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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 CONTENT OF ROAST BEEF PRODUCED IN A COOK/FREEZE FOODSERVICE SYSTEM 1 2  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  In p r e s e n t i n g  this  thesis  in partial  f u l f i l m e n t of the  r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e of B r i t i s h Columbia, I agree that it  freely  the L i b r a r y s h a l l  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 .  agree that p e r m i s s i o n for  University  f o r extensive  s c h o l a r l y p u r p o s e s may  for  financial  shall  of  The U n i v e r s i t y o f B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Columbia  my  It is thesis  n o t be a l l o w e d w i t h o u t my  permission.  Department  thesis  be g r a n t e d by t h e h e a d o f  copying or p u b l i c a t i o n of t h i s  gain  further  copying of t h i s  d e p a r t m e n t 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 . understood that  I  make  written  ABSTRACT  The purpose of this  study was to determine the effect of  microwave and convection reheating on the vitamin B^ content of roast 2  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. retentions  in the two oven  types  as  well  The differences between 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  destruction of the vitamin was not significant. 82.45 +_ 3.57 % to 103.3 + 4.6 %.  The  Retentions ranged from  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-^ present in the drip was found to be related 2  to the amount of drip released from the sample. 625 watt microwave released  Samples reheated in the  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  12  retention.  -•iii-  TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF TABLES  v  LIST OF FIGURES  vii  LIST OF APPENDICES  viii  ACKNOWLEDGEMENTS  ix  I.  INTRODUCTION  -  1  II.  LITERATURE REVIEW 1. Cook/Freeze Catering Systems 2. The Effect of Convection Ovens on the Nutrient Composition of Foods 3. The Effect of Microwave Ovens on the Nutrient Composition of Foods 3.1 Moisture 3.2 Fat Soluble Vitamins 3.3 Water Soluble Vitamins 3.3.1 Thiamin 3.3.2 Riboflavin and Niacin 3.3.3 Pyridoxine and Folacin 3.3.A Ascorbic Acid A. Effects of Heating Methods on the Vitamin Content of Frozen Foods. 5. Nomenclature and Function of Vitamin Bj_ 6. Destruction of Vitamin B]_ 7. The Vitamin B Content of Beef 2  2  12  III.  MATERIALS AND METHODS 1. Extraction of Vitamin B]. 1.1 Extracting Solution 1.2 Phosphate Buffer 1.3 Glassware l.A Procedure 2. Analysis of Vitamin B]_ 2.1 Standard Curve Preparation 2.2 Calculations 3. Determination of the Effect of Microwave Radiation on Vitamin B i at t=0-C 3.1 Sample Preparation 3.2 Sample Treatment 3.3 Expression of Data 2  2  2  A A 7 7 7 10 10 10 15 17 18 20 2A 2 9  30 32 32 32 32 32 33 33 35 36 A3 A3 AA AA  - i v-  4. Determination of the Effect of Microwave and Convection Heating on Vitamin B12 in a Buffer System... 4.1 Sample Preparation 4.2 Sample Treatment 4.3 Expression of Data 5. Determination of the Effect of Reheating on Vitamin B12 o a Beef 5.1 Sample Preparation 5.2 Sample Treatment 5.3 Crude Nitrogen Analysis 5.4 Expression of Data 6. Statistical Analysis i n  IV.  R  45 45 46 47 48 48  RESULTS AND DISCUSSION 1. Preliminary Experiments 2. Determination of the Effect of Microwave Radiation on Vitamin Bj_ in a Buffer Solution at t=0-C 3. Determination of the Effect of Microwave and Convection Heating on Vitamin B12 in a Buffer System... 4. Determination of the Effect of Reheating on Vitamin Bi_2 in Roast Beef 5. General Discussion  50 50  CONCLUSIONS  98  2  V.  st  44 44 45 45  59 70 79 94  REFERENCES CITED  100  APPENDIX A B  106 113  -V-  LIST OF TABLES Page Table I. Table II. Table III. Table IV. Table V. Table VI.  Moisture losses in microwave and conventionally cooked foods Thiamin losses in microwave and conventionally cooked foods  12  Riboflavin and niacin losses in microwave and conventionally cooked foods  16  Ascorbic acid losses i n microwave and conventionally cooked vegetables  19  • The effect of various reheating methods on the nutrient content of precooked frozen foods  9  21  Typical data derived from a vitamin B12 RID assay of meat samples  37  Recovery study of cyanocobalamin in an aqueous solution  52  Recovery study of different concentrations of cyanocobalamin added to meat extracts  53  Repeatability of the RID assay using subsamples of A meat samples (Bj_ expressed as ug/100 g sample). 2  57  Table X.  Interassay variability  58  Table XI.  Retention of Vitamin Bj. i n buffer solution A after exposure to varying periods of radiation in a 625 watt domestic microwave oven  60  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  Retention of Vitamin B i n buffer solution A after exposure to varying periods of radiation in a 1300 watt commercial microwave oven  62  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  Table VII. Table VIII. Table IX.  Table XII.  Table XIII.  Table XIV.  2  12  -viTable 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  Time in seconds for 250 mL buffer samples to come to temperature in three oven types  78  Table XVII.  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. Table XX.  Nitrogen content (%) of meat and drip from meat samples heated in three oven types  85  Retention of Vitamin B12 i 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  Contribution of drip from heated meat samples to the total B_2 content and total sample weight  91  Number of treatment replications needed to detect a significant difference of 28 % (p<0.05)  97  n  Table XXI. Table XXII.  0  -vi i LIST OF FIGURES Page Figure 1.  Conventional and cook/freeze catering operations  Figure 2.  The structure of a cobalamin molecule  Figure 3.  Meat and buffer standard curves expressed as CPM vs. B 1 2 pg/mL Meat and buffer standard curves expressed as logit % B/Bo vs. log B 1 2  Figure 4. Figure 5.  Figure 6.  Figure 7.  Figure 8.  5 25 38 39  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  Retention of vitamin B12 i- buffer solution B after exposure to 625 and 1300 watts of microwave radiation for 15 minute time periods  65  Retention of vitamin B12 i- 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  Retention of vitamin B 1 2 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  n  n  Figure 9.  Vitamin B 1 2 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 B 1 2 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^ 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 2  -vi i i -  LIST OF APPENDICES Page Appendix A.  S t a t i s t i c a l Analysis 1. Time experiments 2. Temperature - S o l u t i o n A - Solution B 3. Meat (ug/100 g sample) - Animal 1 - Animal 2 A. Meat (ug/100 g N) - Animal 1 - Animal 2  Appendix B.  Example of c a l c u l a t i o n to convert vitamin concentrations from ug/100 g sample to ug/100 g N  106 106 107 108 109 110 I l l 112 113  -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  catering.  as for use in hotels,  restaurants,  and airline  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).  -2The 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 reheating on vitamin B the  diet,  necessary  1 2  .  of microwave and convection  Vitamin B^ * 2  s  a n  essential component of  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 ^ content in beef of 2 ug/100 g, the daily consumption 2  of vitamin B^ from beef is 0.9 ug, or 30 % of the recommended dietary 2  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  2)  17  in a buffer system.  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^ content of precooked, frozen and thawed 2  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 a l . (1963). study,  of the foodservices  This  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  utilization of labor and equipment.  very  inefficient with respect to  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 catering.  The  most  out to develop new methods of hospital  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  Service Conventional Catering  I  Freezing I Cold Storage X Transport FROZEN Food  1  Reconstitution 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 a l .  (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. (19A7), reported losses of ascorbic acid  Branion et a l .  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 nutrient  losses,  the  system,  in order to minimize  freezing process should achieve  penetration rate of 0.5 inches per hour (Glew, 1970).  