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