydration through evaporation. 3.2 Fat-Soluble Vitamins Very few data are available on the effect of microwave cooking on fat soluble vitamins. Aldor (1964), reported no s i g n i f i c a n t l y greater losses of vitamin A from microwave-cooked meat than from conventionally cooked meat. 3.3 Water-Soluble Vitamins Of the eleven water soluble vitamins discovered, only thiamine, r i b o f l a v i n , n i a c i n , pyridoxine, f o l i c acid and ascorbic acid have been studied v i s a v i s the effect on them of microwave cooking. 3.3.1 Thiamine Thiamine i s a water-soluble, heat-labile vitamin which i s destroyed by oxidation. I t i s leached from food i n proportion to the amount of available water, the extent of agitation during cooking and the surface area of the food exposed to water (Guthrie, 1979). Cross and Fung (1982) stated that the combined effects of heat, water and rapid b o i l i n g i n conventional cooking suggest that microwave cooking might be less destructive to thiamine than other methods. However, the l i t e r a t u r e contains no conlusive data to support t h i s hypothesis. -11-The effect of microwaves on buffered solutions was studied by Van Zante and Johnson (1970), and Goldblith et al . (1968). Van Zante and Johnson observed slightly higher thiamine retention in conventionally heated solutions than in those heated by microwave but the difference was not significant. This appeared to be the result of varying end temperatures rather than of the microwave energy itself. Goldblith et al. (1968), concluded that microwave energy has no destructive effect on thiamine. Any destruction noted resulted from the effects of accelerated temperatures in both systems. Table II summarizes the literature on the effects of microwaves on thiamine in food systems. Apgar et al. (1959), found that thiamine loss from pork patties and roasts was similar, regardless of the cooking method. They observed significantly lower thiamine loss from chops when cooked in a commercial microwave as compared to a domestic microwave or a conventional oven. The amount of thiamine recovered in the drip was similar for all cooking methods. Apgar et al . (1959) concluded that despite the lack of significant differences in thiamine loss between cooking methods, in a given time period, electronic cooking may be more destructive to thiamine than conventional cooking. The authors suggested that this may be the result of higher internal temperatures reached after exposure to microwave cooking. Baldwin (1976), found a significantly greater loss of thiamine from beef cooked in a domestic microwave compared with that cooked in a commercial microwave or conventional oven. No significant difference in thiamine loss was observed in the Table II. Thiamine losses in microwave and conventionally cooked foods. Reference Sample Treatment Internal Temperature Thiamine Loss Recovered (%) in Drip (%) Apgar et al.(1959) pork patties microwave-domestic 87.8 50.4 — microwave-commercial 87.8 50.2 -conventional 87.8 51.8 -pork roasts microwave-domestic 87.8 34.8 -microwave-commercial 87.8 39.6 -conventional 87.8 36.8 -pork chops microwave-domestic 87.8 44.9 27.6 microwave-commercial 87.8 37.2 . 26.1 conventional 87.8 49.8 27.8 Baldwin et al . beef roast microwave-domestic 70 51.0 (1976) microwave-commercial 70 39.0 conventional 70 31.0 — pork roast microwave-domestic 70 33.0 -microwave-commercial 70 27.0 -conventional 70 28.0 -lamb roast microwave-domestic 70 51.0 -microwave-commercial 70 48.0 -conventional 70 48.0 -Bowers and Fryer turkey breast microwave 68 20.64 (1972) conventional 80 20.73 -Hall and Lin chicken halves microwave-800W 92 22.4 (1981) microwave-1600W 92 22.4 — conventional-121•C 82 28.2 _ conventional-204-C 82 23.4 — Table II. continued. Reference Sample Treatment Internal Temperature Thiamin Loss Recovered (•O (X) in Drip (%) Kylen et al.(1964) beef roasts microwave 57 42.0 13.0 conventional 58 20.0 2.0 pork roasts microwave 86 40.0 31.0 conventional 82 39.0 19.0 beef loaves microwave 85 20.0 — conventional 85 24.0 -ham loaves microwave - 9.0 — conventional - 13.0 -Noble and Gomez roast lamb microwave 73 43.0 _ (1962) conventional 73 46.0 -Ziprin and Carlin beef loaf microwave 74 30.9 (1976) conventional 74 17.5 -beef-soy flour microwave 74 35.4 -loaf conventional 74 24.0 -beef-soy microwave 74 33.3 -concentrate loaf conventional 74 25.4 -Thomas(1949) beef roast microwave 74 37.0 9.0 conventional 74 25.0 6.0 beef patties microwave 79 11.0 8.0 conventional 79 45.0 -pork patties microwave 79 9.0 2.0 conventional 79 21.0 — -14-case of pork or lamb cooked by any of these methods. Bowers and Fryer (1972), found no significant difference in thiamine loss from turkey breasts cooked by microwave or conventional ovens. Hall and Lin (1981), found that thiamine retention was the same in chicken cooked in a domestic microwave, a commercial microwave or a conventional oven set at 204"C. A significantly greater loss was noted when the chicken was cooked in a conventional oven set at 121* C. Kylen et al. (1963), found significantly greater thiamine losses in microwave-cooked beef roasts and pork roasts than in conventionally cooked roasts but insignificant differences when beef and ham loaves were similarly treated. Significantly greater amounts of thiamine were recovered from the drip loss of microwave cooked beef and pork roasts. These authors attributed the greater thiamine recovery in the drip to the fact that significantly more drip was recovered from the microwaved meat and the drip was not exposed to as high a temperature for as long a period of time as the drip from the conventionally cooked meat. Noble and Gomez (1962), observed no significant difference in the thiamine lost from roasts of lamb when microwave or conventionally cooked. Ziprin and Carlin (1976), observed significantly greater losses of thiamine from microwaved beef loaves, beef-soy flour loaves and beef-soy concentrate loaves than from those conventionally cooked. Thomas (1949), found significantly greater losses of thiamine in microwaved roast beef but significantly lower losses in beef patties and pork patties. It was suggested that the prolonged cooking time required to reach appropriate internal temperatures in the roast was responsible for the thiamine loss in microwave-heated meat -15-(Cross and Fung, 1982). The results of the above studies indicate that thiamine destruction in food systems is related to heat levels rather than to exposure to microwave irradiation (Cross and Fung, 1982). 3.3.2 Riboflavin and Niacin Riboflavin and niacin are relatively heat stable vitamins. Riboflavin, however, is unstable in the presence of alkali and light. Both vitamins are slightly water soluble (Cross and Fung, 1982). Because of their stability, very few studies have been undertaken on the effects of microwaves on these vitamins. Van Zante and Johnson (1970), found no significant destruction of riboflavin in a citrate buffer solution heated in a microwave oven. Table III summarizes the literature on the effects of microwaves on riboflavin and niacin in food systems. Stevens and Fenton (1951), found a slightly greater loss of riboflavin in microwave-cooked peas than in peas boiled in water but this was not a significant difference. Bowers and Fryer (1972), and Noble and Gomez (1962), concluded that oven-type did not significantly affect riboflavin in turkey breasts or legs of lamb. The losses occuring from microwave cooking were only slightly greater than those occuring during conventional cooking. Baldwin et al . (1976), found significantly greater losses of riboflavin and niacin from beef cooked in a domestic microwave oven and of niacin from pork cooked in a domestic oven. Slightly greater losses of riboflavin and niacin from beef, pork and lamb were observed when the products were cooked in a commercial microwave oven as compared to a conventional oven. No significant difference in losses was noted when these meats were cooked in domestic microwave ovens compared with commercial microwave ovens. Thomas et al. (1949), found no appreciable difference in the retention of riboflavin and niacin when beef and pork Table III. Riboflavin and niacin losses in microwave and conventionally cooked foods. Reference Sample Treatment Internal Temperature Riboflavin Loss Niacin Loss (-C) (%) (%) Stevens and frozen peas microwave 68 2.0 -Fenton (1951) conventional 80 0.0 -Bowers and Fryer turkey breast microwave 68 13.5 _ (1972) conventional 80 8.2 -Noble and Gomez roast lamb microwave 79 25.0 (1962) conventional 79 16.0 -Baldwin et al. beef roast microwave-commercial 70 2.0 6.0 (1976) microwave-domestic 70 17.0 14.0 conventional 70 1.0 -4.0 pork roast microwave-commercial 70 19.0 13.0 microwave-domestic 70 18.0 21.0 conventional 70 4.0 -1.0 lamb roast microwave-commercial 70 12.0 29.0 microwave-domestic 70 27.0 36.0 conventional 70 2.0 14.0 Thomas (1949) beef patties microwave 79 0.0 10.0 conventional 79 0.0 10 0 pork patties microwave 79 0.0 15.0 conventional 79 0.0 15.0 beef roast microwave 74 15.0 27.0 conventional 74 15.0 19.0 -17-patties were cooked by microwave and conventional methods. Significantly more niacin was lost from beef roasts cooked by microwaves than when cooked by conventional methods. Due to the variability of the data from these studies, no conclusion can be drawn concerning the effect of microwaves on riboflavin and niacin (Cross and Fung, 1982). 3.3.3 Pyridoxine and Folacin Very few studies have been done on these two vitamins. Pyridoxine is relatively stable to heat and acid but it is labile to alkali, oxidizing agents and ultraviolet light (Guthrie, 1979). Bowers et al . (1974), cooked turkey breasts by microwave and conventional methods. The pyridoxine losses incurred were 16.7 ug/g of dried muscle and 16.5 ug/g of dried muscle respectively, a difference which is not significant. They also recorded no significant differences in pyridoxine lost from pork loin when cooked in a microwave oven or when conventionally cooked. Compared with turkey breasts, pork loins lost less pyridoxine when microwaved than when conventionally cooked (12.3 ug/g of dried muscle and 13.2 ug/g of dried muscle respectively). Wing and Alexander (1972), observed significantly greater retention of pyridoxine in microwave cooked chicken breasts than in conventionally cooked breasts, 92.5 % and 88.4 % respectively. They suggested that time rather than temperature was responsible for the decreased losses in microwave cooking. Cross and Fung (1982) concluded from these three studies that microwave applications do not result in increased loss of pyridoxine. -18-Folacin, a water soluble vitamin, has been reported to have cooking losses as high as 50-100 % using conventional cooking methods (Cross and Fung, 1982). Klein and Van Duyne (1979), reported no significant differences in the folacin retention of microwave and conventionally cooked spinach and frozen peas. Retention in both cases was reported to be 80 %. More studies are needed on this vitamin before any conclusion can be drawn. 3.3.A Ascorbic Acid Ascorbic acid is lost in large amounts during normal cooking practices since it is readily water soluble and labile to heat. It is destroyed when subjected to alkalies and to oxidizing agents (Guthrie, 1979). A large number of studies have been done on the effect of microwaves on ascorbic acid but no conlusion can be drawn due to the variability of the results. Table IV summarizes some of the literature on this subject. Stevens and Fenton (1950), found a slightly greater loss of ascorbic acid in peas cooked by microwaves than by boiling water, however, this difference was not significant. Kylen et al. (1960), subjected 7 vegetables to both cooking methods and found no significant difference in the ascorbic acid loss in each. Significantly lower amounts were recovered from the cooking water when cabbage, peas, and green beans were cooked in the microwave. Eheart and Gott (1964), found Table IV. Ascorbic acid losses i n microwave and conventionally cooked vegetables. Reference Sample Treatment Cooking time Ascorbic Acid loss (%) % Recovered Stevens and frozen peas microwave 3 min, 45 sec. 17.0 Fenton (1950) conventional 9 min. 14.0 -Kylen et a l . b r o c c o l i microwave 21.0 11.0 (1960) conventional 17.0 10.0 cabbage microwave 28.0 11.0 conventional 31.0 14.0 c a u l i f l o w e r microwave 13.0 6.0 conventional 8.0 7.0 peas microwave 26.0 15.0 conventional 27.0 23.0 green beans microwave 22.0 7.0 conventional 26.0 9.0 soy beans microwave 24.0 10.0 conventional 21.0 11.0 spinach microwave 44.0 9.0 conventional 39.0 5.0 Eheart and Gott b r o c c o l i microwave with water 23.6 _ (1964) microwave without water - 17.8 _ conventional 25.2 -peas microwave with water 37.3 -microwave without water - 35.0 _ conventional 31.6 _ potatoes microwave with water 23.5 -microwave without water - 25.6 _ conventional 20.1 -spinach microwave with water 35.3 — microwave without water - 32.8 _ conventional 50.7 -Campbell et a l . b r o c c o l i - f r e s h microwave 32.0 (1958) conventional 42.3 _ b r o c c o l i - f r o z e n microwave 21.4 _ conventional 26.5 _ green peas-frozen microwave 6.9 _ conventional 55.2 --20-no significant differences in the ascorbic acid content of peas, spinach, broccoli and potatoes, when cooked in the microwave with or without water. Microwave cooked peas, broccoli and potatoes showed no significant difference in retention of ascorbic acid when compared to amounts retained by the same vegetable cooked by conventional methods. Spinach retained a significantly greater amount of ascorbic acid when cooked by microwaves than when cooked by boiling in water. Campbell e_t al. (1958), showed that microwaved fresh broccoli and frozen peas lost significantly less ascorbic acid than conventionally cooked vegetables. Frozen broccoli lost less ascorbic acid but the difference was not significant. These studies indicate that no appreciably greater losses of ascorbic acid occur when vegetables are microwave cooked as compared to conventionally cooked. Klyen et al. (1960), concluded that the amount of cooking water, and to some extent, the cooking time, have a greater influence on ascorbic acid retention than has the cooking method. 