THIAMINE AND RIBOFLAVIN CONCENTRATION IN FERMENTED MAIZE

ABSTRACT

This study evaluated the Thiamine and Riboflavin concentrations in fermented maize using lactic acid bacteria. A total of four (4) different species of Lactic acid bacteria were isolated from the nunu milk samples using biochemical identification, Gram staining, outcomes of microscopic analysis and carbohydrate fermentation pattern. The total lactic acid bacteria count observed in this study ranged from 4.7×104cfu/ml to 5.4 ×104cfu/ml with sample B4 giving the highest count of 5.4 ×104cfu/ml whereas sample B1 had the lowest lactic acid bacterial count of 4.7×104cfu/ml. The results on assimilation of different carbon sources revealed that all lactic acid bacterial isolates can assimilate different carbon sources like glucose, dextrose, sucrose, fructose and lactose. The Thiamine (vitamin B1) concentration in the fermenting maize recorded 0.016mg/ml at the beginning of fermentation (steeping period), then increased to 0.025mg/ml at the end of the fermentation. In the same sequence, The Riboflavin (vitamin B2) concentration in the fermenting maize recorded 0.0033mg/ml at the beginning of fermentation (steeping period), then increased to 0.065mg/ml at the end of the fermentation process. Thiamine and Riboflavin quantification of the fermenting maize slurry showed that lactic acid bacteria isolates were responsible for Thiamine and Riboflavin production in the fermenting slurry as there was an increase in the Thiamine and Riboflavin concentrations of the slurry. From the result obtained, it can be concluded that vitamin-producing lactic acid bacteria can be used as a cost-effective alternative to current vitamin fortification programmes and for the elaboration of novel vitamin-enriched cereals products. 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                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          viii

List of Figures                                                                                                                         ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aims and Objectives                                                                                                  5

CHAPTER TWO

2.0       Literature Review                                                                                                       6

2.1       Lactic Acid Bacteria Producing B-Group Vitamins: A Great Potential

For Functional Cereals Products                                                                                7

2.1.1    Riboflavin (Vitamin B2) and Functional Bread                                                         7

2.1,2    Folate (Vitamin B11): Endogeneous Bacteria and Sourdough Microbiota                  10

2.1.3    Cobalamin (Vitamin B12)                                                                                          13

2.2       Origin of Maize                                                                                                          16

2.2.1    Types of Maize Grain                                                                                                 17

2.2.2    Uses of maize grain                                                                                                    17

2.3       Properties of Pap                                                                                                         18

2.3.1    Physical properties of Pap                                                                                          18

2.3.2    Nutritional and Chemical Properties of Pap                                                               18

2.3.3    Microbial Properties of Pap                                                                                        19

2.4       Benefits of Fermentation                                                                                            20

CHAPTER THREE

3.0       Materials and Methods                                                                                               21

3.1       Collection of Samples                                                                                                21

3.2       Fermentation of Maize-Pigeon Pea Blends                                                                21

3.3       Normal Saline Preparation                                                                                         21

3.4       Media Preparation for Isolation of Lactic Acid Bacteria from the Samples                      22

3.5       Isolation of Lactic Acid Bacteria                                                                               22

3.6       Sub-Culturing                                                                                                             22

3.7       Characterization and Identification of Lactic Acid Bacterial Isolates                        22

3.7.1    Gram Staining Techniques                                                                                         23

3.7.2    Biochemical Test                                                                                                        23

3.7.3.1 Motility test                                                                                                                23

3.7.3.2 Catalase test                                                                                                                24

3.7.3.3 Coagulase test                                                                                                             24

3.7.3.4 Methyl red test                                                                                                            24

3.7.3.5 Voges-proskaeur test                                                                                                  24

3.7.3.6 Indole test                                                                                                                   25

3.7.3.7 Citrate test                                                                                                                   25

3.7.3.8 Oxidase test                                                                                                                25

3.8       Analysis of Vitamin B1 (Thiamin)                                                                            27

3.9       Analysis of Vitamin B2 (Riboflavin)                                                                         27

CHAPTER FOUR

4.0       Results                                                                                                                        29

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                         38

5.1       Discussion                                                                                                                   38

5.2       Conclusion                                                                                                                   40

References                                                                                                                  41

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 (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). 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 thiamine and riboflavin concentration in fermented maize

The objectives are;

1.     To isolate and characterize lactic acid bacteria from nunu milk

2.     To analyze the vitamin B1 (thiamin) concentration in fermented maize

3.     To analyze the vitamin B2 (riboflavin) concentration in fermented maize

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