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Vitamin B₁₂ and Ascorbic Acid in Kimchi with Propionibacterium

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Vitamin B₁₂ and Ascorbic Acid in Kimchi with Propionibacterium N ABSTRACT OF THE THESISOF SOOKJA LIM no for the degree of DOCTOR OF PHILOSOPHY in Foods and Nutritionpresented on June 2, 1978 Title: VITAMIN B12 AND ASCORBICACID IN KIMCHI WITH PROPIONIBACTERIUM SHEPMANII Redacted for privacy Abstract approved: Vitamin B12 reacheda maximum of 47 ng/100 g during the fermentation at 4°Cof kimchi, a Korean style fermented b Chinese cabbage with saltedshrimp and spices. Inoculation of the kimchi withPropionibacteriurn sherrnanii (ATCC 13673) increased the vitaminproduction to a maximum of 102 ng/100 g at 1 week,of fermentation. Soy flour (0.5%) or beef extract (0.05%), whichwere regarded as protein sources, added to the inoculated kimchifurther increased the vitamin B12 activityto 197 and 203 ng/l00g at 1 week of fermentation. After this point, the vitamin inall of the kirnchi productsdecreased continuously to the end of the 5 weeksof fermentation. After 5 weeks of fermenta- tion, no further decreasewas observed. The change in ascorbicacid content during kimchi fermentation did not differsignificantly between the control and inoculatedkimchi with or withoutsoy flour or beef extract. The fresh, unfermented kirnchi contained 16.8 mg/l0O g of ascorbic acid. The content of ascorbic acid decreased continuouslyduring the first 5 weeks of fermentation. The correlation coefficient,r, for the ascorbic acid levels and thefermentation time was signi- ficant at the 1% level. It was found that lactobacilliare important in kimchi fermentation. By 3 weeks of fermentation, the lactobacilli (2.3 x i08 per g) constitutedmost of the anaerobic micro- organisms (2.7x108per g). Propionibacterium shermannii, whichwas inoculated into kixnchi, did not increase innumbers but survived dur- ing the fermentation. This species was not found in the control kimchi. Five species of propionibacteriawere identified from the control kimchi, includingP. freuden- reichii, which has been knownto produce vitamin B12. All of the kimchi sampleswere optimally fermented after 3 weeks with a pH of 4.8 and total acidityof 0.35% as lactic acid. In. triangular tests, no signficant differencein flavor was found by 9 Korean panel membersbetween the con- trol and inoculated kirnchi with addedsoy flour or beef extract. Canning of kimchi at the end of 1week of fermentation gives promise in preliminarytests of reducing the loss of vitamin B12. Vitamin B12 and Ascorbic Acid in 1'Cirnchi with Propionibacterium shermanii by Sookja Lim Ro A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed June, 1978 Commencement June, 1979 APPROVED: Redacted for privacy ion esso in charge of major Redacted for privacy ot De5rment of Foods and Nutrition Redacted for privacy Date thesis is presented June 2, 1978 Typed by Deanna L. Cramer for Sockja Lim Ro ACKNOWLEDGEMENTS Sincere gratitude is expressedto Dr. Margy Woodburn, Head of the Departmentof Foods and Nutrition, for her wonderful guidance, good suggestions,and coritinous encouragement during my graduate studiesat O.S.tJ. Special appreciation is givento Dr. William E. Sandine, professor of theDepartment of Microbiology, who allowed me to work in hislaboratory and provided unfailing guidance in the laboratory work. My warm thanks is ex- pressed to the graduate studentsin the Dairy Microbiology laboratory for their givingme a nice and comfortable atmosphere to work and sharinggood knowledges. Special thanks is extended tomy husband, In Kyu, for his sound support, understanding,and, encouragement throughout my study. TABLE OF CONTENTS Page INTRODtJCT ION ..... 1 REVIEWOFLITERATtJRE ............. 4 Historical Background of Vitamin 12 ....... 4 Structure and Stability of VitaminB12...... 5 Microbiological Assay of VitaminB12 inFoods . 13 Microbial Production of VitaminB12 inFoods. 18 EXPERIMENTAL PROCEDUBES................ 25 Preparation of Kimchi Sample........... 25 Inoculation with Vitamin B12-ProducingOrganism 27 Determination of Vitamin B12........... 28 Test Organism................ 28 Calibration of Spectronic 20........ 29 Cyanocobalamin Standard Solutio'i...... 31 Media and Buffer Solutions........ 31 Assay Procedure............... 33 Effect of Ascorbic Acidon Vitamin B12Assay. 35 Correction for Vitamin B12 Analogs........ 37 Determination of Ascorbic Acid........... 37 Microbial Counts................. 38 Identification of Propionibacteria........ 39 pH and Total Acidity Measurements......... 40 Sensory Evaluation.................. 40 Statistical Analysis............... 41 RESULTS AND DISCUSSION................ 42 Development of Assay Method........... 42 Production of Vitamin 12 in Kimchi Fermentation 45 Content of Ascorbic Acid in Kimchi........ 