4. EFFECTS OF HEATING METHODS ON THE VITAMIN CONTENT OF FROZEN FOODS Very little information is available regarding the effect of various heating methods on the vitamin content of precooked frozen foods. Table V summarizes the literature that has been published on the subject. Bowers and Fryer (1972), found a slightly greater loss of thiamine and a slightly lower loss of riboflavin from turkey breasts reheated in a microwave to 40"C as compared to those reheated in a gas oven to 55"C. Ang et al . (1975), demonstrated an inverse relationship Table V. The e f f e c t of various reheating methods on the n u t r i e n t content of precooked frozen foods. Vitamin Loss, (%) Reference Sample Treatment Thiamine R i b o f l a v i n Ascorbic ^-carotene Acid Bowers and Fryer (1972) Ang et a l . (1975) turkey breast microwave-AO'C 25.A 9.8 - -conventional-55-C 2A.6 10.2 - -mashed potatoes fresh-held 1/2 hour 3.3 2.A 33. 9 fresh-held 1 1/2 hours 9.2 2.0 A9. 0 — fresh-held 3 hours 18.A 2.7 60. A -frozen-reheated convection 1/2 hr 11.7 3.5 6A. ,3 -frozen-reheated microwave 1/2 hr 8.0 3.5 75. 9 -peas and onions fresh-held 1/2 hour 5.9 7.5 fresh-held 1 1/2 hours 8.1 9.3 - -fresh-held 3 hours 11.8 8.5 - -frozen-reheated convection 1/2 hr 10.3 6.0 - -frozen-reheated microwave 1/2 hr 10.0 6.8 - -c a r r o t s fresh-held 1/2 hour 6.1 _ — 7. 8 fresh-held 1 1/2 hours 11.5 - - 12. 0 fresh-held 3 hours 16.7 - - 8. 2 frozen-reheated convection 1/2 hr 8.A - - 7. 6 frozen-reheated microwave 1/2 hr 2.7 - - 5. 7 pot roast fresh-held 1/2 hour 6.9 11.A _ and gravy fresh-held 1 1/2 hours 8.3 9.0 - -fresh-held 3 hours 16.6 17.5 - -frozen-reheated convection 1/2 hr 13.1 8.5 - -frozen-reheated microwave 1/2 hr 12.A 11.3 - -beans and fresh-held 1/2 hour 6.6 1.8 _ f r a n k f u r t e r s fresh-held 1 1/2 hours 1A.0 7.0 - -fresh-held 3 hours 18.2 5.3 - -frozen-reheated convection 1/2 hr 7.A 3.1 - -frozen-reheated microwave 1/2 hr 9.8 0.9 — -Table V. continued Reference Sample Treatment Thiamine R i b o f l a v i n Vitamin Loss^ (%) Ascorbic ^-carotene Acid Ang et a l . f r i e d f i s h (19757 fresh-held 1/2 hours 0.0 fresh-held 1 1/2 hours 8.7 fresh-held 3 hours 22.8 frozen-reheated convection 1/2 hr 0.0 frozen-reheated microwave 1/2 hr 4.2 Dahl-Sawyer beef l o a f et al.(1982) conduction-18 min convection-25 min microwave-68 sec 31.9 32.9 36.3 peas conduction-18 min convection-25 min microwave-68 sec 15.0 14.2 14.5 potatoes conduction-18 min convection-25 min microwave-68 sec 77.9 82.4 84.8 Dahl and Matthews (1980) beef l o a f microwave-20 sec microwave-50 sec microwave-80 sec microwave-110 sec 27.8 25.0 28.2 29.9 Kahn and beef stew Livingston (1970) fresh held 1 hour @82-C fresh held 2 hour @82-C fresh held 3 hour @82-C microwave heated to 90-C 26.5 32.0 37.0 5.0 chicken fresh held 1 hour @82-C a l a king fresh held 2 hour H82-C fresh held 3 hour (182-C microwave heated to 90-C 25.0 30.0 37.0 6.0 Table V. continued. Vitamin Loss, (%) Reference Sample Treatment Thiamine R i b o f l a v i n Ascorbic \*> -carotene Acid Kahn and Livingston (1970) Ang et a l . (1978) shrimp newburg fresh held 1 hour ©82-C 24.0 fresh held 2 hour ©82-C 27.0 — fresh held 3 hour ©82-C 34.0 -microwave heated to 90 «C 7.5 -peas i n cream fresh held 1 hour ©82-C 13.0 _ sauce fresh held 2 hour ©82-C 17.0 -fresh held 3 hour ©82-C 24.0 -microwave heated to 90-C 7.0 -f r i e d chicken convection cooked 20-25 min 10.2 9.5 convection heated held 1/2 hr 11.2 12.0 convection heated held 1 1/2 hrs 11.7 13.0 convection heated held 3 hrs 26.2 9.8 microwave heated held 1/2.hr 7.9 7.2 char b r o i l e d convection heated held 1/2 hr 5.0 3.8 pa t t i e s microwave heated held 1/2 hr 9.6 0.9 -24-between hot-holding time and thiamine retention. The average loss of thiamine from the six products they studied was 17.4 % after 3 hours of hot-holding, compared to 7.8 % after microwave reheating. These results are comparable with those of Kahn and Livingston (1970), who found an average thiamine loss of 32.6 % when beef stew, chicken a la king, shrimp newburg and peas in cream sauce were held at 180 *F for 3 hours and only a 7.4 % loss when the four products were reheated in a microwave oven. These significantly lower losses were attributed to the shorter heating times in the microwave oven (Ang et a l . , 1975). Dahl-Sawyer et al . (1982), found no significant differences in the thiamine and ascorbic acid contents of beef loaf, peas and potatoes when reheated by convection or microwave ovens. Dahl and Matthews (1980), found slightly greater loss of thiamine from beef loaves with increased microwave heating times but the differences were not significant. No literature is available on the effects of various reheating methods on the other B vitamins or the fat soluble vitamins. 5. NOMENCLATURE AND FUNCTION OF VITAMIN B12 Vitamin B^2 activity was first recognized in 1926 by Minot and Murphy as a cure for pernicious anemia (Guthrie, 1979). In 1948, it was isolated in the crystalline form, simultaneously by L. Smith of England and E. Rickes and K. Folkers of the United States. It then took ten years to determine the complex structure of the small red crystals, using X-ray diffraction analysis (Lehninger, 1982). The substance was found to -25-contain 4 % of its weight as the metal cobalt, located in the centre of a complex molecule known as a corrinoid. This corrin ring is chemically related to the porphyrin ring systems of hemoglobin and chlorophyll. This structure led to Vitamin B 1 2 being called, "cobalamin" (Guthrie, 1979). Figure 2 shows the structure of the cobalamin molecule. HO Ok Figure 2. The structure of a cobalamin molecule. -26-A number of biologically active forms of the vitamin exist, these being; hydroxocobalamin, cyanocobalamin, nitritocobalamin, sulphitocobalamin, methylcobalamin and adenosylcobalamin. The active vitamin forms, hydroxocobalamin and methylcobalamin and an active coenzyme, 5'deoxyadenosylcobalamin, are found stored in human tissue. Adenosylcobalamin and hydroxocobalamin are the predominant forms found in foods, along with smaller amounts of cyanocobalamin and sulphitocobalamin (Farquharson and Adams, 1976). In foods, the vitamin is found bound to protein by a peptide linkage. In the body, cyanocobalamin and hydroxocobalamin can be converted into the active coenzyme, 5'deoxyadenosylcobalamin (Guthrie, 1979). The utilization of cobalamin and its role has documented by Guthrie (1979), and Lehninger (1982). One to three percent of ingested cobalamin is absorbed by diffusion. The major portion of that gaining entry to the body is taken up by an active transport system which carries it across the intestinal membrane. This system is dependent on a mucoprotein, known as intrinsic factor, which is secreted from the parietal cells lining the stomach. Proteolytic enzymes act in the digestive tract to release the cobalamin molecule from its protein complex. A new complex of intrinsic factor and cobalamin is formed. This complex then binds to receptors present in the cells lining the ileum or small intestine, in a reaction which is catalyzed by calcium. Intrinsic factor is then released from this complex by the action of the pancreatic enzymes and the cobalamin is absorbed through the intestine into the bloodstream. Once in the bloodstream, the vitamin is bound to -27-one of three transport proteins known as transcobalamins. This protein bound form is transported to the tissues for use or to the liver for storage. The body can store as much as 2 mg of vitamin B-^* enough to last at least 6 years. Failure at any of these stages can result in the vitamin being unavailable for use by the body. Vitamin B-^ is necessary for the formation of red blood cells and the normal growth and maintenance of nerve tissue. It functions in the form of the coenzyme 5'deoxyadenosylcobalamin. In the bone marrow, the coenzyme is required to provide a methyl group for the erythrocyte precursors (erythroblasts), to be used in the synthesis of DNA. Without DNA synthesis, the erythroblasts continue to produce RNA and protein. They grow in size and become large, immature cells known as megaloblasts. The red blood cells produced by these megaloblasts are the large immature macrocytes characteristic of pernicious anemia, the disease associated with a deficiency of vitamin B-^. In the nervous system, the vitamin plays a role in the metabolism of carbohydrate by keeping glutathione, an essential component of the enzymes involved in carbohydrate metabolism, in its biologically active form. In the event of a deficiency of vitamin B^2, a 50 to 100 % increase in the intermediary products, pyruvic acid and lactic acid, occurs suggesting a block in glucose metabolism. Vitamin B-^ has also been shown to play an indirect role in the oxidation of fatty acids with an odd number of carbon atoms. In this process, a three carbon propionyl CoA molecule remains after two carbon acetyl CoA molecules have been split off from the original fatty acid. -28-The propionyl CoA molecule is then oxidized via a series of reactions in which the enzyme methylmalonyl CoA mutase, containing the coenzyme 5'deoxyadenosylcobalamin, catalyzes the production of succinyl CoA, an intermediate in the TCA cycle. Vitamin B.^ activity has also been noted in the metabolism of the vitamin folic acid and the amino acids methionine, valine and isoleucine. The recommended daily intake of vitamin B^2 is 3 ug, of which only 1 to 1.5 ug are absorbed. An average western diet provides from 7 to 30 ug a day. The vitamin is found widely distributed in foods of animal origin. Meat and eggs have been shown to contribute 78.3 % of the daily intake, dairy products 20.1 % and flours and cereals, 1.5 %. The vitamin is naturally synthesized by microorganisms located in the rumen of polygastric animals following injestion of plant foods. The vitamin is absorbed from the rumen and excess amounts are stored in the muscle tissue bound to proteins by peptide linkages. Pernicious anemia is the disease associated with vitamin B-^ deficiency. The symptoms associated with the disease include shortness of breath, weakness, and mental deterioration. Large immature macrocytes are present in the blood and the vitamin B-^ level is less than 100 ug per mL. A deficiency state resulting from inadequate intake is rare. The main causes of B-^ 2 deficiency are, inadequate production of intrinsic factor to bind the vitamin in the intestine, partial or complete removal of the stomach, lack of the transport proteins (transcobalamins) in the blood, or an intestinal infestation with tape worm. -29-The two groups with no genetic defects who risk deficiency are vegetarians who consume no animal products, and the elderly. The elderly are at risk because they have an increased frequency of diseases such as atrophy of the gastric mucous membrane with resultant decrease in intrinsic factor production. 