an ice  front  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  convection  little  information  oven heating  is  on nutrients  available in foods.  on  the  effects  of  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).  -8Table 1 summarizes the published reports effects  of microwaves  on moisture  conventional cooking methods.  which compare the  in various  foods with those of  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 a l . (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 a l l 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  Marshall (1960)  Sample  Top round roasts of beef  Internal Temperature (-C)  microwave conventional  80 80  microwave conventional microwave conventional microwave conventional  87.8 87.8 87.8 87.8 87.8 87.8  microwave conventional microwave conventional microwave conventional microwave conventional  57 58 86 82 85 85  microwave conventional beef-soy flour microwave loaves conventional beef-soy microwave conventional concentrate  74 74 74 74 74 74  Apgar et a l . pork patties (1959) pork roasts pork chops Kylen et a l . beef roasts (1964) pork roasts beef loaf pork loaf Ziprin and arlin (1976)  Treatment  beef loaves  -  -  Headley and lamb roasts Jacobson(1960)  microwave conventional  65 82  Wing and chicken Alexander(1972)  microwave conventional  88 88  Moisture  % Loss Weight Drip  Evaporation  60.6 34.7  13.8 6.5  28.4 19.7  37.2 36.3 26.5 25.0 17.7 26.4  32.6 22.1 12.4 9.1 14.7 16.8  4.6 14.3 14.3 15.7 2.9 17.7  26.9 14.8 25.5 21.4 5.8 5.8 12.1 5.7  38.8 17.5 37.3 34.1 26.9 24.3 28.3 18.2  18.8 7.4 17.1 12.3 8.9 9.1 5.6 5.2  20.0 10.2 20.2 21.9 18.0 15.2 22.8 13.Q  14.0 5.0 14.0 5.0 14.0 5.0  27.0 19.0 24.0 14.0 24.0 14.0  5.0 12.0 2.0 8.0 2.0 6.0  22.0 7.0 22.0 7.0 22.0 7.0  43.0 35.0  15.0 17.0  27.0 19.0  -  -  _  11.5 8.8  26.6 22.7  2.69 3.95  -  -10The reason f o r the observed increase i n moisture l o s s and weight l o s s 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 l o s s may observed  be a r e s u l t of the higher i n t e r n a l post-cooking temperature after  microwave  cooking,  causing  dehydration  rise  through  evaporation.  3.2 Fat-Soluble Vitamins Very  few data are a v a i l a b l e on the e f f e c t of microwave cooking  on f a t s o l u b l e 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 e f f e c t on them of microwave cooking. 3.3.1  Thiamine Thiamine  is  a  destroyed by o x i d a t i o n .  water-soluble, I t i s leached  heat-labile from  vitamin  which  is  food i n proportion to the  amount of a v a i l a b l e water, the extent of a g i t a t i o n during cooking and the surface area of the food exposed to water  (Guthrie, 1979).  Fung (1982) stated that the combined e f f e c t s of heat, water b o i l i n g i n conventional cooking  Cross and  and rapid  suggest that microwave cooking might be  l e s s d e s t r u c t i v e 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.  -11The effect of microwaves on buffered solutions was studied by Van Zante and Johnson (1970), and Goldblith et a l . (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. al. (1968), concluded that microwave energy thiamine.  Goldblith et  has no destructive effect on  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 a l . (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 a l l cooking methods.  Apgar et a l . (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. greater  loss  Baldwin (1976), found a significantly  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  Apgar et al.(1959) pork patties pork roasts pork chops Baldwin et a l . (1976)  beef roast pork roast lamb roast  Treatment  Internal Temperature  Thiamine Loss (%)  microwave-domestic microwave-commercial conventional microwave-domestic microwave-commercial conventional microwave-domestic microwave-commercial conventional  87.8 87.8 87.8 87.8 87.8 87.8 87.8 87.8 87.8  50.4 50.2 51.8 34.8 39.6 36.8 44.9 37.2 . 49.8  microwave-domestic microwave-commercial conventional microwave-domestic microwave-commercial conventional microwave-domestic microwave-commercial conventional  70 70 70 70 70 70 70 70 70  51.0 39.0 31.0 33.0 27.0 28.0 51.0 48.0 48.0  Bowers and Fryer (1972)  turkey breast  microwave conventional  68 80  20.64 20.73  Hall and Lin (1981)  chicken halves  microwave-800W microwave-1600W conventional-121•C conventional-204-C  92 92 82 82  22.4 22.4 28.2 23.4  Recovered in Drip (%) —  -  27.6 26.1 27.8  —  —  _ —  Table II. continued. Reference Sample Kylen et al.(1964) beef roasts pork roasts beef loaves ham loaves  Treatment microwave conventional microwave conventional microwave conventional microwave conventional  Internal Temperature (•O 57 58 86 82 85 85  Thiamin Loss (X)  -  42.0 20.0 40.0 39.0 20.0 24.0 9.0 13.0  Noble and Gomez (1962)  roast lamb  microwave conventional  73 73  43.0 46.0  Ziprin and Carlin (1976)  beef loaf  microwave conventional microwave conventional microwave conventional  74 74 74 74 74 74  30.9 17.5 35.4 24.0 33.3 25.4  microwave conventional microwave conventional microwave conventional  74 74 79 79 79 79  37.0 25.0 11.0 45.0 9.0 21.0  Thomas(1949)  beef-soy flour loaf beef-soy concentrate loaf beef roast beef patties pork patties  Recovered in Drip (%) 13.0 2.0 31.0 19.0 —  —  _  -  -  9.0 6.0 8.0  -  2.0 —  -14case 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. found that thiamine retention  Hall and Lin (1981),  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 a l . (1963), found  significantly greater thiamine losses in microwave-cooked beef roasts and pork roasts  than  in conventionally  differences  when  beef  cooked roasts  and ham loaves  were  but insignificant 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. slightly  The losses occuring from microwave cooking were only  greater  than  those  occuring  during  conventional cooking.  Baldwin et a l . (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 commercial microwave ovens.  microwave ovens compared with  Thomas et a l . (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  Stevens and Fenton (1951)  frozen peas  microwave conventional  68 80  2.0 0.0  -  Bowers and Fryer (1972)  turkey breast  microwave conventional  68 80  13.5 8.2  _  Noble and Gomez (1962)  roast lamb  microwave conventional  79 79  25.0 16.0  Baldwin et a l . (1976)  beef roast  microwave-commercial microwave-domestic conventional  70 70 70  2.0 17.0 1.0  6.0 14.0 -4.0  pork roast  microwave-commercial microwave-domestic conventional  70 70 70  19.0 18.0 4.0  13.0 21.0 -1.0  lamb roast  microwave-commercial microwave-domestic conventional  70 70 70  12.0 27.0 2.0  29.0 36.0 14.0  beef patties  microwave conventional microwave conventional microwave conventional  79 79 79 79 74 74  0.0 0.0 0.0 0.0 15.0 15.0  10.0 10 0 15.0 15.0 27.0 19.0  Thomas (1949)  pork patties beef roast  Internal Temperature (-C)  Riboflavin Loss (%)  Niacin Loss (%)  -  -  -  -17patties were cooked by microwave and conventional methods.  Significantly  more niacin was lost from beef roasts cooked by microwaves than when cooked by conventional methods. these studies,  Due to the variability of the data from  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 i t is labile to alkali, oxidizing agents and ultraviolet light (Guthrie, 1979). et  a l . (1974),  methods.  cooked turkey  breasts by microwave  and conventional  The pyridoxine losses incurred were 16.7 ug/g of dried muscle  and 16.5 ug/g of dried muscle respectively, significant. lost  Bowers  from  a difference which is not  They also recorded no significant differences in pyridoxine pork  loin  conventionally cooked.  when  cooked  in a  microwave  oven  or when  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). and  Alexander  (1972),  observed  significantly  greater  retention  Wing of  pyridoxine in microwave cooked chicken breasts than in conventionally cooked breasts, 92.5 % and 88.4 % respectively. rather than temperature was responsible microwave cooking.  They suggested that time  for the decreased losses in  Cross and Fung (1982) concluded from these three  studies that microwave applications do not result in increased loss of pyridoxine.  -18Folacin, a water soluble vitamin, has been reported  to have  cooking losses as high as 50-100 % using conventional cooking methods (Cross  and Fung,  significant  1982).  differences  Klein  and Van Duyne (1979),  in the folacin  retention  conventionally cooked spinach and frozen peas.  reported no  of microwave and  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. on this subject.  