CYANOCOBALAMIN AND FOLATE LEVELS OF MAIZE DURING IT’S FERMENTATION TO AKAMU OR OGI BY LACTIC ACID BACTERIUM

                                                           ABSTRACT

This study evaluated the cyanocobalamin and folate levels in fermenting maize to akamu using lactic acid bacteria. A total of four (4) different species of Lactic acid bacteria were isolated from the nunu milk samples. The details of these Lactic Acid Bacterial isolates comprises of L casei, L plantarum, L fermentum, and L acidophilus. The Total Lactic Acid bacteria count observed in this study ranged from 4.7×104cfu/ml to 5.4 ×104cfu/ml with sample A4 giving the highest count of 5.4 ×104cfu/ml whereas sample A1 had the lowest lactic acid bacterial count of 4.7×104cfu/ml. it was observed that L. acidophilus is the most frequently occurring isolates with a percentage occurrence of (40.0%), followed by L. casei with a percentage occurrence of (30.0%), then  L. plantarum with a percentage occurrence of (20.0%) whereas L. fermentum has the least percentage occurrence of (10.0%). The results of the determination of Cobalamin (vitamin B12) and Folate (vitamin B9) levels in fermenting maize as represented in table 5 indicates that the Cobalamin (vitamin B12) level in the fermenting maize recorded 0.037mg/ml at the beginning of fermentation (steeping period), then increased to 0.11mg/ml at the end of the fermentation. Contrary to this, The Folate (vitamin B9) level in the fermenting maize recorded 0.0031mg/ml at the beginning of fermentation (steeping period), then reduced to0.0012mg/ml at the end of the fermentation process. Increasing levels of B-group vitamins in fermented cereals are possible through the selection of microbial species and implementation of fermentation conditions. Different strategies may be applied to improve microbial production of vitamins in cereals fermented products. These strategies include (1) selection of natural overproducers using chemicals, (2) strains selection whitin culture collections, (3) increasing vitamins bioavailability and (4) use of genetically modified LAB (GM-LAB)

TABLE OF CONTENTS

Title Page                                                                                                                                 i

Certification                                                                                                                           ii

Dedication                                                                                                                              iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

List of Figures                                                                                                                         viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aims and Objectives                                                                                                  5

CHAPTER TWO

2.0       Literature Review                                                                                                       6

2.1       Lactic Acid Bacteria                                                                                                   6

2.2       Vitamins                                                                                                                     7

2.3       B-Group Vitamin Production by Lactic Acid Bacteria                                              9

2.3.1    Folates                                                                                                                        9

2.3.2    Vitamin B12                                                                                                               15

2.3.3    Other B-group vitamins                                                                                              19

2.3.4    Other vitamins                                                                                                            20

2.4       Origin of Maize                                                                                                          21

2.4.1    Types of Maize Grain                                                                                                 21

2.4.2    Uses of maize grain                                                                                                    22

2.5       Properties of Pap                                                                                                         22

2.5.1    Physical properties of Pap                                                                                          22

2.5.2    Nutritional and Chemical Properties of Pap                                                               23

2.5.3    Microbial Properties of Pap                                                                                        24

2.6       Benefits of Fermentation                                                                                            24

CHAPTER THREE

3.0       Materials and Methods                                                                                               26

3.1       Collection of Samples                                                                                                26

3.2       Media Preparation for Isolation of Lactic Acid Bacteria from the Samples                       26

3.3       Isolation of Lactic Acid Bacteria                                                                               26

3.4       Sub-Culturing                                                                                                             27

3.5       Characterization and Identification of Lactic Acid Bacterial Isolates                        27

3.5.1    Gram Staining Techniques                                                                                         27

3.5.2    Biochemical Test                                                                                                        28

3.5.3.1 Motility test                                                                                                                28

3.5.3.2 Catalase test                                                                                                                28

3.5.3.3 Coagulase test                                                                                                             28

3.5.3.4 Methyl red test                                                                                                            28

3.5.3.5 Voges-proskaeur test                                                                                                  29

3.5.3.6 Indole test                                                                                                                   29

3.5.3.7 Citrate test                                                                                                                   29

3.6.3.8 Oxidase test                                                                                                                30

3.7       Fermentation of Maize-Pigeon Pea Blends                                                                30

3.8       Isolation of Folate-Producing Lactic Acid Bacteria                                                  32

3.9       Phenotypic Characterization of the Lab Isolates                                                        32

3.10     Screening of Folate Production and Quantification in Culture Medium                      32

3.11     Determination of Folate Concentration                                                                      33

3.12     Vitamin B12 Analysis                                                                                                33

3.12.1  Extraction                                                                                                                   33

3.12.2  Immunoaffinity purification                                                                                       34

3.12.3  UHPLC and LC–MS analyses                                                                                    34

3.12.4  Microbiological assay                                                                                                 35

CHAPTER FOUR

4.0       Results                                                                                                                        36

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                         47

5.1       Discussion                                                                                                                   47

5.2       Conclusion                                                                                                                  49

References                                                                                                                  50

LIST OF TABLES

LIST OF FIGURES

CHAPTER ONE

1.0       INTRODUCTION

Ogi is consumed by adults and children as breakfast meals, and it also serves as a weaning diet (Ashaye and Kehinde, 2000; Amusa et al., 2005). After 5–6 months, breast-feeding is no longer sufficient to satisfy the nutritional requirements of the growing infant. Beginning from this period, the child needs solid foods to meet increasing nutritional needs. This period is the weaning period and in Nigeria, ogi (alternatively called pap or akamu) is introduced gradually to the child’s diet to supplement nutrition. Fermented maize is very widely utilized as food in African countries and in fact cereals account for as much as 77% of total caloric consumption (Osungbaro, 2009). Maize is rich in carbohydrates and minerals, including potassium and magnesium. It contains trace amounts of lysine and tryptophan, contributing to the low content of protein, and trace amounts of B-vitamins.