6. DESTRUCTION OF VITAMIN B12 Most methods of cooking do not destroy vitamin B^2 (Guthrie, 1979). The vitamin itself is heat stable at pH 4-7 with maximum stability in the pH range of 4.5 to 5 (Merck Index, 1976). Vitamin solutions adjusted to a pH of 4.5 with a .05M citric acid buffer have been shown to be stable and can be autoclaved without a detectable loss of the vitamin (Macek and Feller, 1952). Bartilucci and Ross (1954) showed that 50 % destruction of a vitamin solution at pH 10.3 occurred in one week when held at 40*C. Deterioration of the vitamin has been shown to occur in the presence of acacia, aldehydes, ferrous gluconate, ferrous sulphate, vanillin, hydrogen peroxide, cysteine hydrochloride, hydroquinone, thioglycollic acid, decomposition products of ascorbic acid, and the thiazole moiety of thiamine hydrochloric acid (Merck Index, 1976; Bartilucci and Ross, 1954; Macek and Feller, 1952). Under conditions of exposure to bright light, photolysis occurs, producing hydroxocobalamin (Farquharson and Adams, 1976). Aqueous solutions of vitamin B 1 2 a r e relatively stable during the light exposure which occurs in normal sample handling for analysis (Hartley et a l . , 1950). The main loss of vitamin -30-B^2 occurs due to its water solubility. As much as 30 % can be recovered in the cooking juices of meats (Bender, 1978). 7. THE VITAMIN B12 CONTENT OF BEEF The vitamin B-^ 2 content of beef has been reported by a number of researchers. Adams et al . (1973) reported a range of 0.41-0.79 ug of B-^ 2 per kilogram of cooked roast beef, using the micro-organism Euglena  gracilis for the assay. Hoppner et_ al. (1978) reported finding 1.8 ug of Bj 2 per 100 g of cooked steak and beef using the micro-organism, Lactobacillus leichmannii as the test organism. In a review article by Grasbeck and Salonen (1976), values of 1.24-2.7 ug of B 1 2 P e r 1 0 0 9 were reported for rib roasts and 1.3-2.0 ug per 100 g for steak, using microbial assay methods. Bennink and Ono (1982) did a comprehensive study of the B 1 2 content of various cuts and grades of beef in the cooked and uncooked states using a RID assay. Their results indicated that no significant difference exists in the B-^ content of the primal cuts or the carcass grades. When the vitamin Bj 2 content was expressed per 100 g, wet weight, no difference was noted in the raw or cooked state, regardless of the cooking method. However, when the B-^ content was expressed on the basis of nitrogen, a 27-33 % decrease was noted in the cooked meat. This agrees with the findings of Lawrie (1974), Bender (1975), Ensminger (1976), and Grasbeck and Salonen (1976) who reported -31-losses of 10-40 % in cooked meat. Bennink and Ono (1982) found an average of 3.17 ug of B-^ per 100 g of raw and cooked meat. This value is slightly higher than those reported by other researchers. The difference was attributed to the use of a RID assay instead of a microbial assay. The sensitivity of this assay was attributed to enhanced extraction and stability (Van Tonder et a l . , 1975; Beck, 1979). -32-III. MATERIALS AND METHODS 1. EXTRACTION OF VITAMIN B12 All samples were extracted according to the standard AOAC vitamin B 1 2 extraction method (43.108, 12th ed.). 1.1 Extracting solution - (pH 4.0) 13 g of anhydrous sodium phosphate dibasic (BOH Chemicals, Toronto, Ont.), 12 g of citric acid monohydrate (Mallinckrodt Inc., St. Louis, MO.) and 10 g of sodium metabisulfite (Mallinckrodt Inc., St. Louis MO.) were dissolved in distilled water and the volume brought up to 1 L. This was prepared fresh for each run. 1.2 Phosphate buffer - (pH 7.0) 9.1 g anhydrous potassium phosphate monobasic (Mallinckrodt Inc., St. Louis, MO.) and 18.9 g of anhydrous sodium phosphate dibasic (BDH Chemicals, Toronto, Ont.), were dissolved in distilled water and the volume was brought up to 1 L. 1.3 Glassware All glassware was cleaned for 1 hr in an ultrasonic cleaner (Mettler Electronics, Model* ME5.5) containing a 2 % solution of Extran 300 phosphate-free cleaning solution (BDH Chemicals, Toronto, Ont.). This was followed by rinsing for 15 min in a laboratory dishwasher (Heinick Instruments Co., Model* HWB2000-S) and drying in a laboratory oven set at 100'C (Blue M Electric Company, Model* 0V-490A-Z). -33-1.4 Procedure All meat samples were ground in a Cuisinart Food Processor for 30 sec to obtain a homogeneous sample. Of this homogenate, 3 g were weighed into a 100 mL low actinic flask. Up to 5 mL of the drip from the cooked meat samples, and 1 mL of the buffer samples were pipetted into 100 mL low actinic volumetric flasks. The samples were then diluted to 50 mL with the extracting solution. The flasks were agitated for 15 sec to disperse the sample and the sides were washed down with distilled water. The samples were then autoclaved for 10 min at 121-123*C , cooled rapidly to room temperature in a water bath and diluted to volume with the phosphate buffer. After the flasks were inverted 10 times to mix, the solutions were filtered through Whatman No. 2 filter paper. Aliquots of each extract were then frozen at -10*C until they could be analyzed. Each sample was extracted in duplicate. 2. ANALYSIS OF VITAMIN B12 The analysis was done using a Quantaphase B-^ Radioisotope dilution test kit (Bio Rad Laboratories, Mississauga, Ont.). All samples and reagents were allowed to come to room temperature before use. A 200 uL aliquot of each sample and standard were pipetted in duplicate into the appropriate 12 X 75 mm polypropylene reaction tubes (Western Scientific, San Franscisco, CA.). A 1 mL aliquot of a working solution, containing dithiothrietol, a borate buffer, potassium cyanide and radioactively labelled (57Co) vitamin B-^, was added to each tube. The tubes were vortexed to mix the solutions, -34-covered with aluminum foil , and heated in a boiling water bath at 100 *C (General Electric Silverstone Frying Pan) for 20 min. This allowed the various forms of vitamin B^2 to dissociate from the binding proteins naturally present in the meat and converted them to a single, stable form of the vitamin, cyanocobalamin. After heating, the tubes were cooled to room temperature by placing them in a cold water bath for 5 min and then letting them sit at room temperature for 5 min. A 100 ul aliquot of a Microbead reagent, containing affinity purified porcine intrinsic factor covalently coupled to polymer beads, was added to each tube. The tubes were vortexed to mix the contents and allowed to incubate for 1 hr at room temperature. Following incubation, the tubes were centrifuged at 1500xg for 10 min (Sorvall, General Lab Centrifuge 1, Newtown, CN.) to pack the Microbead reagent and the bound vitamin B-^ 2 into the bottom of the tube. The supernatant was immediately decanted by inverting the tubes into a beaker and the last drop was removed by blotting the tubes 57 on a paper towel. The Co decay in the precipitate was then counted for 1 min using a Packard Auto Gamma 500 counter. The vitamin B 1 2 content of the samples was then determined from a standard curve. A fresh standard curve was run with each assay. A blank tube was included in each assay to correct for non-specific binding or trapping of the labelled tracer within the solid phase matrix of the Microbead reagent. -35-2.1 Standard curve preparation 2.1.1 Meat samples Standards provided in the Quantaphase kit, containing concentrations equivalent to 0, 100, 250, 500, 1000, and 2000 pg of B 1 2 per mL of human serum albumin solution, were used as the standard curve for the analysis of the meat samples. 2.1.2 Buffer samples A standard curve, from samples containing no protein, was prepared for the analysis of the buffer system. It was found in preliminary experiments that the absence of protein in the samples interfered with the binding of the labelled vitamin B 1 2 to the intrinsic factor, and resulted in falsely high concentrations of vitamin Bj^ 2 being detected in the buffer samples. This phenomenon was also noted also by Raven et al . (1968), Newmark and Pater (1971), and Hillman (1969). They speculated that the intrinsic factor bound to side of the test tube in the absence of protein and bound Vitamin B 1 2 dissociated from the complex. To make the standard curve, a solution of 100 ug of vitamin B12/mL was prepared by dissolving 50 mg of cyanocobalamin (Sigma Chemical Company, St. Louis, M0.) in 25 % ethanol and diluting it to 500 mL in a low actinic flask. A 5 ug/mL solution was then prepared by pipetting 10 mL of the 100 ug/mL solution into a 200 mL low actinic -36-volumetric flask and diluting it to volume with 25 % ethanol. One mL of this 5 ug/mL solution was pipetted into a 250 mL low actinic Erlenmyer flask and diluted with 249 mL of a sodium phosphate-citric acid buffer at pH 5.6. Zero, 0.5 mL, 1 mL, 2.5 mL, 5 mL and 10 mL of this solution were then pipetted into 100 mL volumetric flasks and extracted as described in Section 1. 2.2 Calculations The data generated from this assay can be converted into vitamin B^2 values in a number of ways. For this experiment, the data were graphically displayed on log-logit paper as % B/Bo, or percent binding, vs the concentration of vitamin B-^ 2 in the standards. Table VI is an illustration of typical data derived from this assay. Figure 3 illustrates the shape of the standard curve when the data are plotted in the 'raw' form (CPM vs. B^2, pg/ml). Figure 4 illustrates the shape of the standard curve after the 'raw' data are converted to % B/Bo and are plotted on log-logit paper. To calculate the percent of bound labelled vitamin B-^ 2 associated with each of the standards and samples, the average corrected (sample CPM - blank CPM) counts per minute (CPM) of each is divided by the average corrected CPM of the zero standard and then the quotient is multiplied by 100. % B/Bo= Avg. Corrected CPM of Standard or Sample X 100 Avg. Corrected CPM of Zero Standard Table V I . T y p i c a l data dervied from a vi tamin B12 R I ° assay of meat samples. Tube CPM Average CPM Average Corrected %B/Bo CPM 1 L o g i t %B/Bo Log B_2 Vitamin Bj2 c o n c e n t r a t i o n (pq/ml) Blank 498 626 562 0-standard 12441 13061 12751 12189 100 100-standard 10678 10599 10638 10076 82.67 1.562 2 250-standard 7773 7918 7845 7283 59.75 0.395 2.3979 500-standard 5465 5362 5413 4851 39.80 -0.414 2.6989 1000-standard 3219 3535 3377 2815 23.09 -1.203 3 2000-standard 2180 2329 2254 1692 13.88 -1.825 3.3010 Sample l a 8365 9692 9028 8466 69.46 0.822 2.187 153.97 Sample l b 8687 8560 8623 8061 66.13 0.669 2.248 177.42 Sample 2a 5037 4775 4906 4344 35.63 -0.591 2.756 569.94 Sample 2b 4983 4989 4986 4424 36.29 -0.562 2.744 554.84 1. Average Corrected CPM = Average bound CPM-Average Blank CPM 14000 -39-Figure 4. Meat and buffer standard curves expressed as l o g i t % B/Bo vs. l o g B 1 2 . . -40-The calculated % B/Bo of each of the standards was plotted on the logit axis against the concentration of vitamin B^2 on the log axis of log-logit paper. A best fit straight line was drawn between the plotted points, and the level of vitamin B^2 in each of the samples was determined from the plot (Figure 4). To simplify the analysis, linear regression analysis was done on a Texas Instrument Programmable 59 computer utilizing the program, RIAPROG, (Faden et a l . , 1980) to determine the equation of the line for the standard curve. The results were then calculated as vitamin B-^ pg/mL using the equation : y = mx+b where: y = logit % B/Bo, (logit % B/Bo = In (B/Bo)/(100-B/Bo) ) m = slope of the line x = log B 1 2 b = y intercept -41-To express the results as vitamin B^2, ug/100 g of sample, the following equation was used: vitamin B, 7 , ug/100 g = C/(100xW) where: C = concentration, pg/mL W = sample weight, g 100 = a combined factor that takes into account the 100 mL extract, the level of Bj^ reported per 100 g of sample and conversion of pg to ug. The meat samples were also expressed as the concentration of vitamin B 1 2 in ug/100 g of nitrogen (N). This was done by dividing the vitamin B-^ content of 100 g of sample (wet weight) by the N content of that sample in grams and multiplying by 100 to give ug of B-^/lOO g of N. ug B12/100 q sample X 100 = B 1 2 ug/100 g N g N/100 g sample -42-To determine the vitamin B^2 content of 100 g of total sample (meat plus drip), the following procedure was used: 1) A x B = C 100 where : A = g of nitrogen in 100 g of drip B = g of drip in 100 g of total sample C = grams of nitrogen in the drip fraction of 100 g of total sample 2) X x Y = Z 100 where: X = g of nitrogen in 100 g of meat Y = g of cooked meat in 100 g of total sample Z = g of nitrogen in the meat fraction of 100 g of total sample -43-3) C + Z = g of nitrogen in 100 g of total sample 4) ug B12/100 q of sample X 100 = ug Bl2/100 g N g N/100 g of sample 3. DETERMINATION OF THE EFFECT OF MICROWAVE RADIATION ON VITAMIN B12  (t=Q-C) 3.1 Sample preparation Two concentrations of vitamin B^2 io a sodium phosphate-citric acid buffer at pH 5.6 were examined. Solution A (200 pg/mL - nominal) was chosen as it represents the average B^2 content of 100 g of beef. Solution B (1500 pg/mL - nominal) represents a high concentration of vitamin B^2 within the upper limits of the standard curve. The pH, 5.6, was chosen as it represents the usual pH of beef. To prepare Solution A, 10 mL of the 100 ug/mL solution, used to prepare the standard curve were diluted to 200 mL with 25 % ethanol. One mL of this solution was then diluted to 250 mL in the sodium phosphate-citric acid buffer. To prepare Solution B, 75 mL of the 100 ug/mL solution were diluted to 200 mL in 25 % ethanol and then 1 mL of this was diluted to 250 mLs in the buffer. All flasks used were low actinic. -44-3.2 Sample treatment One and a half mL of the solutions were pipetted into 2 mL amber 12X37 mm vials (Wheaton Custom Lab Apparatus, Millville, NJ). For each replication 10 vials were prepared. Eight vials were placed horizontally on 2 kg of broken ice in a beaker and exposed to microwave radiation in either a 625 watt oven (Hotpoint GE Dual Wave Oven, RK-93001, Canco Inc., Orangeville, Ont.) or a 1300 watt oven (Litton Moffat Menumaster, system 70/50) for a period of 15 minutes. Two vials were then removed and the melted ice was replaced with fresh ice. This was repeated after 30, 45 and 60 minutes. One mL from each vial was removed and extracted as described in Section 1. The B^2 content of the irradiated samples was compared to that of the unirradiated controls. Each condition was repeated in triplicate. 