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California State University, Northridge CALIFORNIA STATE UNIVERSITY, NORTHRIDGE VITAMIN B-12 CONTENT OF BEERS A thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Home Economics by Mark Stuart Meskin May, 1983 The Thesis of Mark Stuart Meskin is approved; Tung-Shan Chen, Ph.D. Christine Hamilton Smith, Ph.D. Chairperson California State University, Northridge ii DEDICATION For my mother, whom I often miss, DIANE BANGHART MESKIN 1933-1981 She taught me the joy of learning and was my greatest teacher iii ACKNOWLEDGMENTS The completion of this thesis represents only a small part of a much longer process of growth and maturation. I have not only acquired a competent working knowledge of basic nutrition, I have also developed the skills and self­ confidence to teach nutrition and to exchange ideas with experts in my field. The completion of this degree could not have happened without excellent teachers and without the care, love, cooperation and encouragement of my friends and family. I must begin by thanking my wife, Ilana. The thesis itself could not have been completed without her excellent typing and editorial skills but her influence was much more profound than the physical product she helped to produce. She gave me the love, freedom and time to complete my de­ gree in my own way and at my own pace. She was my friend, confidante, cheerleader and therapist. I plan to colla­ borate with her for many years to come. I am deeply ~ndebted and grateful to Dr. Christine H. Smith, my committee chairperson, my teacher and my friend. She has been involved with my education from my first day at CSUN through the completion of this thesis. She is re­ sponsiblefor much of what I know about nutrition and I hope iv to continue to learn from her in the future. I thank her for her wisdom, keen insight and the many precious hours of time she has given me. I would like to thank Dr. Tung-Shan Chen for his ad­ vice and participation in this study. My laboratory skills were developed in his chemistry courses and the environment he created in the food chemistry laboratory made graduate study enjoyable. The combination of his keen critical abilities and his knowledge of microbiolog­ ical vitamin assays proved invaluable in discovering flaws and solving problems in this study. Special thanks go to my friend Arlene Kirsch who constantly shared her time and knowledge with me in the laboratory. She helped solve many of the critical day to day problems that emerged during the hours of laboratory work involved in this study. I am grateful to Dr. Margaret Holzer for reading this thesis and for serving as a member of my committee. Finally, I would like to thank my father, Mort Meskin, and my brother, Joel Meskin, for their love, encouragement and unconditional support throughout my education. v TABLE OF CONTENTS APPROVAL PAGE • • . .. • . • . • . • . • . • . • • . • . • . • • • • • • • • • . ii DEDICATION • • •.. • • • • • . • . • . • . • • • . • . • . • .. • . • • • • • iii ACKNOWLEDGMENTS . • • • . • • • . • . • . • • • . • • . • . • • • . • . • • • i v LIST OF TABLES • . • • • • • • . • . • . • • • . • . • • • . • . • . • • • • • . • • ix LIST OF FIGURES ••••.•.•••••.•..•.••.•.•••••.•..•• xi ABSTRACT •• ·- • • • • • . • • • • • .• • • • • • • • . • . • • • • • • • • • • • • • • • • xi i CHAPTER 1. INTRODUCTION .••..•.•••..••.••.•••..••• 1 CHAPTER 2. REVIEW OF LITERATURE ••••...••.••.•••• 7 Vitamin B-12 (Cyanocobalamin) 7 History . .. ~ . 7 Structure and Terminology •.••.•.•••••.• 8 Chemistry . 11 Metabolism . ... .. 12 Vitamin B-12 Deficiency: Causes and Occurrence • . • . • • . • • . • . • • • • • . • • • 14 Recommended Dietary Intakes •.•••••.••.. 16 Food Sources . 18 Vitamin B-12 Nutriture of Vegans 20 Non-meat Product Sources of Vitamin B-12.................... 22 Human Ability to Store Vitamin B-12 26 High Folacin Intake in Vegans 26 Serum Vitamin B-12 Levels in Vegetarians ••..••.•••••••.••••• 27 Assay Methods • . • • • • • • • • . • • • • • . • . • • • . • • • 28 Beer . 32 vi Historical Background of Beer.......... 32 The Brewing Process •...•••••.•.•••.•••. 33 Types of Beer . • . • . • . • . • • • . • . ..• . • . • 36 Lager Beer •.•.••.••••.•.•.•.•.•.•• 36 Dark Beer •.•.••..••••.••.•.•.••••• 36 Low-Calorie (Light) Beer.......... 37 Ale . • .. • . • . .. 3 7 Malt Liquor ••.. ~.................. 37 Porter . 3 8 Stout . 38 Consumption of Beer.................... 38 Nutritive Value of Beer 39 CHAPTER 3. MATERIALS AND METHODS 44 Materials . 44 Beer Samples .......................... 44 Assay Microorganism.................... 54 Chemical Reagents and Microbiological Media 54 Glassware Maintenance ••..••.•.•••••.••• 54 Equipment . 55 ~~tlloCls • • • • • • • • . • • . • • • • • . • • • • • • . • • • • • • . • • • • • 57 Sample Preparation ••.•••••.