ENUMERATION OF LACTIC ACID BACTERIA FROM FERMENTED AFRICAN OIL BEAN (PENTACLETHRA MACROPHYLLA BENTH) SEEDS FOR NIACIN AND PANTOTHENIC ACID PRODUCTION

ABSTRACT

The enumeration of Lactic Acid Bacteria (LAB) from fermented African Oil Bean  (Pentaclethra macrophylla Benth) Seeds for niacin and pantothenic acid production was studied. Locally produced samples of Pentaclethra macrophylla were procured from Ubani main market, Umuahia, Abia State. The samples were plated on M17 agar, MRS (de Man Rogosa and Sharp) agar and on Nutrient Agar. All the media used for the isolation of these organisms were prepared aseptically according to the manufacturer’s specification. Morphological and biochemical characteristics and sugar fermentation pattern were employed to identify lactic acid bacteria. Sixteen (16) lactic acid bacterial isolates which were found to be Gram positive, non-spore formers and catalase negative were isolated. The identified organisms include Lactobacillus plantarum, Streptococcus lactis, Lactobacillus fermentum, Pediococcus spp., and Leuconostoc mesenteroides. The organisms isolated were able to grow at temperature of 45oC but varied at 15oC. Majority of the isolates survived at a salt concentration of 4 to 6%. The High Performance Liquid Chromatography (HPLC) analysis results revealed that the concentration of vitamins (niacin and pantothenic acid) increased in the fermented samples. Niacin increased from 0.1367mg/ml in the unfermented Pentaclethra macrophylla sample to 0.300mg/ml in the fermented Pentaclethra macrophylla (Ugba) sample, while pantothenic acid increased from 0.5877mg/ml in the unfermented Pentaclethra macrophylla sample to 0.8843mg/ml in the fermented Pentaclethra macrophylla (Ugba) sample. This study has shown the Niacin and Pantothenic acid production potential of lactic acid bacteria from fermented African Oil bean seeds. Hence, vitamin-producing LAB should be employed in the production of vitamin rich fermented foods and drugs in other to enrich the food matrices and improve human health.

TABLE OF CONTENTS

Title page                                                                                                                              i

Certification                                                                                                                          ii

Dedication                                                                                                                            iii

Acknowledgements                                                                                                              iv

Table of contents                                                                                                                   v

List of Tables                                                                                                                         viii

List of Figures ix

List of Plates x

List of Appendices xi

Abstract                                                                                                                                  xii