3.3 Expression of data After analysis of the vitamin as described in Section 2, the data were expressed as the concentration of vitamin B-^, pg/mL. A direct comparison was made between the heated samples and the controls and expressed as percent retention of the vitamin. 4. DETERMINATION OF THE EFFECT OF MICROWAVE AND CONVECTION HEATING  ON VITAMIN B12 IN A BUFFER SYSTEM 4.1 Sample preparation Solutions A and B were prepared as described in Section 3.1. -45-4.2 Sample treatment The samples were randomly heated individually to 50"C, 55'C, 60* C, 65* C, or 70 *C in either a 625 watt oven( Hotpoint GE Dual Wave Oven RK-93001, Canco Inc., Orangeville, Ontario), a 1300 watt (Litton Moffat Menumaster microwave system 70/50), or in a convection oven (Despatch Oven Company, Minneapolis, MN) set at 180*C . After heating, 1 mL of the sample was extracted according to the previously outlined procedure (Section 1). The B^2 content of the heated sample was compared to an unheated control. Each treatment was done in triplicate. 4.3 Expression of data After analysis of the vitamin as described in Section 2, the data were expressed as the concentration of vitamin B- 2^, P9/mL- °f sample. A direct comparison was made between the heated samples and the controls for percent retention of the vitamin. 5. DETERMINATION OF THE EFFECT OF REHEATING ON VITAMIN B12 IN  ROAST BEEF 5.1 Sample preparation Four top round roasts were purchased from Intercontinental Packers, Vancouver. Roasts A and B came from the right and left sides respectively of one animal and each weighed 11 pounds (Animal 1). Similarly roasts C and D came from one animal and weighed 9 pounds each (Animal 2). The beef was purchased the day after slaughter, tied into -46-roasts and frozen at -10"C. Before the roasts were cooked, they were allowed to thaw in the refrigerator for 18 hrs, and then at room temperature for 3 hrs. Cooking took place uncovered, in a Westinghouse Domestic Oven (KIH 3D) set at 150'C , until an internal temperature of 40"C was reached. The roasts were then allowed to sit until the internal temperature stopped rising. They were then immediately sliced into 3mm slices using a Hobart Meat Slicer (Hobart Corp., Troy, OH). The outside slices were discarded. The sample slices were trimmed to be of uniform size , shape and weight (90 g), and packaged in a polyethylene-aluminum-polypropylene retort pouches (6" X 9") (Reynolds Metals Company, Richmond, VA). A vacuum was drawn on the pouches using a Multivac Vacuum Sealer (#AG5, Germany, SeppHaggenmuller, KG). The samples were randomly numbered, frozen and stored at -35"C. 5.2 Sample treatment Twelve hrs before the samples were to be reheated, they were removed from frozen storage and allowed to thaw at regrigerator temperature. Three samples from each of the four roasts were reheated in each oven. Before heating, each sample was weighed and covered with aluminum foil or plastic wrap. After heating, the samples were immediately reweighed, the drip was measured, and up to 5 mL were pipetted into a 100 mL low actinic volumetric flask for extraction (Section 1). The meat was immediately ground in a food processor and extracted as described in Section 1. Samples of drip and ground meat -47-from each roast, after each treatment, were frozen and later analyzed for their nitrogen content (Section 5.3). 5.2.1 Convection Oven The oven was preheated to 180*C (nominal). Four samples at a time were heated for 30 min. Each sample was covered with aluminum foil . The samples were then treated as in Section 5.2. 5.2.2 Microwave Ovens The samples were covered with Saran wrap and individually heated until an internal temperature of 70"C was reached. The time to reach this temperature was recorded for each sample. The samples were then treated as in Section 5.2. 5.3 Crude Nitrogen Analysis Nitrogen analysis was carried out using a micro-Kjeldahl technique of Concon and Soltess (1973). The frozen samples were prepared for analysis by first freeze-drying in a Labconco Freeze Drier (#75018, Labconco Corp., Kansas City, MO) and then grinding with a mortar and pestle. Using a Mettler M3 analytical balance (Mettler Instrumente. AG. Zurich), 10.4 mg of the meat samples and 21.4 mg of the drip samples were weighed into 30 mL Kjeldahl flasks. A catalyst, 2.3 g of a K2S0A (BDH Chemicals, Toronto, Ontario) - HgO (Matheson Coleman and Bell Manufacturing -48-Chemists, Norwood, OH) mixture (190:4, w/w) was added, followed by 2.3 mL of concentrated H2S0^. The mixture was heated rapidly on an electric heater (Labconco 60011) until foaming occured and the foam was starting to escape up the neck of the flasks. The flasks were removed from the heat and 1 mL of hydrogen peroxide was pipetted down the side. They were then heated until all the organic material was oxidized as indicated by heavy fuming and clearing of the digest. The mixture was heated for a further 5 min and then removed from the heat. After cooling to room temperature, the digest was diluted to 25 mL with distilled deionized water. An aliquot of this was analyzed for % nitrogen using a Technicon Autoanalyzer II (Technicon Industrial Systems, Tarrytown, NY). All samples were analyzed in triplicate. 5.4 Expression of data The concentration of B^2 i p the meat samples was expressed both as ug B-^/lOO g of sample and as ug B-^/lOO g of N as described in Section 2.2. The heated samples were compared to the unheated controls for absolute and percent retention of the vitamin. The meat minus the drip was also compared to the total heated sample for absolute loss of B^2 to the drip and percent loss. 6. STATISTICAL ANALYSIS Basic statistical analysis, carried out on the raw data to determine means and standard errors of the means, was performed using a Hewlett Packard, HP-75C computer. -49-Linear regression analysis, to determine the best fit equation of the line for the standard curves was carried out on a Texas Instrument TP-59 calculator, utilizing the program RIAPROG (Faden et a l . , 1980). One way analysis of variance on the meat samples data (ug/100 g sample-moist basis), utilized the One Way Analysis of Variance program written for a Hewlett Packard HP-75C computer. Multiple analysis of variance was carried out on the buffer samples and the meat samples (ug/lOOg N), using the UBC MFAV program package (Le, 1978) available for use on the UBC Amdahl 470 V/8 computer. The Student-Newman-Keuls multiple range test (Zar, 1984) was used to perform multiple comparisons among means. -50-IV. RESULTS AND DISCUSSION 1. PRELIMINARY EXPERIMENTS - performance characteristics of the assay Preliminary experiments were done to determine whether the Quantaphase B-^ radioassay kit (Bio-Rad Laboratories), designed for the determination of vitamin B^2 in serum or plasma, could be used to quantitatively analyze vitamin B-^ in meat samples and buffer solutions. The principle behind the mechanism of the RID assay is saturation analysis, first described by Ekins in 1960. The technique involves the dissociation of the various forms of the vitamin from the binding proteins present in the sample and conversion of the vitamin to a single form, cyanocobalamin, in the presence of cyanide and radioactively labelled 5 7Co B^2. The mixture is then incubated with intrinsic factor, a glycoprotein, which serves as a non-discriminative binding agent for both labelled and unlabelled cyanocobalamin. During the incubation process, labelled and unlabelled cyanocobalamin compete for binding sites on the intrinsic factor on the basis of their concentrations. Non-labelled vitamin B^2 competitively inhibits the binding of labelled B-^ 2 to the binding sites. An inverse relationship is seen between the amount of radioactivity bound to the intrinsic factor and increasing concentrations of non-radioactive cobalamins (Newmark and Patel, 1971). -51-Initial experiments were carried out to determine how accurately the kit could recover various amounts of cyanocobalamin in aqueous solutions. The standard curve provided with the kit was used to analyze the data. Table VII shows the results of these experiments. The mean recovery was 169.9 +_ 7.7 %. A second study was carried out in which various concentrations of a cyanocobalamin solution were added to extracts from meat samples. The results from these samples, shown in Table VIII were derived by comparing the raw data to the standard curve provided with the kit. This study showed an average recovery of 103.2 _+ 1.4 %, when cyanocobalamin was added to meat samples, compared to an average recovery of 169.9 % from aqueous solutions. The 103.2 % recovery from the meat samples compared favorably with the results of other researchers. Using RID assays, Rothenberg (1968), reported a range of 84-118 % recovery from blood samples; Lau et al. (1965), 98.3-103.3 % recovery from blood; Van Tonder (1975), 95-106 % from tissue and Bennink and Ono (1982), 95-104 % from beef when a known quantity was added. Casey et al. (1982), using the Quanta Count II test kit from Bio-Rad Laboratories, reported recoveries ranging from 93.9-103.3 % from 8 different products. Bio-Rad Laboratories reports recovery levels of 103-109 % from blood using the Quantaphase B 1 2 kit-Since both study 1 and 2 were carried out under conditions to minimize microbial contamination, it appeared that the apparent excess of vitamin B ^ recovered from the aqueous samples was an artifact resulting from incomplete binding of labelled B, ? to the intrinsic -52-Table VII. Recovery study of cyanocobalamin in aqueous solutions. Sample B12 added tpg/mL) B j 9 recovered tpg/mL) % Recovery 1 0 1861 -2 125 2032 162.4 3 250 4673 186.8 4 500 8823 176.4 5 1000 1530A 153.0 6 2000 _ 5 -Mean 169.9 + 7.76 1. mean of 5 samples. 2. mean of 2 samples. 3. mean of 4 samples, 2 off end of standard curve. 4. mean of 2 samples, 4 off standard curve. 5. mean of 6 samples, all off standard curve. 6. mean + SE of mean. -53-Table VIII. Recovery study of different concentrations of cyanocobalamin added to meat extracts. Sample B^ 2 added (pg/mL) Expected Value (pg/mL) Recovered Value (pg/mL)-'-% Recovery Meat 1 0 - 721 -1 300 1021 1026 100.5 1 500 1221 1256 102.9 1 1000 1721 1807 104.9 Meat 2 0 - 824 -2 300 1124 1111 98.8 2 500 1324 1370 103.5 2 1000 1824 1976 108.3 Mean 103.2 + 1.42 1. mean of 4 assays. 