•••.•••.•••• 57 Microbiological Assay for Vitamin B-12 • 59 Maintenance of Oahromonas malhamensis ••....•••.•..••.•••• 59 Assay Medium . • . • • • . • . • . • . • . • . • • • 60 Inocul urn . • . • . 61 vii Standard Curve Preparation .••••••• 61 Sample Flask Preparation.......... 62 Sterilization, Inoculation, and Incubation •..•••••.•.•••••• 62 Absorbance Measurement ••.•••.••••• 66 Treatment of Data •.••••••••..•••.•.••.• 67 Standard Curve Construction 67 Calculation of Vitamin B-12 Concentration in Beer Samples 67 Statistical Analysis ••..••••••.••• 67 CHAPTER 4. RESULTS AND DISCUSSION............... 70 Vitamin B-12 Content of Different Brands of Beer Within Each Beer Type ••..••••.••• 70 Domestic Lager Beer 70 Imported Lager Beer 75 Dark Beer .•...•.•....•. ·-·.............. 77 Low-Calorie (Light) Beer ..••.•••••••.•• 77 Ale . • . • . • . • . 8 0 Malt Liquor . • . 8 2 Porter . • . 8 2 Stout . • . 82 Vitamin B-12 Content of Eight Types of Beer • 85 CHAPTER 5. CONCLUSION AND RECOMMENDATIONS ••••••• 91 LITERATURE CITED ..•••...•.•••.•.•.•••.•••.••••••• 95 APPENDICES . 102 viii LIST OF TABLES Table Page 1. Recommended Daily Intake of Vitamin B-12 ••.• 17 2. Some Microorganisms Known to Be Able to Synthesize Vitamin B-12 .........•. 19 3. Common Food Sources of Vitamin B-12 21 4. Non-Meat Product Sources of Vitamin B-12 23 5. Growth Response of Various Assay Micro­ organisms to Clinically Active Molecules ("True B-12") vs. Clinically Inactive Molecules (Analogs With Little or No Activity) ••••••••••.•.••••..•••••..•••••• 31 6. Selected Nutrients in Lager Beer Composition per 355 ml (12 oz.) 41 7. Vitamin B-12 and Alcohol Content of Beers ••• 43 8. Domestic Lager Beers 46 9. Imported Lager Beers . 47 10. Dark Beers.................................. 48 11. Low-Calorie (Light) Beer •••••.•••.••.••.•••• 49 12 . Ale . 50 13. Malt Liquor . 51 14. Porter . 52 15. Stout . 53 16. Protocol of Vitamin B-12 Standard Curve 63 17. Protocol of Vitamin B-12 Assay Flasks for Beer Samples •.•••••.•••••••.•••.••••• 64 18. Vitamin B-12 Content of Domestic Lager Beers •.•••••••.•.•.•.•.••••..•.•••• 71 19. Vitamin B-12 Content of Imported Lager Beers ••..•••.••.••.•••••••.•••.•..• 76 ix Table Page 20. Vitamin B-12 Content of Dark Beers .•..•••••• 78 21. Vitamin B-12 Content of Low-Calorie (Light) Beers •••••.••.••••.••.•.••••••••• 79 22. Vitamin B-12 Content of Ales . 81 23. Vitamin B-12 Contents of Malt Liquors . 83 24. Vitamin B-12 Content of Porters . 84 25. Vitamin B-12 Content of Stouts . 86 26. Average Vitamin B-12 Content of Beers . 88 X LIST OF FIGURES Figure Page 1. Structure of Vitamin B-12 (Cyanocobalamin) • . • . • • • . • • • • • • • . • . • . • • . • . • 9 2. Schematic Outline of Vitamin B-12 Assay Procedure . 58 3. Typical Vitamin B-12 Standard Curve......... 68 . ; xi ABSTRACT VITAMIN B-12 CONTENT OF: ',BEERS by Mark Stuart Meskin Master of Science in Home Economics The vitamin B-12 content of beers regularly available in local retail stores was determined. Eight types and 58 brands of beer were assayed. A microbiological assay em­ ploying the protozoan Ochromonas malhamensis was utilized because it is the most sensitive and specific way to mea­ sure the metabolically and clinically active forms of vi­ tamin B-12 in foods. Vegetable products are not likely sources of vitamin B-12 and researchers have largely ig­ nored the assay of these foods for vitamin B-12. The re­ sults of this study demonstrate that at least one vegetable product, beer, contains vitamin B-12. Small, measurable amounts of vitamin B~l2 were found in all beers assayed. The average values for vitamin B-12 content of beers rang­ ed from a low of 38.3 ng per 355 ml (one 12-ounce bottle) in low-calorie beers to a high of 94.0 ng per 355 ml in xii dark beers. The mean value for all beers assayed was 67.8 ng per 355 ml. A number of individual brands of beer con­ tained at least 100 ng of vitamin B-12 per 355 ml. The data showed that there were significant differences among the various brands of beer in each category but there were not significant differences, in general, among the cate­ gories. These data indicate that the most important fac­ tor in determining the vitamin B-12 content of a beer in this study is the manufacturer and not the actual category in which the beer is found. Animal products are the best sources of vitamin B-12 but for people who do not regular­ ly consume these products, beer could contribute small but significant amounts of vitamin B-12 to their diets as part of a well-balanced diet. xiii Chapter 1 INTRODUCTION Vitamin B-12 or cyanocobalamin is a water soluble B complex vitamin essential for the adequate functioning of all mammalian cells. The dominant forms of vitamin B-12 in mammalian tissues are coenzyme B-12 (5'-deoxyadenosylcobal­
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