Table IV summarizes some of the literature  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 a l . (1960),  subjected 7 vegetables to both cooking methods and found no significant difference  in the ascorbic  amounts were recovered  acid loss  in each.  Significantly lower  from the cooking water when cabbage, peas, and  green beans were cooked in the microwave.  Eheart and Gott (1964), found  Table IV. A s c o r b i c a c i d l o s s e s i n microwave and c o n v e n t i o n a l l y cooked v e g e t a b l e s . Reference  Sample  Treatment  Cooking time  Stevens and Fenton (1950)  f r o z e n peas  microwave conventional  3 min, 45 s e c . 9 min.  Kylen e t a l . (1960)  broccoli  microwave conventional microwave conventional microwave conventional microwave conventional microwave conventional microwave conventional microwave conventional  cabbage cauliflower peas green beans soy beans spinach Eheart and Gott  (1964)  broccoli peas potatoes spinach  Campbell e t a l . (1958)  broccoli-fresh broccoli-frozen green peas-frozen  microwave with water microwave without water conventional microwave with water microwave without water conventional microwave with water microwave without water conventional microwave with water microwave without water conventional microwave conventional microwave conventional microwave conventional  Ascorbic Acid loss  (%)  17.0 14.0 21.0 17.0 28.0 31.0 13.0 8.0 26.0 27.0 22.0 26.0 24.0 21.0 44.0 39.0  -  23.6 17.8 25.2 37.3 35.0 31.6 23.5 25.6 20.1 35.3 32.8 50.7 32.0 42.3 21.4 26.5 6.9 55.2  % Recovered  11.0 10.0 11.0 14.0 6.0 7.0 15.0 23.0 7.0 9.0 10.0 11.0 9.0 5.0 _ _  -_ _  -_ —  _  _ _ _ _  -  -20no 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  significant difference in retention of ascorbic  showed no  acid when compared to  amounts retained by the same vegetable cooked by conventional methods. Spinach retained a significantly greater amount of ascorbic cooked by microwaves than when cooked by boiling in water. al.  acid when Campbell e_t  (1958), showed that microwaved fresh broccoli and frozen peas lost  significantly less ascorbic acid than conventionally cooked vegetables. Frozen broccoli significant.  lost  less ascorbic  acid but the difference  was not  These studies indicate that no appreciably greater losses  of ascorbic acid occur when vegetables are microwave cooked as compared to conventionally cooked.  Klyen  et a l . (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 l i t t l e  various heating foods. subject.  information is available regarding the effect of  methods on the vitamin content of precooked  frozen  Table V summarizes the literature that has been published on the 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 a l . (1975), demonstrated an inverse relationship  Table V. The e f f e c t of v a r i o u s r e h e a t i n g methods on the n u t r i e n t content of precooked f r o z e n foods. Sample  Treatment  Bowers and F r y e r (1972)  turkey b r e a s t  microwave-AO'C conventional-55-C  25.A 2A.6  9.8 10.2  --  Ang et a l . (1975)  mashed potatoes  f r e s h - h e l d 1/2 hour f r e s h - h e l d 1 1/2 hours f r e s h - h e l d 3 hours f r o z e n - r e h e a t e d c o n v e c t i o n 1/2 hr frozen-reheated microwave 1/2 hr  3.3 9.2 18.A 11.7 8.0  2.A 2.0 2.7 3.5 3.5  33. 9 A9. 0 60. A 6A. ,3 75. 9  f r e s h - h e l d 1/2 hour f r e s h - h e l d 1 1/2 hours f r e s h - h e l d 3 hours f r o z e n - r e h e a t e d c o n v e c t i o n 1/2 hr f r o z e n - r e h e a t e d microwave 1/2 h r  5.9 8.1 11.8 10.3 10.0  7.5 9.3 8.5 6.0 6.8  f r e s h - h e l d 1/2 hour f r e s h - h e l d 1 1/2 hours f r e s h - h e l d 3 hours frozen-reheated c o n v e c t i o n 1/2 hr frozen-reheated microwave 1/2 hr  6.1 11.5 16.7 8.A 2.7  _  pot r o a s t and gravy  f r e s h - h e l d 1/2 hour f r e s h - h e l d 1 1/2 hours f r e s h - h e l d 3 hours frozen-reheated c o n v e c t i o n 1/2 hr frozen-reheated microwave 1/2 hr  6.9 8.3 16.6 13.1 12.A  11.A 9.0 17.5 8.5 11.3  beans and frankfurters  f r e s h - h e l d 1/2 hour f r e s h - h e l d 1 1/2 hours f r e s h - h e l d 3 hours frozen-reheated c o n v e c t i o n 1/2 hr frozen-reheated microwave 1/2 hr  6.6 1A.0 18.2 7.A 9.8  1.8 7.0 5.3 3.1 0.9  peas and  onions  carrots  Thiamine  V i t a m i n L o s s , (%) R i b o f l a v i n Ascorbic ^-carotene Acid  Reference  -  -  -  —  -  -  —  7. 8 12. 0 8. 2 7. 6 5. 7 _  -  _  -  -  —  -  Table V. continued Reference  Sample  Ang e t a l . (19757  fried  Treatment fish  Dahl-Sawyer beef l o a f et al.(1982)  Thiamine  Vitamin Loss^ (%) Riboflavin Ascorbic ^-carotene Acid  fresh-held 1/2 hours 0.0 f r e s h - h e l d 1 1/2 hours 8.7 f r e s h - h e l d 3 hours 22.8 frozen-reheated convection 1/2 h r 0.0 frozen-reheated microwave 1/2 hr 4.2 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  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 Livingston (1970)  beef stew  f r e s h held 1 hour @82-C fresh held 2 hour @82-C f r e s h held 3 hour @82-C microwave heated to 90-C  26.5 32.0 37.0 5.0  chicken a l a king  fresh held 1 hour @82-C f r e s h held 2 hour H82-C f r e s h held 3 hour (182-C microwave heated t o 90-C  25.0 30.0 37.0 6.0  77.9 82.4 84.8  Table V. continued. Reference  Sample  Treatment  Thiamine  Kahn and Livingston (1970)  shrimp newburg  f r e s h held 1 hour ©82-C f r e s h held 2 hour ©82-C fresh held 3 hour ©82-C microwave heated t o 90 «C  24.0 27.0 34.0 7.5  peas i n cream sauce  f r e s h held 1 hour ©82-C f r e s h held 2 hour ©82-C f r e s h held 3 hour ©82-C microwave heated t o 90-C  13.0 17.0 24.0 7.0  _  f r i e d chicken  convection cooked 20-25 min convection heated held 1/2 hr convection heated held 1 1/2 h r s convection heated held 3 h r s microwave heated held 1/2.hr  10.2 11.2 11.7 26.2 7.9  9.5 12.0 13.0 9.8 7.2  char b r o i l e d patties  convection heated held 1/2 hr microwave heated held 1/2 hr  5.0 9.6  3.8 0.9  Ang et a l . (1978)  Riboflavin  —  -  -  Vitamin Loss, (%) A s c o r b i c \*> -carotene Acid  -24between 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 a l .  (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^ activity was first recognized in 1926 by Minot and 2  Murphy as a cure for pernicious anemia (Guthrie, 1979).  In 1948, i t was  isolated in the crystalline form, simultaneously by L.  Smith of England  and E.  It then took ten  Rickes and K. Folkers of the United States.  years to determine the complex structure of the small red crystals, using X-ray diffraction analysis (Lehninger, 1982).  The substance was found to  -25contain 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 1979).  12  being called,  "cobalamin" (Guthrie,  Figure 2 shows the structure of the cobalamin molecule.  HO Ok Figure 2. The structure of a cobalamin molecule.  -26A number of biologically active forms of the vitamin exist, these  being;  hydroxocobalamin,  sulphitocobalamin, vitamin  forms,  cyanocobalamin,  nitritocobalamin,  methylcobalamin and adenosylcobalamin.  hydroxocobalamin  and methylcobalamin  coenzyme, 5'deoxyadenosylcobalamin,  The active  and an active  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). protein  by a  hydroxocobalamin  peptide can  In foods, the vitamin is found bound to  linkage. be  In the  converted  into  body, the  cyanocobalamin and active  coenzyme,  5'deoxyadenosylcobalamin (Guthrie, 1979). The utilization of cobalamin and its role has documented by Guthrie (1979), and Lehninger (1982). cobalamin is absorbed by diffusion.  One to three percent of ingested The major portion of that gaining  entry to the body is taken up by an active transport system which carries it  across the intestinal  mucoprotein, parietal  membrane.  known as intrinsic  cells  lining  This system  factor,  the stomach.  is dependent  which is secreted  Proteolytic  on a  from the  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  -27one 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. the form of the coenzyme 5'deoxyadenosylcobalamin.  It functions in  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  megaloblasts. large  in  size  and  become  large,  immature  cells  known  as  The red blood cells produced by these megaloblasts are the  immature macrocytes  characteristic  of  pernicious  disease associated with a deficiency of vitamin B-^.  anemia,  the  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. of  a deficiency of  vitamin B^ , 2  a 50  to  100  In the event  % 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.  -28The propionyl CoA molecule is then oxidized via a series of reactions in which the  enzyme methylmalonyl CoA mutase,  5'deoxyadenosylcobalamin,  containing  the  coenzyme  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^ is 3 ug, of which 2  only 1 to 1.5 ug are absorbed. to 30 ug a day. animal origin.  An average western diet provides from 7  The vitamin is found widely distributed in foods of  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 deficiency.  with vitamin B-^  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. The  A deficiency state resulting from inadequate intake is rare.  main causes  intrinsic  factor  complete  removal  (transcobalamins) worm.  of to of  B-^  2  deficiency  bind the the  are,  inadequate  vitamin in the  stomach,  lack  of  production  intestine, the  partial  transport  of or  proteins  in the blood, or an intestinal infestation with tape  -29The 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^ (Guthrie, 2  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, vanillin,  hydrogen  thioglycollic acid, thiazole  moiety  aldehydes, peroxide,  ferrous  gluconate,  cysteine  ferrous  hydrochloride,  decomposition products of ascorbic  of thiamine  hydrochloric  acid  Bartilucci and Ross, 1954; Macek and Feller, 1952). exposure to bright light, photolysis occurs, (Farquharson and Adams, 1976).  (Merck  sulphate,  hydroquinone, acid,  and the  Index,  1976;  Under conditions of  producing hydroxocobalamin  Aqueous solutions of vitamin B  12  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  -30B^  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-^ content of beef has been reported by a number 2  of researchers.  Adams et a l . (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. Bj  2  Hoppner et_ a l . (1978) reported finding 1.8 ug of  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  er  1 0 0  9  were reported for rib roasts and 1.3-2.0 ug per 100 g for steak, using microbial assay methods. study of the B  1 2  Bennink and Ono (1982) did a comprehensive  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 B j content was expressed 2  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  -31losses 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. is  slightly  higher  than those reported  difference was attributed microbial  assay.  by other  This value  researchers.  to the use of a RID assay instead  The sensitivity  of this  assay  The of a  was attributed  enhanced extraction and stability (Van Tonder et a l . , 1975; Beck, 1979).  to  -32III.  MATERIALS AND METHODS  1. EXTRACTION OF VITAMIN B12 All vitamin B  12  samples were extracted  according  to  the  standard AOAC  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).  -331.4 Procedure  All meat samples were ground in a Cuisinart Food Processor for 30 sec to obtain a homogeneous sample. weighed into a 100 mL low actinic flask.  Of this homogenate, 3 g were 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. 50 mL with the extracting solution.  The samples were then diluted to  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 ( Co) vitamin B-^, was 57  added to each tube.  The tubes were vortexed to mix the solutions,  -34covered with aluminum f o i l , and heated in a boiling water bath at 100 *C (General Electric Silverstone Frying Pan) for 20 min. various forms of vitamin B^  2  to dissociate  This allowed the  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-^ into the bottom of 2  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  content of the samples was then determined from a standard curve. fresh standard curve was run with each assay.  12  A  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.  -352.1 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 prepared  curve,  for the analysis  preliminary experiments interfered  with  from samples  containing no protein, was  of the buffer system.  It  was found in  that the absence of protein in the samples  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 a l . (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  12  dissociated  from the complex. To make the standard curve, a solution of 100 ug of vitamin B /mL 12  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  -36volumetric 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-^ in the standards. 2  illustration  of  typical  data  derived  from this  Table VI is an  assay.  Figure  3  illustrates the shape of the standard curve when the data are plotted in the 'raw' form (CPM vs. B^ , pg/ml). 2  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 Avg. Corrected CPM of Zero Standard  X 100  T a b l e V I . T y p i c a l data d e r v i e d from a v i t a m i n B12 Tube  CPM  Average CPM  R  I ° assay o f meat  Average Corrected  %B/Bo CPM  1  samples. Logit %B/Bo  Log B_2  Vitamin Bj2 concentration (pq/ml)  Blank  498 626  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 C o r r e c t e d  562  CPM = Average bound CPM-Average Blank CPM  14000  -39-  F i g u r e 4.  Meat and b u f f e r standard curves expressed as l o g i t % B/Bo logB  1 2  ..  vs.  -40The calculated % B/Bo of each of the standards was plotted on the logit axis against the concentration of vitamin B^ on the log axis 2  of  log-logit paper.  A best fit straight  line was drawn between the  plotted points, and the level of vitamin B^ in each of the samples was 2  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  12  b = y intercept  -41To express the results as vitamin B^ , ug/100 g of sample, the 2  following equation was used:  vitamin B , , ug/100 g = C/(100xW) 7  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  12  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 g N/100 g sample  12  ug/100 g N  -42-  To determine the vitamin B^ content of 100 g of total sample 2  (meat plus drip), the following procedure was used:  1)  Ax 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 B /100 g N l2  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^ io a sodium phosphate-citric 2  acid buffer at pH 5.6 were examined.  Solution A (200 pg/mL - nominal)  was chosen as it represents the average B^ content of 100 g of beef. 2  Solution B (1500 pg/mL - nominal) represents  a high concentration of  vitamin B^ within the upper limits of the standard curve. 2  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  phosphate-citric acid buffer.  diluted to  250  mL in the sodium  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. actinic.  A l l flasks used were low  -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). replication 10 vials were prepared.  For each  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. and 60 minutes.  One mL from each vial was removed and extracted as  described in Section 1. compared to  that  This was repeated after 30, 45  The B^ content of the irradiated samples was  of the  2  unirradiated controls.  Each condition was  repeated in triplicate. 3.3 Expression of data After analysis of the vitamin as described in Section 2, data were expressed  as the concentration  of vitamin B-^,  the  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, 60* C,  65* C,  or  70 *C in either  Wave Oven RK-93001,  Canco Inc.,  a 625  watt oven(  55'C,  Hotpoint GE Dual  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). was compared  to  an unheated  The B^ content of the heated sample 2  control.  Each treatment  was done in  triplicate. 4.3 Expression of data After analysis of the vitamin as described in Section 2, data were expressed sample.  as  the  concentration  of vitamin B-^ , 2  P9/ mL  the °f  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 Packers, Vancouver. respectively  round roasts  were purchased  from  Intercontinental  Roasts A and B came from the right and left sides  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 temperature for 3 hrs.  for 18 hrs,  and then at room  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  internal temperature stopped rising.  sit  until  the  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  weight  size  ,  shape  and  (90  g),  and  packaged  polyethylene-aluminum-polypropylene retort pouches (6" X 9")  in  a  (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 (Section 1).  volumetric flask for  extraction  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). time were heated for 30 min. foil.  Four samples at a  Each sample was covered with aluminum  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. this temperature was recorded for each sample.  The time to reach  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. analytical balance (Mettler Instrumente.  