2. mean + SE of mean. -54-factor. Since there is an inverse relationship between the amount of radioactive labelled cyanocobalamin bound to the binding protein and the concentration of unlabelled cyanocobalamin in the sample, incomplete binding would be interpreted as a falsely high vitamin content. This is particularly evident in the case of sample 1 in Table VII where 186 pg were recovered in 1 mL of sample, when in fact no B^2 had been added. The phenomenon of incomplete binding to the binding protein has been documented by a number of researchers. Rothenberg (1963), Raven et al. (1968), Rothenberg (1968), and Hillman et al. (1969), all observed an increase in the binding efficiency of the intrinsic factor in the presence of human serum albumin solution. Raven et al. (1968), found an average increase of 23.54 % in the binding of labelled B^2 to eleven intrinsic factor solutions after a 5 % solution of B-^ 2 deficient serum had been added. It has been suggested by a number of researchers, that the presence of serum or albumin prevents the adsorption of intrinsic factor to the sides of the test tube. Newmark and Patel (1971), found that at a low pH, adsorption of intrinsic factor to the side of the tube was favored. The addition of serum or albumin resulted in an increase in pH, and subsequently, adsorption was decreased. The addition of protein also resulted in a larger complex being formed from intrinsic factor, protein, and cyanocobalamin. A larger complex would result in easier separation of the bound fraction from the unbound fraction with centrifugation. However, the exact mechanism of the phenomenon of enhanced binding in the presence of protein is unknown. -55-For the purpose of this study, a serum free standard curve was prepared to analyze the buffer samples. This was prepared as described in Methods Section 2.1.2. The standard curve provided in the kit, containing human serum albumin, was used to analyze the meat samples. The reproducability of the assay was determined by analyzing subsamples of 4 samples of meat. The results from this study are shown in Table IX. The mean coefficient of variation for the 4 samples was 5.8 %. This value is slightly lower than that reported by other researchers. Casey et al. (1982), using a similar kit, reported reproducability results with a mean coefficient of variation of 6.5 % for 8 fortified samples and 8.3 % for 12 unfortified samples. Bennink and Ono (1982), reported an 8.2 % coefficient of variation for 8 subsamples of raw meat. Rothenberg (1968), reported a mean variation of 7.4 % between duplicate assays of 15 serum samples. Van Tonder (1975), found a mean variation of 9.5 % between duplicate assays of 7 different liver samples. Throughout the course of this study, samples were run as controls to test for interassay variability. Table X shows the results from this analysis. Nine assays were run to analyze the buffer samples. Two samples were assayed as controls. Sample 1 had a mean B-^ content of 466.8 +_ 8.5 pg/mL and a coefficient of variation of 5.4 %. Sample 2 had a mean content of 543.9 +_ 14.7 pg/mL and a coefficient of variation of 8.1 %. The mean interassay variability for the buffer assays was 6.7 %. Six assays were run for the meat samples. Sample 3 was used as a -56-control for these assays and had a mean B-^ content of 457.2 +_ 8.2 pg/mL and a coefficient of variation of 4.5 %. The mean interassay variability for the kit throughout the study was 6.0 %. Bio-Rad Laboratories reported interassay variabilities of 2.7-8.3 % for 4 samples run 10 times. The mean interassay variability was 4.6 %. The intra assay variability, determined from 60 randomLy chosen samples, extracted and assayed in duplicate, was 8.0 %. This value is considerably higher than the mean value of 3.8 % reported by the manufacturer, however, it is not unusual for RID assays. Dawson et al . (1980) reported that a coefficient of variation of 10 % is usually achievable with radioisotope assays. -57-Table IX. Repeatability of the assay using subsamples of A meat samples (B12 expressed as ug/100 g sample). Replication Sample 1* Sample 21 Sample 3^  Sample A^-1 1.989 2.028 0.388 0.357 2 1.9A2 1.867 0.386 0.378 3 2.052 1.779 0.3A8 0.3A5 A 2.108 1.937 0.A00 0.300 5 2.033 1.863 0.382 -6 1.912 2.022 0.383 — Mean 2.006 1.906 0.381 0.3A5 SE 0.029 0.0AA 0.007 0.016 CV (%) 3.58 5.61 A.A6 9.53 1. Mean of 2 assays ( each extract assayed in duplicate). - 5 8 -Table X. Interassay variability. Run Sample 1 (buffer pg/mL)-1-Sample 2 (buffer pg/mL)1 Sample 3 (meat pg/mL)1 1 456.4 535.3 428.5 2 451.9 486.9 451.7 3 428.5 545.4 471.5 4 468.0 555.5 466.5 5 512.3 496.6 483.2 6 479.1 511.2 444.2 7 462.1 522.8 -8 494.5 626.5 -9 448.6 584.9 -Mean 466.8 540.6 457.2 SE 8.4 14.7 8.2 C.V. (%) 5.4 8.0 4.5 1. n=2 samples (each sample extracted in duplicate and each extract assayed in duplicate). -59-2. DETERMINATION OF THE EFFECT OF MICROWAVE RADIATION ON VITAMIN B12 IN  A BUFFER SOLUTION (t=Q'C) - Experiment 1 Tables XI to XIV show the mean absolute and per cent retention of vitamin B-j^ in buffer solutions A and B after exposure to blocks of 15 min of microwave radiation in a 625 watt or a 1300 watt microwave oven. These data are also expressed graphically in Figures 5 and 6. The variability of the data collected is such that it is difficult to determine a trend. As shown in Figure 5, the means of the data collected from solution A appear to decline after exposure to radiation for increasing lengths of time in both the 625 watt oven and the 1300 watt oven. However, an analysis of variance (ANOVA) done on the data indicates that there is not enough difference to determine if this decline is real and not due to chance. This is particularly evident in the case of the 1300 watt oven, where the treated sample means plus the standard error are equal to or greater than the mean of the controls plus the standard error. This indicates that for each time period, at least one replicate had a vitamin concentration equal to or greater than the mean of the control samples. As Figure 6 shows, there is even less evidence of a trend among the samples from solution B. After exposure to 15 minutes of radiation in either microwave oven, a decline in the vitamin concentration was observed. However, further exposure to radiation in the 1300 watt oven resulted in an increase in vitamin concentration, to the point where samples exposed to 30 and 45 minutes of radiation appeared to contain a mean vitamin content greater than that of the controls. In both cases, at least one of the replicates was within -60-Table XI. Retention of Vitamin B^ 2 i n buffer solution A after exposure to varying periods of radiation in a 625 watt domestic microwave oven. Total time of B12 retention-1- Bio retention exposure (min) (pg/mL) (%) 0 (control) 150.7 + 3.3 a -15 143.9 + 3.9 a 95.5 + 0.5 30 137.2 + 1.0 a 91.1 + 1.9 45 141.9 + 2.7 a 94.3 + 3.5 60 140.9 + 3.4 a 93.7 + 4.4 1. Mean of 3 samples each extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B^ 2 percent retentions were calculated from time zero. a. Means in a column followed by the same letter are not significantly different as determined by the Student-Newman-Kuels multiple range test. - 6 1 -Table XII. Retention of Vitamin in buffer solution B after exposure to varying periods of microwave radiation in a 625 watt oven. Total time of B12 retention1 Bio retention2 exposure (min) (pg/mL) (%) 0 (control) 1454 + 74a -15 1263 + 112a 86.6 + 3.8 30 1302 + 93a 89.5 + 3.5 45 1452 + 55a 100.5 + 6.6 60 1391 + 50a 96.1 + 5.5 1. Mean of 3 samples, each extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B^2 percent retentions were calculated from time zero. a. Means in a column followed by the same letter are not significantly different as determined by Student-Newman-Kuels multiple range test. -62-Table XIII. Retention of Vitamin B i 2 in buffer solution A after exposure to varying periods of microwave radiation in a 1300 watt microwave oven. Total time o f B 1 2 r e t e n t i o n 1 B i o retention-exposure (min) (pg/mL) {.%) 0 (control) 188.9 + 23.3 a -15 189.8 + 25.9 a 100.1 + 1.3 30 184.0 + 28.6 a 96.9 + 4.6 45 184.6 + 28.2 a 97.0 + 2.9 60 180.2 + 34.5 a 94.1 + 6.2 1. Mean of three samples, each extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B^2 percent retentions calculated from time zero. a. Means in a column followed by the same letter are not significantly different as determined by Student-Newman-Kuels multiple range test. -63-Table XIV. Retention of Vitamin B12 i n buffer solution B after exposure to varying periods of radiation i n a 1300 watt microwave oven. Total time of B12 retention- 1 Bio retention exposure (min) (pg/mL) (%) 0 (control) 1270 + 62 a -15 1230 + 114 a 96.6 + 5.9 30 1407 + 127 a 110.3 + 4.9 45 1325 + 85 a 104.2 + 2.1 60 1227 + l l l a 96.3 + 5.1 1. Mean of three samples, each extracted i n duplicate and each extract assayed i n duplicate. 2. Vitamin B12 percent retentions calculated from time zero. a. Means i n a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i fferent as determined by the Student-Newman-Kuels multiple range t e s t . pg B12/mL 160 150 140 130 120 110 100 0 a) 625 watt microwave 210 200 190 180 170 pg B12/mL 1 6 0 150 140 130 -120 110 100 0 —1—j 1—1—1 i ~ r —=t=— 0* ralign of v a r i a t i o n i n assay 15 30 45 Time (minutes) 60 8% range oT variation in assay b) 1300 watt microwave 15 30 45 Time (minutes) 60 F i g u r e 5. Retention of vitamin B i n b u f f e r s o l u t i o n A a f t e r exposure to 625 and 1300 watts of microwave r a d i a t i o n f o r 15 minute time p e r i o d s . 1600 -• 1500 1400 •• pg B12/inL 1300 •• 1200 •• 1100 -• 1000 0 a) 625 watt microwave 15 30 45 Time (minutes) 60 1500 _. 8% range of v a r i a t i o n i n assay 1400 1300 --pg B12/mL 1200 -• 1100 1000 .. b) 1300 watt microwave 15 30 45 Time (minutes) 60 8% range of v a r i a t i o n i n assay F i g u r e 6. R e t e n t i o n of v i t a m i n i n b u f f e r s o l u t i o n B a f t e r exposure to 625 and 1300 watts of microwave r a d i a t i o n f o r 15 minute t ime p e r i o d s . -66-the range of the mean of the controls, plus or minus the standard error of the mean. An analysis of variance done on these data indicated that a significant difference was not detected between the vitamin concentrations of the samples after different lengths of exposure to microwave radiation. The ANOVA also indicated that the difference between the 625 watt domestic microwave oven and the 1300 watt commercial microwave oven, was not significant when both solutions A and B were included in the analysis. It must be recognized that the sensitivity and the confidence placed in the ANOVA is limited by the variability of the data and the inherent variability within the assay. To determine the sensitivity or the power of the ANOVA, the following formula was used (documented by Zar, 1984): (p = n<^ 2ks2 where t7j = phi £ = the minimum detectable difference between n sample means k = the number of treatments n = the number of sample replications within each treatment s = the variability within k treatments (J) is then converted to the power of the ANOVA using power and -67-sample size tables (Zar, 1984), where power equals 1 - >^ = the probability of committing a type II error). The power indicates the probability of rejecting a null hypothesis when it is false and should be rejected. If the null hypothesis is that the sample populations are the same, the power of the ANOVA indicates the probability that a real difference will be detected between sample means. The same formula can be used to determine the number of sample replications that need to be done with a given sample variability to be 95 % confident that a false null hypothesis will be rejected. (See Appendix A for ANOVA tables and the sensitivity of the analysis). In order to be 95 % confident of detecting a true difference between treatments in this experiment, 300 replications of each treatment would have to have been carried out with solution A and 30 with solution B. As can be seen in Figures 5 and 6, the means of the B solution samples and the standard error of the means are within the 8 % variability range of the assay, with the exception of the samples from solution B exposed to 15 and 30 minutes of radiation in a 625 watt oven. For both of these sets of samples, the mean concentration was below the 8 % range of the controls, however, the upper limit of the standard error of the mean was within this range. The low mean values can be attributed to 2 separate samples which appeared to lose 20.76 % and 17.46 % of the initial vitamin content after exposure to 15 minutes and 30 minutes of radiation, respectively. Subsequent analysis after 30 and 45 minutes of exposure, indicated increases of 18.95 % and 36.11 % in the concentration -68-respectively. This leads one to believe that the initial low concentrations may have resulted from sampling errors or an error in the analysis. All samples of solution A, treated in the 1300 watt oven, exhibited standard errors of the mean outside the 8 % range of the control mean. This can be attributed to one set of samples which exhibited concentrations 22.3-36.4 % higher than the mean of the samples over the 5 time periods. Since the samples were randomly assayed, this indicates that a sampling error must have occurred. The large amount of variability of these samples increases the number of replications which should be done in order to be 95 % confident of detecting a true difference between samples. The results from this experiment indicate that there is not enough evidence to show that microwave radiation exposure at intensities of 625 watts or 1300 watts, for time periods of up to one hour, destroys vitamin B 1 2 i n a buffer solution. This is similar to the results of Goldblith et al . (1968), who found that microwave irradiation of samples held at 0*C for extended periods of time had no influence on the destruction of thiamine. Rosen (1972) documented the effects of microwaves on foods and related materials. He concluded that microwave radiation does not induce chemical changes in materials. Chemical changes that occur when radiation is absorbed