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. Although, the vitamin requirement of the body is usually adequately supplied by a balanced diet, significant subgroups in most European populations are still subjected to the risks associated with low micronutrient intakes (Flynn et al., 2003).

The B group or B-complex vitamins include thiamine (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), pantothenic acid (B5), biotin (B7 or H), folate (B9) 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). B-group vitamins, normally present in many foods, can be easily removed or destroyed during cooking and food processing, so their deficiencies is rather common in human population. For this reason, several countries have adopted laws requiring the fortification of certain foods with specific vitamins and minerals (Burgess et al., 2009).

Cereals contain various B vitamins. Whole cereal products have been identified as a major source of thiamine when flours are optimally processed. Riboflavin is essentially provided by dairy products, but whole wheat bread could provide 20 % of the daily requirements of riboflavin. Cereals are also important sources of folates that are needed to prevent neural tube defects (Katan et al., 2009). Folate is mainly concentrated in the bran fractions and in nations where folic acid fortification is not generally practiced, cereals contribute 43 and 36 % of the total folate intake of men and women, respectively (Kariluoto et al., 2006; Kariluoto et al., 2010). Bread is also a non-negligible source of pyridoxine (about 16 % of daily requirements). Cereal grains apparently lack vitamin B12.

B-vitamins variability in cereal grains and cereals products such as bread depends on several factors. Cultivar, seasoning, growing location, milling or flour extraction rate and genetic determinants are all factors that may influence vitamins concentrations within grains (Batifoulier et al., 2006). Moreover, B-group vitamins are differently distributed in grain tissues. Although the endosperm represents 80–85 % of wheat grain dry mass, it contains only a small proportion of total B vitamins such as pyridoxine (6 %) and thiamine (3 %), with most of the pyridoxine and thiamine (80 %) or riboflavin (42 %) being found in the external layers of wheat grains (Batifoulier et al., 2006). In contrast, the endosperm contains a significant concentration of riboflavin (32 %) which is also present in the germ (26 % of the total grain riboflavin content). Of particular importance is the biological availability of the B-vitamins contained within cereal grains and their content after milling, processing and cooking (Batifoulier et al., 2006). Indeed, B-group vitamins, normally present in cereals products as well as in many other foods, are easily removed or destroyed during milling, food processing or cooking. Therefore, the food industry makes a great effort to develop products based on cereals (e.g., the ‘breakfast cereals’), often containing vitamins and minerals added by the manufacturer to enhance its nutritional value and compensate for any losses that may have occurred during the manufacturing processes.

As an attractive alternative to the chemical synthesis of vitamins, specific biotechnological processes for vitamin inclusion in foods have been developed. Among these, fermentation with food grade lactic acid bacteria (LAB) offers unique opportunities to improve the nutritional value of food products and the development of novel functional foods with an enhanced vitamin content due to bacteria fermentation has been suggested and would even contribute to growing market for these products (Stanton et al., 2005). LAB are an industrially important group of microorganisms used all over the world for a large variety of food fermentations, such as those of dairy, wine, bread and vegetables. 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 European Food Safety Agency (EFSA) has recently introduced a system for a premarket safety assessment of selected taxonomic groups of microorganisms leading to a ‘Qualified Presumption of Safety’ (QPS), a European equivalent of the Generally Recognized As Safe status. Several species of food-associated LAB have obtained a QPS status. 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 ingredients can be macronutrients, micronutrients (such as vitamins) or nonnutritive 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 (LeBlanc et al., 2011). Certain fermented milks have shown high levels of B-group vitamins due to LAB biosynthesis. Many industrially important LAB such as Lactococcus lactis and Streptococcus thermophilus have the ability to synthesize folate (vitamin B11) and folate biosynthesis by yogurt starter cultures can increase the “natural” folate levels in this product (Laino et al., 2012). The genes involved in the riboflavin (vitamin B2) biosynthesis by LAB, have been identified in several species and some application of riboflavin-producing LAB in dairy and cereals-based products have also been reported (Capozzi et al., 2011). Cobalamin (vitamin B12), a complex corrin compound, was found to be produced by strains of Lactobacillus reuteri awell-recognized probiotic species. 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. Therefore, a deep knowledge on genes/operones 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.

1.1       AIMS AND OBJECTIVES

The aim of this study is to determine the cobalamin and folate concentration in fermented maize by lactic acid bacteria

The objectives are;

1.     To isolate and characterize folate and cobalamin producing lactic acid bacteria from fermented maize samples

2.     To analyze the vitamin B11 (folate) level in fermented maize

3.     To analyze the vitamin B12 (cobalamin) level in fermented maize

Buyers has the right to create dispute within seven (7) days of purchase for 100% refund request when you experience issue with the file received. 

Dispute can only be created when you receive a corrupt file, a wrong file or irregularities in the table of contents and content of the file you received. 

ProjectShelve.com shall either provide the appropriate file within 48hrs or send refund excluding your bank transaction charges. Term and Conditions are applied.

Buyers are expected to confirm that the material you are paying for is available on our website ProjectShelve.com and you have selected the right material, you have also gone through the preliminary pages and it interests you before payment. DO NOT MAKE BANK PAYMENT IF YOUR TOPIC IS NOT ON THE WEBSITE.

In case of payment for a material not available on ProjectShelve.com, the management of ProjectShelve.com has the right to keep your money until you send a topic that is available on our website within 48 hours.

You cannot change topic after receiving material of the topic you ordered and paid for.