Using a Mettler M3  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,  Toronto,  Ontario)  -  2.3  HgO (Matheson  g of  a  K S0 2  A  (BDH Chemicals,  Coleman and Bell Manufacturing  -48-  Chemists, Norwood, OH) mixture (190:4, w/w) was added, followed by 2.3 mL of concentrated H S0^. 2  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 a l l 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. temperature, water.  After cooling to room  the digest was diluted to 25 mL with distilled deionized  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^ i 2  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^ to the drip and percent loss. 2  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 f i t 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 used to perform multiple comparisons among means.  (Zar, 1984) was  -50-  IV. RESULTS AND DISCUSSION 1. PRELIMINARY EXPERIMENTS - performance characteristics of the assay Preliminary  experiments  were done to  determine  Quantaphase B-^ radioassay kit (Bio-Rad Laboratories), determination of vitamin B^  2  whether  the  designed for the  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  saturation analysis, first described by Ekins in 1960.  is  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 factor, agent  57  Co  B^ . 2  The mixture  a glycoprotein, for  is  then  incubated  with  intrinsic  which serves as a non-discriminative binding  both labelled and unlabelled cyanocobalamin.  During  the  incubation process,  labelled and unlabelled cyanocobalamin compete for  binding  the  sites  concentrations.  on  intrinsic  factor  Non-labelled vitamin B^  2  on  basis  competitively  binding of labelled B-^ to the binding sites. 2  the  of  their  inhibits the  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 a l . (1982), using the Quanta Count II test kit  from  Bio-Rad  Laboratories,  reported  93.9-103.3 % from 8 different products.  recoveries  ranging  from  Bio-Rad Laboratories reports  recovery levels of 103-109 % from blood using the Quantaphase B  12  kit-  Since both study 1 and 2 were carried out under conditions to minimize microbial contamination, it appeared that the apparent excess of vitamin resulting  B^  recovered  from the aqueous  samples  from incomplete binding of labelled B ,  was an ?  artifact  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  186  -  2  125  203  162.4  3  250  467  186.8  4  500  882  176.4  5  1000  1530  6  2000  _ 5  1  2  3  3  A  Mean 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, a l l off standard curve. 6. mean + SE of mean.  153.0  169.9 + 7.7  6  -53Table VIII. Recovery study cyanocobalamin B^2 added Sample (pg/mL)  of different concentrations of added to meat extracts. Expected Recovered % Recovery Value (pg/mL) Value (pg/mL)-'-  Meat 1  0  -  721  -  1  300  1021  1026  100.5  1  500  1221  1256  102.9  1  1000  1721  1807  104.9  0  -  824  -  2  300  1124  1111  98.8  2  500  1324  1370  103.5  2  1000  1824  1976  108.3  Meat 2  Mean 1. mean of 4 assays. 2. mean + SE of mean.  103.2 + 1.4  2  -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^ had been added. 2  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 a l . (1969), a l l observed an increase  in the binding efficiency  of the intrinsic  presence of human serum albumin solution.  factor  in the  Raven et a l . (1968), found an  average increase of 23.54 % in the binding of labelled B^ to eleven 2  intrinsic factor solutions after a 5 % solution of B-^ deficient serum 2  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. in Methods Section 2.1.2.  This was prepared as described  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  researchers.  Casey  et  al.  (1982),  reported  using a similar  by other  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,  controls to test for interassay variability.  samples were run as  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 %. variability  for the kit throughout  the study  The mean interassay 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  manufacturer, however, it is not unusual for RID assays.  by the  Dawson et a l .  (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 2  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  1. Mean of 2 assays ( each extract assayed in duplicate).  -58-  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  difficult to determine a trend. data collected  collected  is  such that  it  is  As shown in Figure 5, the means of the  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 i f 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  radiation in the 1300  was  observed.  However,  further  watt oven resulted in an increase  exposure  to  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 buffer solution A after exposure to varying periods of radiation in a 625 watt domestic microwave oven. i n  Total time of exposure (min)  12 retention- (pg/mL)  B  1  Bio retention (%)  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.  -61-  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 exposure (min)  B12 retention (pg/mL)  0 (control)  1454  +  74  15  1263  +  112  86.6  +  3.8  30  1302  +  93  89.5  +  3.5  45  1452  +  55  100.5  +  6.6  60  1391  +  50  96.1  +  a  a  a  B i o retention (%)  2  -  a  a  1  5.5  1. Mean of 3 samples, each extracted in duplicate and each extract assayed in duplicate. 2. Vitamin B^ percent retentions were calculated from time zero. 2  a. Means in a column followed by the same letter are not significantly different as determined by Student-NewmanKuels multiple range test.  -62-  Table XIII. Retention of Vitamin B i in buffer solution A after exposure to varying periods of microwave radiation in a 1300 watt microwave oven. 2  Total time o f B 1 2 r e t e n t i o n B i o retentionexposure (min) (pg/mL) {% . ) 1  a  -  0 (control)  188.9  + 23.3  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 StudentNewman-Kuels multiple range test.  -63-  Table XIV. Retention of Vitamin B12 i n buffer s o l u t i o n B a f t e r exposure t o varying periods of r a d i a t i o n i n a 1300 watt microwave oven. Total time of exposure (min)  B12 retention(pg/mL)  0 (control)  1270  + 62  15  1230  + 114  30  1407  + 127  45  1325  + 85  60  1227  + l l l  1  Bio r e t e n t i o n (%)  -  a  96.6  a  a  a  a  + 5.9  110.3  + 4.9  104.2  + 2.1  96.3  + 5.1  1. Mean of three samples, each extracted i n duplicate and each extract assayed i n d u p l i c a t e . 2. Vitamin B12 percent retentions c a l c u l a t e d 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 f f e r e n t as determined by the StudentNewman-Kuels m u l t i p l e range t e s t .  160 150  —1—j  1——i— = ~t =r —  140  1  1  0* ralign of v a r i a t i o n i n assay  130  pg B12/mL  120 110 100 0 15 Time  a) 625 watt microwave  30 45 (minutes)  60  210 200 8% range oT v a r i a t i o n i n assay  190 180 170 pg B12/mL  1  6  0  150 140 130 120 110 100 0 15 b) 1300 watt microwave  F i g u r e 5.  Time  30 45 (minutes)  60  Retention of vitamin B i n buffer solution A after e x p o s u r e t o 625 and 1300 w a t t s o f 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 _.  1500  8% range of v a r i a t i o n i n assay 1400  1400 ••  1300  1300 --  ••  8% range of v a r i a t i o n i n assay  pg B12/mL  pg B12/inL 1200  ••  1200 -•  1100  -•  1100  1000  1000  ..  0 15 a) 625 watt microwave  F i g u r e 6.  Time  30 45 (minutes)  15  60 b) 1300 watt microwave  30 Time  45  60  (minutes)  Retention 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 t o 625 and 1300 w a t t s microwave r a d i a t i o n f o r 15 minute time p e r i o d s .  of  -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  concentrations microwave  was  not  detected  between  the  vitamin  of the samples after different lengths of exposure  radiation.  The ANOVA also  indicated that  the  to  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<^ 2ks  2  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  -67sample size tables  (Zar, 1984), where power equals 1 - ^>  probability of committing a type II error).  = the  The power indicates the  probability of rejecting a null hypothesis when i t 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. the sensitivity of the analysis).  (See Appendix A for ANOVA tables and 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 a l . (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. chemical  changes  He concluded that microwave radiation does not induce 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" x l/r\ 4  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^ in buffer solutions A and B after heating to various 2  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 i t 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 a l l 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 8 % range of variability of the assay.  The means are a l l within 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  questionable since samples heated to 70 *C had a  is  somewhat  retention to 98.82 +_  3.61 %, an increase relative to those at 55, 60 and 65*C. heated in the 1300 watt oven showed an opposite trend.  Solution B, 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 a l l 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-^ i ° 2  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).  -72Table 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 (•C)  B^2 retention-'12 retention^ (absolute, pg/mL) (%)  Control  218.2 + 11.6  50 55 60 65 70  209.3 214.1 220.2 214.6 214.7  + + + + +  15.2 10.3 6.8 16.0 16.3  50 55 60 65 70  212.6 193.2 184.8 189.0 215.7  + + + + +  1.8 11.2 8.2 21.5 7.9  Convection 50 55 60 65 70  207.8 200.6 216.2 189.9 179.0  + + + + +  4.0 9.3 24.7 7.9 6.8  Microwave (625)  Microwave (1300)  B  a  a a  a a a  a a  a a  a  a a a a a  95.4 98.1 100.9 98.3 98.4  + + + + +  7.0 4.7 3.1 7.3 7.5  97.4 88.5 84.7 86.6 98.8  + + + + +  0.5 5.1 3.8 9.9 3.6  95.2 91.9 99.0 87.0 82.4  + + + + +  1.8 4.3 11.3 3.6 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 StudentNewman-Kuels multiple range test.  -73Table 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 (•C)  12 retention E$12 retention (absolute, pg/mL) (56)  B  1  -  Control  1236.9 + 95.6 a  Microwave (625)  50 55 60 65 70  1211.1 1259.1 1224.1 1220.7 1213.5  + + + + +  38.2 13.8 19.7 32.8 19.9  a 97.9 + 3.1 a 101.8 + 1.1 a 99.0 + 1.6 a 98.7 + 2.7 a 98.1 + 1.6  Microwave (1300)  50 55 60 65 70  1212.8 1257.0 1277.3 1277.4 1252.6  + + + + +  41.8 19.2 30.9 57.2 11.2  a a a a a  + + + + +  3.4 1.6 2.5 4.6 0.9  Convection 50 55 60 65 70  1236.1 1198.2 1220.9 1254.5 1240.4  + + + + +  17.2 47.5 29.1 52.9 48.0  a 99.9 + a 96.8 + a 98.7 + a 101.4 + a 100.3 +  1.4 3.8 2.4 4.3 3.9  98.0 101.6 103.3 103.3 101.2  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 StudentNewman-Kuels multiple range test.  240 230 4  -r  220 .. pg mL  B12/  j 8 % range of v a r i a t i o n i n assay  210 -• 200  190 -• 180 -• 170 -160 •• 0 C o n t r o l 50  55  60  65  70  C o n t r o l 50  55  60  65  70  C o n t r o l 50  55  60  65  70  Temperature I*C) a) 625 watt  F i g u r e 7.  microwave  b) 1300 watt  microwave  c) C o n v e c t i o n oven  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 h e a t i n g t o 50-70"C i n a 625 watt microwave, a 1300 watt microwave and a c o n v e c t i o n oven s e t a t 180"C.  1360 1340 1320 1300 1280 •• 1260  pg B /mL 12  1 2 4 0  I 8% range of v a r i a t i o n i n assay  1220 • 1200 .. 1180 •• 1160 1140 1120 •1100 0 C o n t r o l 50  55  60  a) 625 watt  microwave  65  70  C o n t r o l 50  55  60  Temperature(  F i g u r e 8.  b) 1300 watt  65  70  C o n t r o l 50  55  60  65  70  C)  microwave  c) C o n v e c t i o n oven  Retention of vitamin B i n b u f f e r s o l u t i o n B a f t e r h e a t i n g t o 50-70"C i n a 625 watt microwave, a 1300 watt microwave and a c o n v e c t i o n oven s e t a t 180'c.  -76The 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. within the food.  With microwave heating,  radiation  is deposited  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. great  variability  standardized  cooking procedures  (Lorenz, 1976). Johnson  in biological materials  (1970),  This is due to the  and failure  to establish  for foods used in research  studies  The one study done in buffer solutions by Van Zante and concluded  that  there  was l i t t l e  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. T e m p e r a t u r e 6 2 5 watt 1 3 0 0 w a t t C o n v e c t i o n oven microwave microwave (180-C, nominal) 1  1  1  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 ^ . designed  Experiment 3 was  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. Laboratories), pg/mL.  According to the manufacturers (Bio-Rad  the minimum concentration detectable  However,  Rothenberg  (1968)  extrapolating a standard curve.  reported  by the kit is 20  errors  of 25 % when  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  significant.  controls.  This  increase,  however,  was  not  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. samples from Animal 1,  The  reheated in the convection oven, appeared to  retain only 82.A +_ 2.1 % of the origional B^ content. 2  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  determined by Kjeldahl analysis, are shown in Table XIX.  conversion,  as  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  observed after heating, ranging from 1.62 to 9.64 %.  in B^  2  w a s  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).  -32Table 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  1  Control  0.A27 + 0.035  la  1  625  0.465 + 0.035  la  108.95 + 8.37  1  Convection  0.352 + 0.009  2b  82.40 + 2.10  1  1300  0.444 + 0.023  2  Control  1.556 + 0.087  2  625  1.669 + 0.043  2  Convection  1.722 + 0.086  3c  110.66 + 5.53  2  1300  1.779 + 0.038  3c  114.38 + 2.46  ug/100 q  la  3c  3c  (%)*»  -  103.92 + 5.34  107.30 + 2.81  1. Mean + SE of themean, 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 StudentNewman-Kuels multiple range test.  -83-  0.5  0.45 .  8% range o f variation in assay  ug B12/ 100 g sample 0.40 .  0.35--  0.3  ..  Control  625 W  Convection  1300 W  Treatment  F i g u r e 9.  Vitamin B r e t e n t i o n (ug/100 g) o f samples from Animal 1 r e h e a t e d t o 70*C i n a 625 watt microwave oven, a 1300 watt microwave oven and a c o n v e c t i o n oven s e t a t 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  C o n v e c t i o n 1300 W  Treatment  F i g u r e 10.  Vitamin B r e t e n t i o n (ug/100 g) o f samples from Animal 2 r e h e a t e d t o 70"C i n a 625 watt microwave oven, a 1300 watt microwave oven and a c o n v e c t i o n oven s e t a t 180"c.  -85-  Table XIX. Nitrogen content (%) of meat and drip from meat samples heated in three oven types. Oven  % N  Meat  Drip  -  Controls  11.300 + 0.499  625  12.028 + 0.156  7.617 + 0.257  Convection  12.133 + 0.145  6.966 + 0.171  1300  12.162 + 0.079  7.064 + 0.271  1  1  2  1. Mean + SE of the mean, n=6. 2. Mean + SE of the mean, n=12. 3. Mean + SE of the mean, n=9.  3  1  2  3  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  12 retention, (absolute), meat and drip B  B]o retention, (%), meat and drip  12 retention (absolute), meat ug/100 g N  B 12 ] o retention  -  -  B  1  Control  3.781 + 0.314la  -  1  625  3.934 + 0.312  104.1  1  Convection  3.009 +_ 0.059  1  1300  3.780 + 0.192i  2  Control  13.77 + 0.77 2c  2  625  14.14 + 0.39  2 c  101.6 + 2.5  13.75 + 0.44 2c  2  Convection  14.93 + 0.73  2 c  108.4 + 5.3  13.57  2  1300  15.10 + 0.22 2c  109.6 + 1.6  14.14 + 0.23 2c  la  3b  a  8.3  3.741 + 0.244  79.6 + 1.6  2.855 + 0.129  100.0 + 5.1  3.590 + 0.2l5  +  -  (%), meat  la  95.4 + 1.3  3b  94.8 + 2.4  la  -  95.0 + 0.9  + 0.69 2c  97.1  +  0.9  90.8 + 1.5 93.7  +  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.  0.5  Control  Meat + Drip  Meat  Control  Meat + Drip  Meat  Control  Meat + Drip  Meat  Treatment a) 625 watt microwave  F i g u r e 11.  b) Convection oven  c) 1300 watt microwave  Vitamin B r e t e n t i o n (ug/100 g N) o f samples from A n i m a l 1 reheated to 70*C i n a 625 watt microwave, a 1300 watt microwave, and a c o n v e c t i o n oven s e t a t 180"C.  15.0  14.0 8% range of v a r i a t i o n i n assay  ug B12/ 100 g N  13.0  12.6 0 Control  Meat + Drip  Meat  a) 625 watt microwave F i g u r e 12.  Control  Meat + Drip Treatment  Meat  b) C o n v e c t i o n oven  Control  Meat + Drip  Meat  c) 1300 w a t t microwave  Vitamin B r e t e n t i o n (ug/100 g N) o f samples from Animal 2 reheated to 70"C a 625 watt microwave, a 1300 watt microwave and a c o n v e c t i o n oven s e t a t 180*C.  l  CO  co i  -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  In order to reproduce conditions that would be found  a hospital catering  samples.  system,  the vitamin was extracted  from hot  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  12  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 amount of drip. B  12  and  the  12  to the drip and at the same time, lost the least  Samples heated in the convection oven lost the most most  drip.  