PROBIOTIC POTENTIALS OF LACTIC ACID BACTERIA ISOLATED FROM LOCALLY FERMENTED MAIZE (AKAMU)

ABSTRACT

Lactic Acid Bacteria (LAB) as probiotic organisms can be sourced from fermented diary and vegetable products. The problem in Nigeria is their affordability to the greater populace, as the common sources are relatively expensive. The study was aimed at identifying the probiotic LAB and screening their probiotic activity involved in a low cost meal which is locally fermented maize (Akamu). This LAB predominates the microflora of fermented products. They produce metabolites that inhibit the growth of food borne pathogen and are beneficial to the GIT. Isolation and identification of LAB from fermented maize (akamu) was evaluated based on the biochemical characteristics and carbohydrate fermentative pattern. The four (4) isolates of lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casie and Enterococcus faecium. The lactic acid bacteria (LAB) strains were tested for their probiotic activity against four test pathogens; Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Klebsiella species. Their resistance to acid and bile was determined. Their optimum antimicrobial activity was judged by their different zones of inhibition. There was significant differences between the samples at P<0.05. Lactobacillus plantarum was significantly higher at (21.67±0.58) for Salmonella followed by Lactobacillus acidophilus. The test LAB demonstrated efficient probiotic properties by inhibiting the test pathogens, tolerating acid at pH 3.0 and withstanding 0.3% bile concentration. The results revealed that akamu is a rich source of probiotic LAB and thus can be recommended as food.

TABLE OF CONTENTS

Title page                                                                                                             i

Certification                                                                                                         ii

Dedication                                                                                                           iii

Acknowledgement                                                                                                iv

Table of contents                                                                                                  v

List of tables                                                                                                        vii

List of figures                                                                                                       viii

Abstract                                                                                                               ix

CHAPTER ONE

1.1          Probiotics                                                                                                  1

1.2          Prebiotics                                                                                                   2

1.3          Synbiotics                                                                                                  3

1.4          Aim and Objectives of the study                                                                 4                                                                                

CHAPTER TWO

2.0      Literature Review                                                                                      5

2.1      Lactic Acid Bacteria                                                                                   5       

2.2      Lactobacillus bacteria                                                                                 6

2.3      Enterococcus                                                                                             9

2.4      Probiotic Potentials of Lactic Acid Bacteria Isolated from Locally

Fermented Maize                                                                                       11

2.4.1   pH and Organic Acid Production                                                                12

2.4.2   Acid and Bile Tolerance                                                                             13

2.4.3   Stimulation of the Immune System                                                             14

2.4.4   Adhesion to Intestine Mucus Layer and Epithahial Cells of Human GIT   15

2.4.5   Resistance of Antibiotics                                                                            16

CHAPTER THREE

MATERIALS AND METHODS

3.1      Collection of Akamu Samples                                                                     17

3.2      Test Organisms                                                                                          17

3.3      Isolation of Lactic Acid Bacteria from Akamu                                             17

3.4      Identification of Laboratory Isolates                                                            18

3.4.1   Gram Staining                                                                                            18

3.5      Biochemical Test                                                                                       18

3.5.1   Catalase Test                                                                                              18

3.5.2   Oxidase Test                                                                                              19

3.6      Sugar Utilization Test                                                                                 19

3.7      Probiotic Activities of the Isolates                                                               19

3.7.1   Antimicrobial Activities                                                                             19

3.7.2   Acid Tolerance                                                                                          20

3.7.3   Bile Tolerance                                                                                           20

CHAPTER FOUR

Result                                                                                                                  22

CHAPTER FIVE

DISCUSSION AND CONCLUSION

5.1      Discussion                                                                                                 30

5.2      Conclusion                                                                                                35

REFERENCES

LIST OF TABLES

Table                      Title                                                         Page

1                   Characteristics of LAB Isolates from Akamu                                        24

2                   Occurrence of Lactic Acid Bacteria in Akamu from Different Markets  25

3                   Antimicrobial Activity of Lactic Acid Bacteria Isolate from Akmu       26

LIST OF FIGURES

Figure      Title                                                                   Page

1                   Zones of Inhibition (mm) of Lactic Acid Bacteria against the test

Pathogen                                                                                         27

2                   Effect of Acid Exposure at pH 3.0 on the Probiotic Lactic Acid

Bacteria Isolated from Akamu                                                          28

3                   Viability of rpobiotic Lactic Acid Bacteria in MRS broths with 0.3%

Bile                                                                                                 29

CHAPTER ONE

INTRODUCTION

1.1      PROBIOTICS

Probiotics are live microorganisms that when administered in adequate amounts confer a health benefits on the host. (FAO/WHO, 2002). Probiotic bacteria may produce various compounds which are inhibitory to the pathogens growth, which include organic acids (lactic acid and acetic acids), bacteriocins and reuterin. The organic acids of the pathogen, but they can also be toxic to the microbes (Tambekar and Bhutada, 2010).