by organic materials are the result of breakage of chemical bonds and the formation of ions or free radicals which react and -69-form secondary products. In order to break bonds, the quantum energy of the radiation must be equal to or greater than the energy of the bond being broken. According to Rosen, microwaves have a quantum energy of 0.000012 eV calculated by the formula: E=1.24 x 10"4 x l/r\ where = wavelength of the radiation in cm and E is expressed in eV The quantum energy is not influenced by the intensity of the radiation. Cobalamin contains chemical bonds primarily of the C-C, C-N, C=N, N-H and C-0 types, with energies of 3.6 eV, 3.0 eV, 6.4 eV 4.0 eV and 3.6 eV respectively. The quantum energy of microwaves falls short by several orders of magnitude of the energy needed to break these bonds. The documentation by Rosen (1972) supports the results of the present study in that the destruction of cyanocobalamin by microwave radiation would not be expected under the experimental conditions used. -70-3. DETERMINATION OF THE EFFECT OF MICROWAVE AND CONVECTION HEATING ON  VITAMIN B12 IN A BUFFER SYSTEM - Experiment 2 Tables XV and XVI show the mean absolute and per cent retention of vitamin B^2 in buffer solutions A and B after heating to various temperatures in a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180*C. These data are also expressed in graphic form in Figures 7 and 8 As with Experiment 1, the variability of the data collected is such that it is difficult to identify a trend. Solution A, seen in Figure 7, appears to decline slightly in vitamin content when heated in the 625 watt oven, with the minimum retention being 95.42 +_ 7.04 % at 50*C (Table XV). However, the standard error of the mean is such that, in all cases, the upper limit of the sample values is greater than the mean concentration of the controls, indicating that some of the samples were within the range of the control samples. The means are all within the 8 % range of variability of the assay. An ANOVA performed on the data indicated that the difference between the unheated controls and the heated samples was not significant. Similar results were found for solution B heated in the 625 watt oven (Figure 8). In the 1300 watt oven, the vitamin content of the solution A appeared to decline after heating to each of the temperature endpoints. The means of the samples heated to 55*C , 60*C and 65'C , were outside the +8 % range of the assay with retentions of 88.54 +_ 5.14 %, 84.67 + 3.78 % and 86.62 + 9.87 %, respectively (Table XV). The upper limit of the standard errors of the means of samples heated to 55'C and 65"C were within the variability range of the assay (Figure 7). The -71-validity of the data for these three temperatures is somewhat questionable since samples heated to 70 *C had a retention to 98.82 +_ 3.61 %, an increase relative to those at 55, 60 and 65*C. Solution B, heated in the 1300 watt oven showed an opposite trend. Retentions of 101.6 +_ 1.6 %, 103.3 + 2.5 % and 103.3 + 4.6 %, were observed for samples heated to 55'C , 60*C and 65'C , respectively (Table XVI). The means of the concentrations of all the samples _+ the standard error of the mean, are within the variability range of the assay (Figure 8). An ANOVA performed on the values for both solutions A and B, heated in the 1300 watt oven, indicated that there is not enough evidence to identify a true difference between the vitamin concentration of the heated samples and the unheated controls. Similarly, the ANOVA done on the data derived from samples of solutions A and B heated in the convection oven, indicated that there is not enough evidence to identify a true difference between the heated samples and the unheated controls. As with the 1300 watt oven, the retention of Vitamin B-^ 2 i° solution B, heated to different temperatures in a convection oven, was very good, ranging from 96.80+3.84 % to 101.4 +4.3 % (Table XVI). All of the means and standard errors of the means were within the 8 % range of the assay (Figure 8). Solution A exhibited retentions ranging from 82.45 + 3.57 % to 99.05 + 11.32 % (Table XV). Samples heated to 65'C and 70"C had mean concentration +_ the standard error of the mean, lower than the range of variation of the assay (Figure 7). -72-Table XV. Vitamin B 1 2 retention in buffer solution A after exposure to heat in a 625 watt microwave, a 1300 watt microwave, and a convection oven set at 180*C . Oven Temperature B^ 2 retention-'- B12 retention^ (•C) (absolute, pg/mL) (%) Control 218.2 + 11.6 a -Microwave 50 209.3 + 15.2 a 95.4 + 7.0 (625) 55 214.1 + 10.3 a 98.1 + 4.7 60 220.2 + 6.8 a 100.9 + 3.1 65 214.6 + 16.0 a 98.3 + 7.3 70 214.7 + 16.3 a 98.4 + 7.5 Microwave 50 212.6 + 1.8 a 97.4 + 0.5 (1300) 55 193.2 + 11.2 a 88.5 + 5.1 60 184.8 + 8.2 a 84.7 + 3.8 65 189.0 + 21.5 a 86.6 + 9.9 70 215.7 + 7.9 a 98.8 + 3.6 Convection 50 207.8 + 4.0 a 95.2 + 1.8 55 200.6 + 9.3 a 91.9 + 4.3 60 216.2 + 24.7 a 99.0 + 11.3 65 189.9 + 7.9 a 87.0 + 3.6 70 179.0 + 6.8 a 82.4 + 3.6 1. Mean of three samples +_ SE of mean. Each sample extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B\2 percent retentions calculated from unheated controls. a. Means in a column followed by the same letter are not significantly different as determined by the Student-Newman-Kuels multiple range test. -73-Table XVI. Vitamin B 1 2 retention in buffer solution B after exposure to heat in a 625 watt microwave, a 1300 watt microwave, and a convection oven set at 180 C-. Oven Temperature B12 retention1 E$12 retention (•C) (absolute, pg/mL) (56) Control 1236.9 + 95.6 a -Microwave 50 1211.1 + 38.2 a 97.9 + 3.1 (625) 55 1259.1 + 13.8 a 101.8 + 1.1 60 1224.1 + 19.7 a 99.0 + 1.6 65 1220.7 + 32.8 a 98.7 + 2.7 70 1213.5 + 19.9 a 98.1 + 1.6 Microwave 50 1212.8 + 41.8 a 98.0 + 3.4 (1300) 55 1257.0 + 19.2 a 101.6 + 1.6 60 1277.3 + 30.9 a 103.3 + 2.5 65 1277.4 + 57.2 a 103.3 + 4.6 70 1252.6 + 11.2 a 101.2 + 0.9 Convection 50 1236.1 + 17.2 a 99.9 + 1.4 55 1198.2 + 47.5 a 96.8 + 3.8 60 1220.9 + 29.1 a 98.7 + 2.4 65 1254.5 + 52.9 a 101.4 + 4.3 70 1240.4 + 48.0 a 100.3 + 3.9 1. Mean of three samples _+ SE of mean. Each sample extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B^ 2 percent retentions calculated from unheated control. a. Means in a column followed by the same letter are not significantly different as determined by the Student-Newman-Kuels multiple range test. 240 230 4 -r 220 .. pg B12/ mL 210 -• 200 190 -• 180 -• 170 --160 •• 0 Control 50 55 60 65 70 a) 625 watt microwave Control 50 55 60 65 Temperature I*C) b) 1300 watt microwave 70 j8% range of v a r i a t i o n i n assay Control 50 55 60 65 70 c) Convection oven Figure 7. Retention of vitamin B i n buffer s o l u t i o n A a f t e r heating to 50-70"C i n a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180"C. 1360 1340 -1320 1300 1280 •• 1260 pg B 1 2/mL 1 2 4 0 I 1220 • 1200 .. 1180 •• 1160 1140 1120 •-1100 0 8% range of v a r i a t i o n i n assay Control 50 55 60 65 70 Control 50 55 60 65 70 Control 50 55 60 65 70 a) 625 watt microwave Temperature( C) b) 1300 watt microwave c) Convection oven Figure 8. Retention of vitamin B i n buffer s o l u t i o n B a f t e r heating to 50-70"C i n a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180'c. -76-The general conclusion that may be drawn from this experiment is that there is insufficient evidence to conclude that a difference exists between heated and unheated samples, or oven types. However, as with Experiment 1, the confidence placed on the ANOVA is limited by the variability of the data. To be 95 % confident of detecting a true difference between the heated and unheated samples, 82 replications of each treatment should have been done with solution A and 205 replications with solution B. To detect a difference between oven types, 21 replications should have been done with solution A and 65 with solution B. The conclusion drawn from this experiment agrees with what is known regarding the nature and stability of vitamin B-^. The Merck Index (1976), describes the vitamin as being heat stable at pH 4-7, with maximum stability in the pH range of 4.5-5. Macek and Feller (1952), demonstrated that 0.05 M citric acid buffer solutions of cyanocobalamin at pH 4.5 were stable and could be autoclaved without a detectable loss of the vitamin. Rosen (1972) documented the difference between conventional heating and microwave heating to be the initial distribution of heat. In ordinary heating, the heat is deposited on the surface of the object and transmitted inward through conduction or in the case of a liquid, through convection currents. With microwave heating, radiation is deposited within the food. As the microwaves penetrate the food, they cause the polar molecules to oscillate in the electric field. These molecules cause intermolecular friction which results in heat being produced. If the thickness of the material is greater than the range of penetration of -77-the microwaves, heating then occurs by conduction or convection. The ability of the microwaves to deposit energy within the food, rather than just on the surface, results in the reduced heating time experienced in microwave ovens (Curnette, 1980). Table XVII illustrates the mean time required for the samples to come to temperature in the three oven types. These data indicate that heating in a 625 watt oven takes roughly twice as long as heating in a 1300 watt oven. Convection heating takes roughly seven and a half times as long as heating in the 625 watt oven and fifteen times as long as in the 1300 watt oven. In the event that vitamin B^2 n a c l been destroyed by heat, and considering the reduced time required to reach certain temperatures in microwave ovens, one would expect to see the greatest retention of the vitamin in the 1300 watt microwave oven, followed by the 625 watt oven, and finally, the convection oven. However, as documented in Section 1, there is very little agreement among data available in the literature comparing microwave heating to conventional heating. This is due to the great variability in biological materials and failure to establish standardized cooking procedures for foods used in research studies (Lorenz, 1976). The one study done in buffer solutions by Van Zante and Johnson (1970), concluded that there was little or no practical difference in the retention of thiamine or riboflavin between heating in an electronic range and a conventional oven. -78-Table XVII. Time in seconds required for 250 mL buffer samples to come up to temperature in three types of ovens. Temperature625 watt 1 1 3 0 0 w a t t 1 C o n v e c t i o n oven1 microwave microwave (180-C, nominal) 50 89.6 + 2.2 44.2 + 0.8 600.0 + 23.1 55 101.2 + 1.3 50.2 + 1.2 769.6 + 10.0 60 119.3 + 1.0 54.3 + 0.4 945.0 + 57.8 65 126.6 + 1.5 63.2 + 0.8 1014.0 + 20.5 70 146.8 + 2.8 71.5 + 1.8 1144.8 + 29.4 1. Mean + SE of mean, n=6. -79-4. DETERMINATION OF THE EFFECT OF REHEATING ON VITAMIN B12 IN ROAST  BEEF - Experiment 3 Experiments 1 and 2 were designed to determine whether microwave radiation and heating had any effect on vitamin B ^ . Experiment 3 was designed to determine whether more vitamin B-^ was lost to the drip of microwave reheated meat than convection reheated meat. The experiment was designed so that 6 samples from each animal were heated in each oven. The data collected indicated that a significant difference (P<^ 0.05) existed between the mean vitamin content of meat from Animal 1 and Animal 2 (in the cooked state). These concentrations were 0.427 _+ 0.035 ug/100 g sample and 1.556 +_ 0.087 ug/100 g sample (wet weight) respectively. These values are within the range of 0.2 and 2.7 ug/100 g sample reported by other researchers (Bennink and Ono, 1982). The variability among the control samples from Animal 1 was extremely high (40.65 %), to the extent that 2 of the samples appeared to contain concentrations of less than 100 pg/mL which is beyond the lower limit of the standard curve. According to the manufacturers (Bio-Rad Laboratories), the minimum concentration detectable by the kit is 20 pg/mL. However, Rothenberg (1968) reported errors of 25 % when extrapolating a standard curve. For the purpose of this study, only those samples within the range of the standard curve were used. This placed some limitations on the conclusions that could be drawn from the data for Animal 1. -80-Table XVIII and Figures 9 and 10, show the data derived from reheated meat samples, expressed as the absolute retention of vitamin 13-^2 (ug/100 g sample, wet weight) and as percent retention . In all cases, except the convection heated samples from Animal 1, the vitamin concentration of the reheated samples appeared to increase over the unheated controls. This increase, however, was not significant. The observed increase may be attributed to the variability