The  significantly less (p<0.05) B  12  625  watt  oven  appeared  to  lose  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-^ appears to be released from the heated meat into the drip, 2  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^ lost 2  to the drip from the heated samples appears to be a function of the amount of drip released.  -91-  Table XXI. Contribution of drip from heated meat samples to the total B 1 2 content and total sample weight. Cow  Oven  Contribution of drip to total Bio (%)  1  625  A. 605 + 1.301  a  A. 307 + 1.117  1  Convection^  6.638 + 2.258  a  8.350 + 1.1A8  1  1300  5.03A + 0.876  8.335 + O.AA7  2  625  2.850 + 0.8A9  A. 667 + 0.913  2  Convection  9.197 + l.A9A  2  1300  6.305 + 0.5A5  1  1  a  b  2  2  2  a  a  Contribution of drip to total weight (%) ab  3  a  d  11.865 + 1.855  a  9.907 + 0.806  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 StudentNewman-Kuels multiple range test.  3  -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 a l . (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. et  a l . (1968),  speculated that the increase  denaturation resulting from internal heating.  Carpenter  may be due to muscle Apgar et a l . (1959), and  Kylen (1964) suggested that the increase in moisture loss may be a result of the increased post-cooking temperature cooking.  rise observed in microwave  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 a l . (1982), reported greater  weight losses from convection reheated beef loaf than from microwave reheated  meat,  but this  evaporation losses.  loss  was not broken down into  drip and  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_ a l . (1971),  observed lower drip losses from turkey reheated in a domestic microwave than in a conventional oven. shorter  This was speculated to be due to the  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 a l . (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  respectively.  times  were  30  minutes  and 49.58  +_ 1.82  seconds,  The loss of drip probably results from the denaturation of  proteins, their subsequent insolubilization and decrease in water binding capacity. temperature.  The extent  of denaturation  is a  function of time and  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,  related to microwave absorption. oven would result  that water  binding is  The increase in power in a microwave  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. suggestion by Ruyack and Paul (1972),  The  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  procedures.  (p <C 0.05)  loss  to be detected  using  statistical  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 microbiological  is  little  evidence  to  believe  that  the  AOAC  assays are any better than RID assays in terms of  variability and sensitivity.  Adams et a l . (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 a l . (1961) reported an assay variability of 9.33 % using L.  leichmanni.  coefficients gracilis.  Mollin et a l . (1976) documented researchers reporting  of variation ranging from 13.5 to 25 % using Euglena  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 a l l 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 mean variation of 5.01  %.  % and 16.11  %.  Solution B had a  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 i f losses of 20 % of the original vitamin B samples and frozen,  12  content occurred when buffer  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.  -97Table XXII. Number of treatment replications needed to detect a significant difference of 20 % (p<0.05). Experiment  Variable  Replications Theoretical Actual  Time (A)  Treatments Oven  11 1  3 3  Time (B)  Treatments Ovens  3 2  3 3  Temperature (A)  Treatments Temperature Ovens  12 2 2  3 3 3  Temperature (B)  Treatments Temperature Ovens  3 1 1  3 3 3  Animal (1)  Treatments Oven Drip  17 3 A  A A A  Animal (2)  Treatments Oven Drip  6 3 A  6 6 6  -98-  V. CONCLUSIONS The objectives of this study were to determine whether vitamin B^ in buffer solutions and meat samples was destroyed in microwave and 2  convection ovens. Microwave radiation, at intensities of 625 watts and 1300 watts, does not cause a significant destruction of vitamin B-^. variability  existed  in the data,  vitamin B^  2  Although some  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  however, this loss was not significant.  vitamin B-^ to 2  the drip,  The percent of total B^ lost 2  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 limitations  of  the  information obtained  study was fulfilled, must  be  recognized.  the 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 i f  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.  -100-  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. Abstract, 34:661.  Nutr.  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. Apgar, J . , Cox, N., Downey, I. and Fenton, electronically. J. Amer. Diet. Assoc., 35:1260.  F. 1959. Cooking pork  Baldwin, R., Korschgen, B., Russel, M. and Mabel, L. 1976. Proximate analysis, free amino acids, vitamin and mineral content of microwave cooked meat. 3. Food Sci., 41:762. Bartilucci, A. and Foss, N.E. 1954. Cyanocobalamin I: A study of the stability of cyanocobalamin and ascorbic acid in liquid formulations. J. Amer. Pharm. Assoc. S c i . , 43:159. Beck, R. 1979. Comparison of two radioassay methods for cyanocobalamin in seafoods. J. Food Sci., 44:1077. Bender, A. 1960. Nutritive effects of food processing. Sci., 13:6. Bender, A. 1975. Nutritional value of meat. London, p.447.  Rev. Nutr. Food  In Meat.  Butterworths,  Bender, A. 1978. Vitamins. In Food Processing and Nutrition. Press, San Francisco, p.27.  Academic  Bennink, M. and Ono, K .1982. Vitamin B12, E and D content of raw and cooked beef. J. Food. S c i . , 47:1786. Bowers, 3. and Fryer, .1972. Thiamin and riboflavin in cooked and frozen reheated turkey. J. Amer. Diet Assoc. Bowers, 3., Fryer, B. and Engler, P. 1974. Vitamin B6 in turkey breast muscles cooked in microwave and conventional ovens. Poultry Sci., 53:844. Bowers, 3., Fryer, B. and Engler, P. 1974. Vitamin B6 in pork muscle cooked in microwave and conventional ovens. J. Food Sci., 39:426. Branion, H . , Roberts, J . , Cameron, C. and McCready, A. 1947. The loss of ascorbic acid in the preparation of old and freshly harvested potatoes. 3. Amer. Diet. Assoc., 23:414.  -101-  Campbell, C , Lin, T. and Proctor, B. 1958. Microwave versus conventional cooking. 1. Reduced and total ascorbic acid in vegetables. J. Amer. Diet. Assoc., 34:365. Carpenter, S. 1968. Tenderness and cooking loss of beef and pork. Amer. Diet. Assoc., 58:38.  J.  Casey, P., Speckman, K., Ebert, F. and Hobbs, W. 1982. Radioisotope dilution technique for determination of vitamin Bj^ in foods. J . Food Sci. Cipra, J . , Bowers, J. and Hooper, A. 1971. Precooking and reheating of turkey. J. Amer. Diet. Assoc., 53:353. Cross, G. and Fung, D. 1982. The effect of microwaves on the nutrient value of foods. Crit. Rev. Food Sci. Nutr., April, p.355. Curnutte, B. 1980. Priciples Protection, 43(8):618.  of microwave  radiation.  J.  of Food  Dahl, C. and Matthews, M. 1980. Effect of microwave heating in cook/chill food service systems. J . Amer Diet Assoc., 77:289. Dahl-Sawyer, C , Jen, J . and Huang, P. 1982. Cook/chill foodservice systems with conduction, convection and microwave reheat subsystems. Nutrient retention in beef loaf, potatoes, and peas. J . Food S c i . , 47:1089. Dawson, D., Delamore, I., Fish, D., Flaherty, T . , Gowenlock, A., Hunt, L., Hyde, K., Maclver, J . , Thornton, J . and Waters, H. 1980. An evaluation of commercial radioisotope methods for the determination of folate and vitamin B12. J . Clin. Pathol., 33:234. Drew, F., Rhea, K. and Carpenter, Z. 1980. Cooking at variable microwave power levels. J . Amer. Diet. Assoc., 77:455. Ensminger, M. 1976. Beef Cattle Publishers, Inc., II.  Science.  The Interstate Printers and  Eddy, T . , Nicholson, A. and Wheeler, E. 1968. Pre-cooked frozen foods: the effects of heating on vitamin C. Nutrition, 22:122. Eheart, M. and Gott, C. 1964. Conventional and microwave cooking of vegetables. J . Amer. Diet. Assoc., 44:116. Ekins, R.P., 1960. The estimation of thyroxine in human plasma by an electrophoretic technique. Clin. Chim Acta, 5:453. Erikson, S.E. and Boyden, R.E., 1947. Kentucky Agricultural Experiment Stations Bulletin 504 (see Harris and von Loesecke, 1960).  -102-  Faden, V., Huston, J. Munson, P. and Robard, D. 1980. RIAPPROG Logit-log radioimmunoassay data processing, Biomedical Computing Technology Information Centre, Nashville, TN. Farquharson, J.and Adams, J. 1976. The forms of vitamin B12 in foods. Br. J. Nutr., 36:127. Feller, B. and Macek, T. 1955. Effect of thiamine hydrochloride on the stability of solutions of crystalline vitamin B12. 3. Amer. Pharm. Assoc. S c i . , 44:662. Glew, G. 1970. Precooked frozen food in hospital catering. Society of Health Journal, 90(3):139.  The Royal  Goldblith, S., Tannenbaum, S. and Wang, D. 1968. Thermal and 2450 Mhz microwave energy effect on the destruction of thiamine. Food Technol., 22:1266. Grasbeck, R. and Salonen, E. 1976. Vitamin B12. 2:193.  Prog. Food Nutr. S c i . ,  Guthrie, H. 1979. Water soluble vitamins. In Introductory Nutrition. C.V. Mosby Company, Toronto, p.270. Hall, K. and Lin, C. 1981. Effect of cooking rates in electic or microwave ovens on cooking losses and retention of thiamine in broilers. J. Food Sci., 46:1292. Hamm, R. and Deatherage, F. 1960. Changes in hydration, solubility and charges of muscle proteins during heating of meat. Food Research, 25:587. Harris, R. 1960. Effects of large scale preparation on nutrients in foods of plant origin. In Nutritional Evaluation of Food Processing, ed. Harris, R. and Von Loesecke, H. John Wiley and Sons, New York. p.418. Harris, R. and Karmas, E. 1977. Effects of food service practices on nutrients. In Nutritional Evaluation of Food Processing. Avi Publishing Company, Inc., Connecticut, p.463. Harris, R.S. and von Loescke, H. 1960. Nutritional Evaluation of Food Processing. John Wiley and Sons, Inc., New York. Hartley, F., Stross, P. and Stuckey, R. 1950. Some pharmaceutical aspects of vitamin B12. J. Pharm. Pharmacol., 2:648. Headley, M. and Jacobson, M. 1960. Electronic and conventional cooking of lamb roasts. Cooking losses and palatability. J. Amer. Diet. Assoc., 36:337.  -103-  Hillman, R., Oakes, M. and Finholt, C. 1969. Hemoglobin coated charcoal radioassay for serum B12: a simple modification to improve intrinsic factor reliability. Blood, 34:3. Hoppner, K., Lampi, B. and Smith, D. 1978. An appraisal of the daily intakes of vitamin B12, pantothenic acid and biotin from a composite Canadian diet. Can. Inst. Food Sci. Technol., 11(2):71. Kahn, L. and Livingston, G. 1970. Effect of heating methods on thiamine retention in fresh or frozen prepared foods. J. Food Sci., 35:358. Klein, B. and Van Duyne, F. 1979. Effects of food preparation practices on nutrient content and food quality. F.S.T.A. Comput. Search, File 51. Korschgen, B. and Baldwin, R. 1978. Moist heat microwave and conventional cooking of round roasts of beef. J. Microwave Power, 13:257. Kylen, A., Charles, V., McGrath, B., Schleter, Duyne, F. 1961. Microwave cooking of vegetables. 39:321.  J . , West, L. and Van J. Amer. Diet. Assoc.,  Kylen, A., McGrath, B., Hallmark, E. and Van Duyne, F. 1964. Microwave and conventional cooking of beef. J. Amer. Diet. Assoc., 39:139. Lachance, P., Ranadivic, A. and Matos, J. 1973. Symposium: Effects of processing, storage, and handling on nutrient retention in foods. Food Technol., 27:36. Lawrie, R. 1974. Meat Science. Pergamon Press. N.Y., p.350 Le, C D . 1978. Centre, UBC.  UBC MFAV. Analysis of variance/covariance. Computing  Lehninger, A. 1982. Principles of Biochemistry. Worth Publishers Inc., New York. Lorenz, K. 1976. Microwave heating of foods-changes in nutrient and chemical composition. Crit. Rev. Food Sci. Nutr., p.339. Macek, T. and Feller, K. 1952. Crystalline vitamin B12 in pharmaceutical preparations. J. Amer. Pharm. Assoc., 41:285. Marshall, N. 1960. Electronic cookery of top rounds of beef. Econ., 52:31.  J. Home  Newmark, P. and Patel, N. 1971. The adsorption of intrinsic factor concentrate to glass: occurrence and prevention with reference to radioisotope dilution assays of vitamin B12. Blood, 38(4):524.  -104-  Noble, T. and Gomez, L. 1962. Vitamin retention electronically. J. Amer. Diet. Assoc., 41:217. Piatt, B., Eddy, T, and Pellett, University Press.  in meat cooked  P. 1963. Food in Hospitals. Oxford  Raven, J . , Robson, M., Walker, P. and Barkham, P. 1968. The effect of cyanide, serum and other factors on the assay of vitamin B12 by a radioisotope method using 57Co B12, intrinsic factor and coated charcoal. Guy's Hospital Reports, 117:89. Rosen, C.G. 1972. Effects of microwaves on food and related materials. Food Technol., 26(7):36. Rothenberg, S. 1963. Radioassay of serum vitamin B12 by quantitating the competition between 57CoB12 and unlabeled B12 for the binding sites of intrinsic factor. J. Clin. Invest., 42(9):1391. Rothenberg, S. 1968. A radioassay for serum B12 using unsaturated Transcobalamin I as the B12 binding protein. Blood, 31(1):44. Ruyack, D.F. and Paul. 1972. Conventional and microwave heating of beef:use of plastic wrap. Home'Econ. Res. J . , 1:98. Stevens, H. and Fenton, F. 1951. Dielectric versus stewpan cookery. Comparison of palatability and vitamin retention in frozen peas. J. Amer. Diet. Assoc., 27:32. Thomas, M., Brenner, S., Eaton, A. and Craig, V. 1949. kEffect of electronic cookery on nutritive value of foods. J. Amer. Diet. Assoc., 25:39. Van Tonder, S., Metz, J. and Green, R. 1975. Tissue vitamin B12 assay by radioisotope dilution technique. Clin. Chem. Acta., 63:285. Van Zante, H. and Johnson, S. 1970. Effect of electronic cookery on thiamine and riboflavin in buffered solutions. J. Amer. Diet. Assoc., 56:133. Wagner. K.H. 1971. On the question of vitamin preservation in food which has been treated according to the Multimet-Multiserv procedure, as compared to the preservation in orthodox thermo containers. Bull CX167 Crown-X, Inc., Cleveland (see Harris and Karmas, 1971). Westerman, B.D. 1948. The Kitchen Reporter, Kelvinator Kitchen, Detroit (see Harris and von Loescke, 1960). Windholz, M. 1976. The Merck Index. Merck and Co., Inc., New Jersey, USA. p.9670.  105-  Wing, R. and Alexander, J. 1972. Effect of microwave heating on vitamin B6 retention in chicken. J. Am Diet. Assoc., 61:661. Zar, J. 1984. Biostatistical Cliffs, New Jersey.  Analysis. Prentice-Hall,  Inc.,  Englewood  Ziprin, Y. and Carlin, S. 1976. Microwave and conventional cooking in relation to quality and nutrient value of beef and beef-soy loaves. 3. Food Sci., 44:4.  APPENDIX A STATISTICAL ANALYSIS 1. TIME EXPERIMENTS SOURCE  DF  SUM SQ  MEAN SQ  F-VALUE  PROBABILITY  Time Oven Cone Ti X Ov Ti X Cn Ov X Cn T X0 X C Error Total  4 1 1 A A 1 A AO 59  36790.0 533A.6 0.20A72E+08 AA805.0 38097 56657 39819 0.5A066E+06 0.2123AE+08  9197.A 533A.6 0.20A72E+08 11201.0 952A.3 56656 995A.7 13517  0.680A5 0.39A67 1514.6 0.82870 0.70A6A A.1917 0.736A8  0.60959 0.53343 0.83267E-16 0.51486 0.59346 0.47230E-01 0.57261  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  Treat 1 Cvstrt 1 Temp 1 Oven 1 Te X Ov Error Total  15 1 4 2 8 32 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  ERROR  F-VALUE  PROB  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  Treat 1 Cvstrt 1 Temp 1 Oven 1 Te X Ov Error Total  15 1 4 2 8 32 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  ERROR  F-VALUE  PROB  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 Error Total  3 12 15  0.039 0.038 0.077  0.013 0.003  4.126  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 Error Total  3 20 23  0.163 0.552 0.715  0.054 0.028  1.96931  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 1 Cvstrt 1 Oven 1 Drip 1 Ov X Drip Error Total  6 1 2 1 2 21 27  5.9615 0.41860 5.3289 0.21395 0.53583E-04 4.3705 10.332  0.99358 0.41860 2.6644 0.21395 0.26792E-04 0.20812  4.7741 2.0114 12.803 1.0280 0.12873E-03  0.32399E-02 0.17079 0.23160E-03 0.32216 0.99987  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 1 Cvstrt 1 Oven 1 Drip 1 Ov X Drip Error Total  6 1 2 1 2 35 41  12.839 1.2924 2.7089 7.4193 1.4189 61.201 74.040  2.1399 1.2924 1.3544 7.4193 0.70947 1.7486  1.2238 0.73913 0.77459 4.2430 0.40574  0.31774 0.39579 0.46863 0.46910E-01 0.66958  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 in 81.43 g of meat. 2) If there are 0.71 ug of B12 i in 5.0g of drip.  n  100 g of meat, then there are 1.32 ug  n  100 g of drip, then there are 0.03 ug  3) If there are 1.35 ug (1.32 + .03) of Bi_ in 86.43 g of sample, then there are 1.56 ug in 100 g of sample. 2  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 the total. o r 2  2  2 5%  o f  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 in 100 g of sample and 11.833 g of N in 100 g of sample, then there are 13.183 ug of B i in 100 g of N. 2  2  

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