Lactobacillus and Bifidobacteria are examples of general of which some of the species are promising probiotics (Saito, 2004). Others with probiotic characteristics include some species of the general Enterococcus, Lactobacillus rhamnosus GG and a variety of L. casei and L. rhamonosus, which is an extensively studied probiotic that has been shown to be effective in reducing the serverity and duration of diarrhoea (Jakobsen and Lei, 2004).

Probiotics play a key role in enhancing resistance to colonization by exogenous, potentially pathogenic organisms (Helland et al., 2004). They do this by producing compounds such as lactic acid, hydrogen peroxide and acetic acid that increase acidity of the intestine and inhibit the reproduction of many harmful bacteria. Probiotics also produce bacteriocins, which act as natural antibiotics that kill undesirable/ pathogenic microorganisms. They are also known to compete the pathogenic microorganisms hence preventing their survival in the gastrointestinal tract (GIT) (Helland et al., 2004). There is increasing evidence that probiotics are beneficial to gastrointestinal disturbances, such as diarrhoea, dysentery, typhoid (Tambekar and Bhutads, 2010).

In addition, the following properties and functions have been attributed to probiotics: they adhere to host epithetial tissue; they are acid resistant and bile tolerant; they are safe, non-pathogenic and non-carcinogenic; they cause improvement of the intestinal micro flora; they have a cholesterol lowering effect; synthesize and enhance the bioavailability of nutrients (Parvez et al., 2006).

1.2      PREBIOTICS

Associated with probiotics are prebiotics. Prebiotics are non-digestible food ingredients that have a beneficial effect on the host by selectively stimulating growth of health promoting bacteria (Desai et al., 2004). They are assimilated by beneficial bacteria such as Bifidobacteria hence improving their growth activity leading to an enhanced intestinal balance. For a food to qualify as a prebiotic, it has to be no-digestible by human enzymes, has to undergo selective fermentation by potentially beneficial bacteria in the colon; should cause an alteration in the composition of the colonic micro biota towards a healthier composition and all these changes should lead to beneficial health to the host (Pariyaporn et al., 2003).

Most commercial prebiotics are carbohydrates, predominantly oligosaccharides and some polysaccharides. The non-digestible character of prebiotics is a feature shared with dietary fibre, but their physiological functions are often different (Macfarlane et al., 2006). The prebiotics are selective in their growth stimulation and at the same time they are reported to supress pathogenic bacteria present in the gastrointestinal tract (GIT) because they can only use the prebiotic ingredient for growth to a limited extent or not at all (Holzaptal and Schillinger, 2002).

Hence, the prebiotic principle is based on selective stimulation of microorganisms able to hydrolyse the prebiotic to carbohydrate monomers and use them for growth in the GIT (Bomba et al., 2002).

Other beneficial effects of prebiotics are improvement of catcium bioavailability and reduction in the risk for cardiovascular diseases, non-insuin-dependent diabetics, obesity, osteoporosis, colon cancer as well as reduction of traveller’s diarrhoea (Macfarlane et al., 2006).

1.3      SYNBIOTICS

Synbiotics refer to nutritional supplements combining probiotics and prebiotics in a form of synergism. The symbiotic concept was first introduced as mixtures of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract (GIT), by selectively stimulating the growth and or by activating the metabolism of one or a limited number of health promoting bacteria, thus improving host welfare (Kentaro et al., 2013).

Indeed, symbiotic combinations are considered to have more beneficial effects on human health than probiotics or prebiotics alone. Recent studies established that synbiotics improve the intestinal microbial environment and activate host immune function, leading to prevention of bacterial translocations synbiotics have also been employed to enhance the health benefits of infact formulae. Administration of synbiotics as a food supplement is safe, simple and convenient.

It could be argued that fermented cereal products, such as koko, are potential symbiotic products in that they contain lactic acid bacteria (potential probiotics) as well as water-soluble fibres, oligosaccharides and resistant starch (potential prebiotics). However, the present study will be limited to the probiotic effect of lactic acid bacteria.

1.4      AIM AND OBJECTIVES OF THE STUDY

Aim

The aim of this work was to determine the probiotic potentials of lactic acid bacteria isolates from fermented maize.

Objectives

1.             To isolate and identify lactic acid bacteria from fermenting maize

2.             To screen for probiotic potentials of the LAB isolates

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