of the assay, to sample variability and partly to the concentration effect or evaporation losses that occur as a result of heating. The samples from Animal 1, reheated in the convection oven, appeared to retain only 82.A +_ 2.1 % of the origional B^2 content. The validity of this observation is questionable though, since data from only 2 samples out of the origional six could be used. To overcome the concentration effect, the data were expressed in terms of ug/100 g N. The data used to make this conversion, as determined by Kjeldahl analysis, are shown in Table XIX. The same values were used for both Animal 1 and Animal 2 since an ANOVA done on the raw data indicated that a significant difference could not be detected between the two animals and the mean variation of the combined samples was 3.28 %. The method used for the conversion of the data from the ug/100 g (wet weight) form to ug/100 g N is described in Appendix B. Table XX illustrates the converted data both as absolute retention and percent retention of the total sample (meat plus drip) in comparison to the unheated control. Also illustrated in Table XX is the -81-absolute retention and percent retention of the meat portion of the heated sample as compared to the total heated samples. This is also illustrated in graphical form in Figures 11 and 12. In the majority of cases, a slight increase in B^2 w a s observed after heating, ranging from 1.62 to 9.64 %. An ANOVA performed on the data, concluded that this increase is not significant. As in experiments 1 and 2, the confidence placed in the ANOVA is limited by the variability of the data. To be 95 % confident of detecting a true difference, 40 replicates for each treatment should have been done. The number of replicates physically possible is limited by the size of the animal and 40 would have been impossible under the experimental conditions carried out. A significant difference (p<Co.05) was detected between samples from Animal 1 heated in the convection oven and samples heated in microwave ovens. These samples appeared to lose 20.41 % of the origional vitamin content, but as previously mentioned, the available data were limited and therefore it is difficult to draw a firm conclusion from this. Except for samples from Animal 2 heated in the convection oven and the 1300 watt microwave oven, the means plus the standard error of the means of the treated samples were within the range of error around the mean of the controls. Convection heated samples from Animal 2, exhibited an overlap between the lower range of error of the mean and the range of error around the controls, indicating that some of the samples were within the range of variability of the controls (Figure 12). -32-Table XVIII. Vitamin B 1 2 retention (ug/100 g moist basis) of meat plus drip samples reheated to 70* C in a 625 watt microwave, 1300 watt microwave and a convection oven (set at 180- C). Animal Oven B12 retention ug/100 q (%)*» 1 Control 0.A27 + 0.035la -1 625 0.465 + 0.035la 108.95 + 8.37 1 Convection 0.352 + 0.0092b 82.40 + 2.10 1 1300 0.444 + 0.023la 103.92 + 5.34 2 Control 1.556 + 0.0873c -2 625 1.669 + 0.0433c 107.30 + 2.81 2 Convection 1.722 + 0.0863c 110.66 + 5.53 2 1300 1.779 + 0.0383c 114.38 + 2.46 1. Mean + SE of the mean, n=4, 2. Mean + SE of the mean, n=2, 3. Mean + SE of the mean, n=6, 4. Vitamin B^2 percent retentions calculated from unheated controls. a,b,c. Means in a column followed by the same letter are not significantly different as determined by Student-Newman-Kuels multiple range test. -83-0.5 0.45 . ug B12/ 100 g sample 0.40 . 0.35--0.3 .. 8% range of v a r i a t i o n i n assay Control 625 W Convection 1300 W Treatment Figure 9. Vitamin B r e t e n t i o n (ug/100 g) of samples from Animal 1 reheated to 70*C i n a 625 watt microwave oven, a 1300 watt microwave oven and a convection oven set at 180*C. -84-1.80 -• 1.70--ug B12/ 100 g sample 1.60--8* range of v a r i a t i o n i n assay 1.50" 1.40.. Control 625 W Convection 1300 W Treatment Figure 10. Vitamin B r e t e n t i o n (ug/100 g) of samples from Animal 2 reheated to 70"C i n a 625 watt microwave oven, a 1300 watt microwave oven and a convection oven set at 180"c. - 8 5 -Table XIX. Nitrogen content (%) of meat and drip from meat samples heated in three oven types. Oven % N Meat Drip Controls 11 .300 + 0.4991 -625 12 .028 + 0.1561 7.617 + 0.2571 Convection 12 .133 + 0.1452 6.966 + 0.1712 1300 12 .162 + 0.0793 7.064 + 0.2713 1. Mean + SE of 2. Mean + SE of 3. Mean + SE of the mean, n=6. the mean, n=12. the mean, n=9. Table XX. Retention of Vitamin B 1 2 in meat and drip samples heated to 70* C in 625 watt microwave, a 1300 watt microwave and a convection oven set at 180* C . Animal Oven B12 retention, (absolute), meat and drip B]o retention, (%), meat and drip B12 retention (absolute), meat ug/100 g N B ] o retention 12 (%), meat 1 Control 3.781 + 0.314la - - -1 625 3.934 + 0.312la 104.1 + 8.3 3.741 + 0.244la 95.4 + 1.3 1 Convection 3.009 +_ 0.0593b 79.6 + 1.6 2.855 + 0.1293b 94.8 + 2.4 1 1300 3.780 + 0.192ia 100.0 + 5.1 3.590 + 0.2l5 l a 95.0 + 0.9 2 Control 13.77 + 0.77 2c - - -2 625 14.14 + 0.39 2 c 101.6 + 2.5 13.75 + 0.44 2c 97.1 + 0.9 2 Convection 14.93 + 0.73 2 c 108.4 + 5.3 13.57 + 0.69 2c 90.8 + 1.5 2 1300 15.10 + 0.22 2c 109.6 + 1.6 14.14 + 0.23 2c 93.7 + 0.5 1. Mean + SE of the mean, n=4. 2. Mean +_ SE of the mean, n=6. 3. Mean +_ SE of the mean, n=2. a,b,c. Means in a column followed by the same letter are not significantly different as determined by Student-Newman-Kuels multiple range test. Control Meat + Meat Control Meat + Meat Control Meat + Meat Drip Drip Drip Treatment a) 625 watt microwave b) Convection oven c) 1300 watt microwave Figure 11. Vitamin B retention (ug/100 g N) of samples from Animal 1 reheated to 70*C i n a 625 watt microwave, a 1300 watt microwave, and a convection oven set at 180"C. 15.0 14.0 ug B12/ 100 g N 8% range of v a r i a t i o n i n assay l CO co i 13.0 12.6 0 Control Meat + Meat Drip Control Meat + Meat Drip Treatment Control Meat + Meat Drip a) 625 watt microwave b) Convection oven c) 1300 watt microwave Figure 12. Vitamin B retention (ug/100 g N) of samples from Animal 2 reheated to 70"C a 625 watt microwave, a 1300 watt microwave and a convection oven set at 180*C. -89-The apparent increase in vitamin content in the samples might have been corrected for i f more accurate methods of measuring evaporation loss had been used. In order to reproduce conditions that would be found in a hospital catering system, the vitamin was extracted from hot samples. The evaporation measured was that which occurred during the heating process only. Although precautions were taken to keep the sample covered and thereby capture the moisture lost during homogenization, it is possible some was not accounted for. Except for samples from Animal 2 heated in the 1300 watt microwave and the upper range of variability of the convection heated samples, the rest are within the 8 % variability range of the assay. This partially accounts for the difficultly in concluding that the observed increase is real and not just due to assay variability. Based on what is known about the heat stability of cyanocobalamin and the results obtained from Experiments 1 and 2, a loss of the vitamin after the reheating of meat samples would not be expected. Also illustrated in Table XX and Figures 11 and 12, is the retention of the vitamin in the meat portion of the sample, or conversly, the loss of vitamin B 1 2 to the drip. Each oven caused a decrease in vitamin retention or a loss to the drip, ranging from 2.86 % to 9.2 %. An ANOVA done on the data, in the absolute form, indicated that significant differences could not be detected between the meat plus drip samples, and the meat alone. As seen in Table XX, only samples from -90-Animal 2 heated in the convection oven incurred losses greater than 8 % (the variability range of the assay). Within this sample group, the variability was 12.36 %, and as a result the ANOVA was limited in its ability to detect this as a true difference. A significant difference was not detected between oven types. Table XXI compares the contribution of the drip to the total B-^ 2 content of samples heated in the 3 ovens as well as the contribution of the drip to the total weight of the samples after they had been heated. A definite trend can be identified here. For both Animal 1 and 2, the samples heated in the 625 watt microwave lost the least amount of B 1 2 to the drip and at the same time, lost the least amount of drip. Samples heated in the convection oven lost the most B 1 2 and the most drip. The 625 watt oven appeared to lose significantly less (p<0.05) B 1 2 than the convection oven and the 1300 watt oven when these values are compared by ANOVA. ANOVA also indicated that significantly less drip was lost from the samples heated in the 625 watt oven than the other ovens (p<Co.05). The general conclusion drawn from this experiment is that vitamin B-^ 2 appears to be released from the heated meat into the drip, however, this loss was not significant when the absolute values were compared. When comparing the percent loss of B-^ 2 to the drip, significantly less appeared to be lost in the 625 watt microwave and significantly less drip was released (p^O.05). The amount of B^2 lost to the drip from the heated samples appears to be a function of the amount of drip released. - 9 1 -Table XXI. Contribution of drip from heated meat samples to the total B 1 2 content and total sample weight. Cow Oven Contribution of Contribution of drip to total Bio drip to total weight (%) (%) 1 6251 A. 605 + 1.301a A. 307 + 1.117ab 1 Convection^ 6.638 + 2.258a 8.350 + 1.1A83 1 13001 5.03A + 0.876a 8.335 + O.AA7a 2 6252 2.850 + 0.8A9b A. 667 + 0.913d 2 Convection2 9.197 + l.A9Aa 11.865 + 1.855a 2 13002 6.305 + 0.5A5a 9.907 + 0.8063 1. Mean + SE of the mean, n=A. 2. Mean +_ SE of the mean, n=6. a,b. Means in a column followed by the same letter are not significantly different as determined by Student-Newman-Kuels multiple range test. -92-There are some discrepancies in the literature regarding the effect of different ovens and different power levels in microwaves ovens on drip losses from cooking. Drew et al. (1980) found significantly higher losses when roasts were cooked in a microwave at the "high" power setting than when cooked in conventional ovens. They found greater losses from roasts cooked at "low" power settings than from roasts cooked conventionally. The difference observed between "high" and "low" power levels was not significant. Hall and Lin (1981), found significantly greater cooking losses from broilers cooked in a 1600 watt microwave as compared to a 800 watt microwave, even though the cooking time in the 800 watt oven was approximately 1.7 times longer than in the 1600 watt oven. It is difficult to make a direct comparison between the results obtained by other researchers, as biological materials differ greatly in their moisture and fat contents and the experimental conditions differ with each study. A number of researchers have offered hypotheses on the mechanism behind the increased drip lost from microwave cooked products. Carpenter et al. (1968), speculated that the increase may be due to muscle denaturation resulting from internal heating. Apgar et al . (1959), and Kylen (1964) suggested that the increase in moisture loss may be a result of the increased post-cooking temperature rise observed in microwave cooking. Ruyack and Paul (1972), suggested the loss may be caused by increased ease of release of water due to oscillation of the water molecules in the alternating electric field. -93-The literature available regarding the effects of reheating on the drip loss from meat is limited. Dahl et al. (1982), reported greater weight losses from convection reheated beef loaf than from microwave reheated meat, but this loss was not broken down into drip and evaporation losses. Dahl and Matthews (1980), observed increases in drip losses with increases in time of exposure to microwave radiation, but no comparison was made to conventional heating. Cipra et_ al. (1971), observed lower drip losses from turkey reheated in a domestic microwave than in a conventional oven. This was speculated to be due to the shorter times required to heat samples in a microwave. Nothing is available comparing drip losses from meats reheated at different power levels in a microwave The results reported by Cipra et al. (1971) agree with the results found in this study. The convection oven samples lost significantly (p<(o.05) more drip than the domestic microwave samples. The heating times were 30 minutes and 49.58 +_ 1.82 seconds, respectively. The loss of drip probably results from the denaturation of proteins, their subsequent insolubilization and decrease in water binding capacity. The extent of denaturation is a function of time and temperature. With the increased time of heating in the convection oven, an increased release of drip would be expected. -94-The greater loss of moisture observed in the samples reheated in the 1300 watt microwave, in this study, agrees with the observations of Hall and Lin (1981). Rosen (1972), suggested that hydrogen bonded structures in biological materials may be affected by very low energies, such as microwave radiation, and, therefore, that water binding is related to microwave absorption. The increase in power in a microwave oven would result in more radiation being emmitted. Theoretically, absorption of the microwaves by the food material would be increased in a given time period, and the water binding capacity would decrease. The suggestion by Ruyack and Paul (1972), that the oscillation of water molecules in an electric field eases the release of water, may apply. As more microwaves are absorbed, more oscillation would occur and subsequently more water would be released. 