HPLC QUANTIFICATION OF RIBOFLAVIN AND COBALAMIN BY PROBIOTIC BACTERIA ISOLATED FROM FERMENTED MILK (NUNU)

ABSTRACT

Vitamins are important micronutrients that are often precursors to enzymes, which all living cells require to perform biochemical reactions. This work studied the vitamin contents of fermented milk produced by lactic acid bacteria (LAB). Four (4) LAB species were isolated from fermented milk in this study of which were Lactococcus lactis, Streptococcus thermophilus, Pediococcus pentosaceus and Lactobacillus plantarum based on morphological, biochemical and physiological characteristics. The possible production of riboflavin and cobalamin by the LAB isolates was analyzed by High Performance Liquid Chromatography (HPLC) analysis. The LAB species which were isolated from nunu were used as a consortium for milk fermentation to determine the production potency of riboflavin and cobalamin by the LAB isolates. Increase in the vitamin contents of the fermented milk was observed with the concentration of riboflavin increasing from 0.0988mg/ml in unfermented nunu to 0.2553mg/ml after fermentation by the LAB isolates. This means there was 61% increase in concentration of vitamin B2 in fermented milk sample when compared to the concentration of vitamin B2 in unfermented milk. Also, cobalamin content increased from 0.0194mg/ml in unfermented nunu to 0.0403mg/ml after LAB fermentation showing 52% increase in the concentration of B12 in fermented milk when compared to the concentration of vitamin B12 in unfermented milk. Therefore, it can be said that the isolated LAB from Nunu have the potency to produce riboflavin and cobalamin. The ability of these LAB to produce B vitamins could supplant the expensive chemical production of these vitamins to enrich food or be improved for in situ fortification of fermented foods.

TABLE OF CONTENTS

Title page ﾿ i

Certification ﾿ ii

Dedication ﾿ iii

Acknowledgment ﾿ iv

Table of contents ﾿ v

List of tables ﾿ vi

List of Figures ﾿ vii

Abstract ﾿ viii ﾿

CHAPTER 1: INTRODUCTION

1.1 ﾿ Introduction ﾿ 1

1.2 ﾿ Aim and  Objectives ﾿ 2

CHAPTER 2: LITERATURE REVIEW

2.1 ﾿ Definition of Fermentation ﾿ 4

2.1.1    ﾿ Benefits of Fermentation ﾿ 4

2.2 ﾿ Nunu ﾿ 8

2.2.1 ﾿ Nunu Production process ﾿ 9

2.3 ﾿ Vitamins ﾿ 12

2.3.1 ﾿ Water-Soluble Vitamins ﾿ 12

2.3.2 ﾿ Riboflavin (B2) ﾿ 13

2.3.2.1 Riboflavin biosynthesis ﾿ 14

2.3.3 ﾿ Cobalamin (B12) ﾿ 16

2.3.3.1 Cobalamin biosynthesis ﾿ 16

2.4 ﾿ Probiotics ﾿ 23

2.5 ﾿ Lactic acid bacteria ﾿ 25

2.5.1 ﾿ History and Occurrence of LAB ﾿ 27

2.5.2 ﾿ Lactic acid bacteria as Probiotics ﾿ 28

2.6 ﾿ High Performance Liquid Chromatography (HPLC) ﾿ 33

CHAPTER 3: MATERIAL AND METHODS

3.1 ﾿ Study Area ﾿ 35

3.2 ﾿ Collection of Samples ﾿ 35

3.3 ﾿ Culture Media preparation and Sterilization ﾿ 35

3.4 ﾿ Preparation of the Nunu Samples ﾿ 35

3.5 ﾿ Inoculation of Samples ﾿ 36

3.6 ﾿ Colony count of isolates ﾿ 36

3.7 ﾿ Characterization and identification of isolates ﾿ 37

3.7.1 ﾿ Isolation and identification of Lactic acid bacteria ﾿ 37

3.7.2 ﾿ Phenotypic Characterization ﾿ 37

3.8 ﾿ Biochemical Characterization ﾿ 37

3.8.1 ﾿ Gram staining ﾿ 37

3.8.2 ﾿ Catalase test ﾿ 38

3.8.3 ﾿ Citrate test ﾿ 38

3.8.4 ﾿ Haemolytic Activity ﾿ 39

3.8.6 ﾿ Spore Stain test ﾿ 39

3.9 ﾿ Characterization of Lactic acid bacteria ﾿ 40

3.9.1    Carbohydrate fermentation test ﾿ 40

3.9.2 ﾿ Gas production from glucose ﾿ 40

3.9.3    Salt Tolerance Test ﾿ 41

3.9.4 ﾿ Growth at different temperatures ﾿ 41

3.9.5 ﾿ Growth at different pH ﾿ 41

3.10 ﾿ Laboratory fermentation of nunu using lab isolates for vitamin production ﾿ 41

3.11 ﾿ Hplc analysis for determination of vitamin ﾿ 42

CHAPTER 4

RESULTS ﾿ 43

CHAPTER 5

5.1 ﾿ Discussion ﾿ 53

5.2 ﾿ Conclusion and Recommendation ﾿ 55

References ﾿ 56

Appendix I ﾿ 63

Appendix II ﾿ 64

Appendix III ﾿ 65

Appendix IV ﾿ 66

Appendix V ﾿ 67

LIST OF TABLES

TABLES

Table 2.1: The benefits of food fermentation ﾿ 7

Table 2.2: Recommended Dietary Intake (RDA) and Adequate Intake (AI) 

Table 2.3: List of some lactic acid bacteria found in milk and milk products ﾿ 31

Table 2.4: Biochemical test for the identification of isolated Latic acid bacteria ﾿ 45

Table 2.5: Sugar fermentation by the isolated LAB ﾿ 46

Table 2.6: Production Potency of Riboflavin and Cobalamin from Unfermented Nunu and Nunu Fermented by Cultures of Lactic Acid Bacteria ﾿ 52

LIST OF FIGURES

FIGURES

Figure 2.1: A Flow diagram of nunu processing ﾿ 11

Figure 4.1: Chromatogram of the blank ﾿ 47

Figure 4.2: Chromatogram of Riboflavin/ vitamin B2 standard ﾿ 48

Figure 4.3: Chromatogram of Riboflavin sample ﾿ 49

Figure 4.4: Chromatogram of Cobalamin standard ﾿ 50

Figure 4.6: Chromatogram of Cobalamin sample ﾿ 51