51 Microbial Counts................. 55 Identification of Propionibacteria........ 59 pH and Total Acidity............... 63 Sensory Evaluation................ 67 Additional Studies, Preliminary......... 68 Preservation by Canning........... 68 Commercial Kimchi.............. 71 Vegetarian Kimchi.............. 71 Applications and Further Research......... 72 S Ut4rARY........................ 74 BIBLIOGRAPHY..... .. .............. LIST OF FIIJPES Figure Page 1 Structure of vitamin B12 and5-deoxyadenosyl cobalamin................... 6 2 Calibration of Spectronic 20..... 30 3 Standard curve basedon values of 20 replications.................. 36 4 Content of vitamin B12 in control and inoculated kimchi, withor without soy flour or beef extract, and fermented at 4°C..... 48 5 Ascorbic acid content ofcontrol arid inoculated kimchi, withor without soy flour or beef extract, and fermented at 4°C..... 54 6 Total anaerobic microorganismsand lacto- bacilli per gram of control andkimchi inoculated with P. shermannii andfermented at 4°C..................... 57 7 Total anaerobic microorganismsand lacto- bacilli per gram of kimchiinoculated with P. shermannii and addedsoy flour or beef extract and fermented at 4°C. 58 8 Total number of propionibacteriaper gram of control and inoculated kimchi,with or without soy flour or beef extract,and feriTtented at 4°C................ 62 9 The pH and total acidity of control arid inoculated kimchL withor without soy flour or beef extract, and fermentedat 4°C...................... 66 LIST OF TABLES Table Page 1 Some organisms used for industrial production of vitaminB12..................20 2 Ingredients of kimchi studied and their dis- tribution . ...............25 3 Effects of buffer solutions and heattreat- ments on vitamin B12 extraction of kirnchi . 32 4 Effect of added ascorbic acid and ironon vitamin B12 assay in kimchi...........43 5 Recovery of vitamin B12 added to kimchi samples . 44 6 Effects of blending and steaming before blend- ing of kinchi on vitamin B12content.......44 7 Content of vitamin B12 in control kimchi and kirnchi inoculated with Propionibacterium shermannii, with and without addedsoy flour or beef extract, and fermented at 4°C......46 8 ANOVA table for vitamin B12 production in kimchi. 50 9 Ascorbic acid content of control and inoculated kimchi, with or withoutsoy flour or beef extract, and fermented at 4°C.......... 52 10 Total counts of anaerobic microorganisms and lactobacilli per gram of control and inoculated kimchi, with or withoutsoy flour or beef extract, and fermented at 4°C...... 56 11 Total counts of propionihacteria per gram of control and inoculated kimchi, withor without soy flour or beef extract, and fermentedat4°C................. 60 12 The pH and total acidity of control and inoculated kimchi, with or without soy flour or beef extract, and fermented at 4°C......64 13 Results of sensory evaluation for control and inoculated kirnchI, with added soy flour or beef extract and fermented at40C ....... 67 List of Tables -- continued Table Page 14 Vitamin B12 and ascorbic acid infresh and canned kirnchi ............. 69 15 Vitamin B12 and ascorbic acid incommercial kimchi. S 71 VITAMIN B12 ANDASCORBIC ACID IN KIMCHI WITH PROPIONIBACTERIUMSIIERMANIl INTRODUCTION Vitamin B12 (cyanocobalainin)is known to bea dietary requirement for the growthof humans, a variety ofanimals, and microorganisms. In the human, it has beenshown to be essential for normal blood formation, proteinsynthesis and thus for normal growth, maintenance ofneural function, and for some metabolic processes in which the vitaminacts as a coenzyme (Wagner andFolkers, 1964) Vitamin B12 is essentialin the human dietas evi- denced by the development of pernicious anemia witha de- ficiency of the vitamin. This generallyoccurs due to the inability to absorbthe vitamin. However, subclinical de- ficiency levels ofserum vitamin (45-193 pg/mi with an average value f ill pg/mi) havebeen found in adults who have subsisted on vegetarian diets for extendedperiods (Wokes et al., 1955) . Parenteral administration ofthe vitamin increasedthe levels to normal. Since the vitaminwas first isolated in crystalline form from liver in 1948 by Rickeset al. (1948a) and by Smith, it has been found inmost animal tissues and muscles, milk, and eggs. Itappears to be absent from most of the higher plants,althoughsome plant roots show small amounts which may derive from contaminationwith microorganisms in the soil (tJngley, 1955) . The vitamin is not synthesized by animals, but it is producedby bacterial or fungal fermenta- tion within the ruminants'own digestive tract and then absorbed.. Humans are unable to utilize the intestinally synthesized vitamin. This exclusively microbial production of the vitamin is uniqueamong the known vitamins (Maugh, 1973) Fermented products of soybeanor fish
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