5. GENERAL DISCUSSION Some questions might be raised regarding the validity of using a RID assay to quantitatively measure vitamin B-^. The inherent variability of this assay requires losses to be greater than 8 % in order for a significant (p <C 0.05) loss to be detected using statistical procedures. Significant losses less than 8 % can be detected, however, the number of replications of each treatment required to increase the confidence of the ANOVA to 95 % would be beyond the scope of this study, considering time, expense, and physical limitations. -95-There is little evidence to believe that the AOAC microbiological assays are any better than RID assays in terms of variability and sensitivity. Adams et al. (1973), reported that 34 % of the samples analyzed had second assay results that varied by greater than 20 % of the first assay, using the organism, Euglena gracilis. Lichlenstein et al . (1961) reported an assay variability of 9.33 % using L. leichmanni. Mollin et al . (1976) documented researchers reporting coefficients of variation ranging from 13.5 to 25 % using Euglena  gracilis. RID assays have the advantage of being simpler, more accurate, more reproducible , more specific and less subject to contamination than microbial assays (Casey et a l . , 1982). A question to be considered when doing a vitamin retention study is the amount of vitamin loss that would be of practical concern to the consumer. The assay must be evaluated on the basis of its ability to detect this loss. For the purposes of this study, a 20 % loss was considered to be significant. With the 8 % inherent variability of the assay, a 28 % loss would have to be detected. Once the variability of the assay and of the samples is known, it is possible to calculate the number of sample replications necessary in order to be 95 % confident that an apparent loss of 28 % is real and not just the result of chance. These data are presented in Table XXII. In all cases, except treatment differences for Solution A and treatment differences for Animal 1, a significant difference of 28 % could be detected with the same number or less than the number of replications done in the study. As previously -96-discussed, Solution A (time treatments), exhibited variabilities as high as 26.27 % when treated in the 1300 watt oven. This resulted from one set of samples being 22.3 - 36.4 % higher than the mean of the samples over the five time periods. This was attributed to a sampling error. Variabilities for Solution B ranged from 5.47 - 13.2 %. Solution A (temperature), exhibited a mean variation of 10.9 %, resulting from 2 sample sets with variations of 16.15 % and 16.11 %. Solution B had a mean variation of 5.01 %. Animal 1 had a mean variation of 17.9 % whereas Animal 2 had a variability of 9.4 %. The large variabilities noted for Solution A and Animal 1 account for the increased number of sample replications required to be 95 % confident that a difference of 28 % is real. The general conclusion drawn from this discussion is that if losses of 20 % of the original vitamin B 1 2 content occurred when buffer samples and frozen, thawed roast beef samples are heated under the experimental conditions carried out in this study, statistical analysis using ANOVA would be able to identify this as a real difference. -97-Table XXII. Number of treatment replications needed to detect a significant difference of 20 % (p<0.05). Experiment Variable Replications Theoretical Actual Time Treatments 11 3 (A) Oven 1 3 Time Treatments 3 3 (B) Ovens 2 3 Temperature Treatments 12 3 (A) Temperature 2 3 Ovens 2 3 Temperature Treatments 3 3 (B) Temperature 1 3 Ovens 1 3 Animal Treatments 17 A (1) Oven 3 A Drip A A Animal Treatments 6 6 (2) Oven 3 6 Drip A 6 -98-V. CONCLUSIONS The objectives of this study were to determine whether vitamin B^2 in buffer solutions and meat samples was destroyed in microwave and convection ovens. Microwave radiation, at intensities of 625 watts and 1300 watts, does not cause a significant destruction of vitamin B-^. Although some variability existed in the data, vitamin B^2 did not appear to be significantly destoyed when buffer samples at pH 5.6 were exposed to radiation for time periods totalling 1 hour. Vitamin B^2 is not subject to significant thermal degradation when exposed to heat for time periods ranging from 44.2 +_ 0.8 sec to 1144 +_ 29 sec (Table XVII). Buffer samples (pH 5.6) heated to temperatures ranging from 50-70 *C in a 625 watt microwave and a 1300 watt microwave and a convection oven set at 180'C (nominal), did not experience significant losses of vitamin B^2« Frozen, thawed, roast beef samples, reheated to 70*C in a 625 watt microwave and a 1300 watt microwave oven or in a convection oven for 30 minutes (180'C , nominal) did not experience significant losses of vitamin B-^. Reheated meat samples lost vitamin B-^ 2 to the drip, however, this loss was not significant. The percent of total B^2 lost to the drip is related to the amount of drip lost from the sample. Samples reheated in the 625 watt microwave lost significantly less drip than samples reheated in the 1300 watt microwave and the convection oven. -99-While the original intent of this study was fulfilled, the limitations of the information obtained must be recognized. The variability of the samples combined with the inherent variability of the RID assay (8 %) and the number of replications, limited the ability of the statistical analysis techniques used to identify small differences as significant. In this study, significant losses may have been detected if more replications of the treatments had been carried out. However, when considering vitamin losses at levels which may be of concern to consumers (20 %), the number of replications done in this study would be sufficient to identify this as a significant loss at the 95% confidence limit. - 1 0 0 -REFERENCES Adams, J . , McEwan, F. and Wilson, A. 1973. The vitamin B12 content of meals and items of diet. Br. J. Nutr., 29:65. Aldor, T. 1964. The behavior of food in the high frequency range. Nutr. Abstract, 34:661. Ang, C , Chang, C. Frey, A. and Livingston, G. 1975. Effects of heating methods on vitamin retention in six fresh and frozen prepared food products. J. Food Sci. , 40:997. 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APPENDIX A STATISTICAL ANALYSIS 1. TIME EXPERIMENTS SOURCE DF SUM SQ MEAN SQ F-VALUE PROBABILITY Time 4 36790.0 9197.A 0.680A5 0.60959 Oven 1 533A.6 533A.6 0.39A67 0.53343 Cone 1 0.20A72E+08 0.20A72E+08 1514.6 0.83267E-16 Ti X Ov A AA805.0 11201.0 0.82870 0.51486 Ti X Cn A 38097 952A.3 0.70A6A 0.59346 Ov X Cn 1 56657 56656 A.1917 0.47230E-01 T X 0 X C A 39819 995A.7 0.736A8 0.57261 Error AO 0.5A066E+06 13517 Total 59 0.2123AE+08 SENSITIVITY OF ANOVA Time: 20 % probability of detecting a real treatment difference 442 replications required for 95 % probability Oven: 20 % probability of detecting a real treatment difference 972 replications required for 95 % probability 2. TEMPERATURE - SOLUTION A SOURCE DF SUM SQ MEAN SQ ERROR F-VALUE PROB Treat 15 1 Cvstrt 1 1 Temp 4 1 Oven 2 1 Te X Ov 8 Error 32 Total 47 8028.5 555.04 754.74 2435.4 4283.3 15608 23637 535.23 555.04 188.69 1217.7 535.41 487.75 1 Te X Ov 1 Te X Ov 1.0973 1.1379 0.35241 2.2743 1.0977 0.39651 0.29407 0.83556 0.16518 0.39030 SENSITIVITY OF ANOVA Treatment: 20 % probability of detecting a real treatment difference 82 replications required for 95 % probability Temperature: 20 % probability of detecting a real treatment difference 29 replications required for 95 % probability Oven: 85 % probability of detecting a real treatment difference 4 replications required for 95 % probability 2. TEMPERATURE - SOLUTION B SOURCE DF SUM SQ MEAN SQ ERROR F-VALUE PROB Treat 15 1 Cvstrt 1 1 Temp 4 1 Oven 2 1 Te X Ov 8 Error 32 Total 47 26064 0.11600E-01 4503.8 7617.6 13943 0.16579E+06 0.19185E+06 1737.6 0.11600E-01 1126.0 3808.8 1742.9 5180.9 1 Te X Ov 1 Te X Ov 0.33539 0.22390E-05 0.64604 2.1854 0.33640 0.98604 0.99882 0.64514 0.17489 0.94520 SENSITIVITY OF ANOVA Treatment: 20 % probability of detecting a real treatment difference 205 replications required for 95 % probatility Temperature: 20 % probability of detecting a real treatment difference 47 replications required for 95 % probability Oven : 35 % probability of detecting a real treatment difference 13 replications required for 95 % probability MEAT (ug/100 g sample) - COW 1 SOURCE DF SUM SQ MEAN SQ F - VALUE Treatment 3 0.039 0.013 4.126 Error 12 0.038 0.003 Total 15 0.077 SENSITIVITY OF ANOVA Treatment: 95 % probability of detecting a real treatment difference Using Student - Newman - Kuels multiple range test - Convection oven was significantly lower than 625 watt or 1300 watt microwave oven MEAT (ug/100 g sample) - COW 2 SOURCE DF SUM SQ MEAN SQ F - VALUE Treatment 3 0.163 0.054 1.96931 Error 20 0.552 0.028 Total 23 0.715 SENSITIVITY OF ANOVA Treatment: 75 % probability of detecting a real treatment difference 16 replications required for 95 % probability MEAT (ug/100 g N) - COW 1 SOURCE DF SUM SQ MEAN SQ F - VALUE PROB Treat 6 5.9615 0.99358 4.7741 0.32399E-02 1 Cvstrt 1 0.41860 0.41860 2.0114 0.17079 1 Oven 2 5.3289 2.6644 12.803 0.23160E-03 1 Drip 1 0.21395 0.21395 1.0280 0.32216 1 Ov X Drip 2 0.53583E-04 0.26792E-04 0.12873E-03 0.99987 Error 21 4.3705 0.20812 Total 27 10.332 SENSITIVITY OF ANOVA Treatment: 95 % probability of detecting a real difference between convection oven samples and microwave samples Oven: 95 % probability of detecting a real difference between convection oven and microwave ovens Drip: 20 % probability of detecting a real treatment difference 43 replications required for 95 % probability MEAT (ug/100 g N) - COW 2 SOURCE DF SUM SQ MEAN SQ F - VALUE PROBABILITY Treat 6 12.839 2.1399 1.2238 0.31774 1 Cvstrt 1 1.2924 1.2924 0.73913 0.39579 1 Oven 2 2.7089 1.3544 0.77459 0.46863 1 Drip 1 7.4193 7.4193 4.2430 0.46910E-01 1 Ov X Drip 2 1.4189 0.70947 0.40574 0.66958 Error 35 61.201 1.7486 Total 41 74.040 SENSITIVITY OF ANOVA Treatment: 41 % probability of detecting a real treatment difference 40 replications required for 95 % probability Ovens: 30 % probability of detecting a real treatment difference 31 replications required for 95 % probability Drip: 92 % probability of detecting a real treatment difference 7 replications required for 95 % probability -113-APPENDIX B Example of calculation to convert vitamin concentrations from ug/lOOg  sample to ug/lOOg N. Sample data: start weight - 87.94 g end weight - 86.43 g meat weight - 81.43 g drip weight - 5.00 g meat B12 content - 1.62 ug/100 g drip B 1 2 content - 0.71 ug/100 g meat N content - 12.133g N/100 g drip N content - 6.966 g N/100 g 1) If there are 1.62 ug of B 1 2 i n 100 g of meat, then there are 1.32 ug in 81.43 g of meat. 2) If there are 0.71 ug of B 1 2 i n 100 g of drip, then there are 0.03 ug in 5.0g of drip. 3) If there are 1.35 ug (1.32 + .03) of Bi_2 in 86.43 g of sample, then there are 1.56 ug in 100 g of sample. 4) If meat contributes 81.43 g to 86.43 g of total sample, then meat contributes 94.2 g to 100 g of sample and drip contributes 5.8 g. 5) If 100 g of meat contains 1.62 ug of B12 then 94.2 g would contain 1.525 ug and contribute 97.75 % of the total B12 in 100 g of sample. 5.8 g of drip would contribute 0.035 ug to the total B i 2 o r 2 - 2 5 % o f the total. 6) If there are 12.l33g of N in 100 g of meat, then there are 11.429 g of N in 94.2 g of meat. 7) If there are 6.966 g of N in 100 g of drip, then there are .404 g in 5.8 g of drip. 8) In 100 g of meat plus drip, there are 11.833g of N (11.429+.404). 9) If there are 1.56 ug of B i 2 in 100 g of sample and 11.833 g of N in 100 g of sample, then there are 13.183 ug of Bi 2 in 100 g of N. 

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