CHAPTER 1

1.1 Introduction 1

1.2 Aim and objectives 3

CHAPTER 2: LITERATURE REVIEW

2.1 African Oil Bean Seeds 4

2.1.1 Nature of the plant and the seeds 5

2.1.2 Chemical composition of African oil bean seeds 6

2.1.3 Microorganisms involved in the fermentation of African oil bean seeds 7

2.1.4 Changes that occur during Ugba fermentation 8

2.1.5 Optimization of ugba fermentation 10

2.2 Vitamins 11

2.2.2 Niacin 13

2.2.2.1 Biosynthesis of Niacin 14

2.2.3 Pantothenic Acid 17

2.3 Lactic Acid Bacteria 18

2.3.1 Fermentation by LAB 19

2.3.2 Microorganisms involved in the lactic acid fermentation 19

2.4 Factors Affecting Fermentation Process 21

2.5 Effects of Fermentation 25

2.5.1 Flavour Enhancement 25

2.5.2 Nutritional Quality 25

2.5.3 Preservative Properties 26

2.5.4 Detoxification 26

2.5.5 Antibiotic Activities 27

2.1.3 Traditionally Fermented Foods in Nigeria 28

CHAPTER 3: MATERIALS AND METHODS

3.1 Source of Raw Material 30

3.2 Materials 30

3.3 Preparation of homogenate Ugba samples 30

3.4 Inoculation of samples 31

3.5 Colony count of isolates 31

3.6 Characterization and identification of isolates 31

3.6.1 Isolation and identification of lactic acid bacteria 32

3.6.2 Phenotypic characterization 32

3.7 Biochemical characterization 32

3.7.1 Gram staining 32

3.7.2 Catalase test 33

3.7.3 Spore Stain Test 33

3.7.4 Citrate Test 33

3.7.5 Amylase test 34

3.8 Characterization of Lactic Acid Bacteria 34

3.8.1 Growth at different temperatures 34

3.8.2 Growth at different pH 34

3.8.3 Salt tolerance test 35

3.8.4 Gas production from glucose 35

3.8.5 Carbohydrate fermentation test 36

3.9 In vitro/ Laboratory Fermentation of Ugba Using LAB 36

      Isolates for Vitamin Production

3.10 HPLC analysis 37

CHAPTER 4

Results 38

CHAPTER 5: DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1 Discussion 47

5.2 Conclusion 50

5.3 Recommendation 50

REFERENCES 51

APPENDICES 58

LIST OF TABLES

LIST OF FIGURES

LIST OF PLATES

LIST OF APPENDICES

CHAPTER 1

1.1 INTRODUCTION

African oil bean (Pentaclethra macrophylla Benth) seeds which are utilized by the Igbos and other ethnic groups in southern Nigeria as a delicacy and food flavoring. The oil bean seed is mainly composed of proteins (42%), lipids (43%) and carbohydrates (15%) (Ogueke and Aririatu, 2004).

The traditional fermentation methods widely practiced in Africa and other developing countries usually involve a spontaneous development of different lactic acid producing bacteria. The final microbiological status of the products so derived by these methods is influenced in part by the raw materials and the process method (Steinkraus, 2006). These process methods have almost always led to the problem of inconsistent product quality and other attendant problems. In Africa, majority of the fermented foods are produced at household level and hygiene is a major concern (Olasupo et al., 2002; Gadaga et al., 2008).

Published studies on the microbiology of the fermentation of African oil bean seeds have identified Bacillus spp as the main microorganisms responsible for its fermentation. The predominant species is Bacillus subtilis but other species like B. pumilus, B. megaterium, B. lichenformis have also been found (Ouoba et al., 2010). The same group of organisms has been implicated in the fermentation of other fermented food condiments like Iru, Dawadawa, Soumbala, Afiyo and Ogiri.

Lactic acid bacteria (LAB) are an industrially important group of microorganisms used all over the world for a large variety of food fermentations. LAB are also natural members of the human gastrointestinal microbiota and several strains are considered beneficial to the host and have been selected for probiotic applications (Bove et al., 2012). The adaptability of LAB to fermentation processes, their biosynthetic capacity and metabolic versatility are some of the principal features that facilitate the application of LAB in foods for producing, releasing and/or increasing specific beneficial compounds. These compounds can be macronutrients, micronutrients (such as vitamins) or non-nutritive compounds (Russo et al., 2012).

Among these, vitamin production by LAB has recently gained the attention of the scientific community (LeBlanc et al., 2011). The proper selection and exploitation of nutraceutical-producing LAB is an interesting strategy to produce novel fermented foods with increased nutritional and/or health-promoting properties (Hugenholtz and Smid 2002; LeBlanc et al., 2011).

Vitamins are micronutrients that are essential for the metabolism of all living organisms. They are found as precursors of intracellular coenzymes that are necessary to regulate vital biochemical reactions in the cell. Humans are incapable of synthesizing most vitamins, which, consequently, have to be obtained exogenously such as from the gut microbiota and the diet.

Certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group (Capozzi et al., 2012). The B group or B-complex vitamins include thiamine (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), pantothenic acid (B5), biotin (B7 or H), folate (B11–B9 or M) and cobalamin (B12). These molecules are water-soluble and play an important role in metabolism, particularly the cellular metabolism of carbohydrates (thiamine), proteins and fats (riboflavin and pyridoxine).

Vitamin production by LAB varies considerably being a species-specific or strain-dependent trait. This feature is generally related to the partial or complete interruption of the genetic information for vitamins biosynthesis (Capozzi et al., 2012). Therefore, a deep knowledge on genes/operons involved in vitamins biosynthesis is essential in order to select suitable vitamin-producing LAB or to design strategies in order to increase vitamin production in food. This work is hence aimed at evaluating lactic acid bacteria from fermented African oil bean seeds for niacin and pantothenic acid production.

1.2 Aim and objectives

The aim of this study was to enumerate lactic acid bacteria from fermented African oil bean seeds for Niacin and Pantothenic acid production.

The specific objectives are:

1. To isolate LAB from fermented African oil bean seeds

2. To identify LAB from fermented African oil bean seeds

To determine the niacin (nicotinic acid, B3) and pantothenic acid (B5) production abilities of LAB isolates using High Performance Liquid Chromatography.

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