CHARACTERIZATION OF LACTIC ACID BACTERIA ISOLATED FROM FERMENTED TIGER NUT MILK

ABSTRACT

Lactic acid bacteria (LAB) are the main group of microorganisms that are responsible for the production of fermented products with a great economic importance. This study was done to characterize and identification of lactic acid bacteria isolated from fermented tiger nut milk. The objectives were to isolate lactic acid bacteria from fermented tiger nut milk, characterize and identify lactic acid bacteria using morphological, physiological and biochemical techniques and to access the technological potentials of lactic acid bacteria isolated from fermented tiger nut milk. The result of the analysis showed that L.brevis 1.32 x 105 was highest at pH 4.0 while L.bugaricus was low at pH 6.5, the results of bile salt tolerance showed that L.mesenteriodes 1.22 x 105  was high at 0.3% bile salt concentration while L. helveticus 0.8 x 105 was low at 0%. It was proven that tiger nut milk contained both homofermentative and hetrofermentative lactic acid bacteria. Lactic acid bacteria also produce exopolysaccharides which are essential as texture formation. It is therefore recommended that lactic acid bacteria also produce exopolysaccharides which are essential as texture formation. Considering the existing reports on several health-promoting properties as well as their generally recognized as safe (GRAS) status of LAB, they can be widely used in the developing of new fermented milk products.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgements                                                                                                                iv

Table of contents                                                                                                                    v

List of Tables                                                                                                                          viii

Abstract                                                                                                                                   ix

CHAPTER ONE

1.0           Introduction                                                                                                                1

1.1       Background of the Study                                                                                            1

1.2       Aims and Objectives                                                                                                   4

CHAPTER TWO

2.0           Literature Review                                                                                                       5

2.1       Classification, Taxonomy and Sources of Lactic Acid Bacteria                                    5

2.1.1    Lactic Acid Bacteria                                                                                                   5

2.1.2    Sources of Lactic Acid Bacteria                                                                                 5

2.1.3    Classification of Lactic Acid Bacteria                                                                       5

2.2       Lactic acid bacteria as source of exopolysaccharides                                                7

2.2.1    Definition and classification of exopolysaccharides                                                  7

2.3       Food fermentation                                                                                                      8

2.3.1    Benefits of Food Fermentation                                                                                   10

2.4       Fermentation of Foods by Lactic Acid Bacteria                                                        12

2.5       Fermentation of Tiger Nut by Lactic Acid Bacteria                                                  14

2.6       Economic and Nutritional Benefits of Tiger Nut                                                       15

CHAPTER THREE

3.0       Materials and Methods                                                                                               17

3.1       Source of Materials                                                                                                    17

3.1.1    Sample Preparation                                                                                                     17

3.2       Glass Wares and Media Used and Their Preparation                                                 17

3.3       pH Determination                                                                                                       18

3.4       Microbiological Analyses                                                                                           22

3.4.1    Enumeration of total bacteria                                                                                     22

3.4.4    Enumeration of Lactic acid bacteria                                                                           24

3.4.5    Isolation of Lactic Acid Bacteria                                                                                24

3.4.6    Characterization of Lactic Acid Bacteria                                                                   25

3.4.6.1  Morphological characterisation                                                                                  26

3.4.6.2    Biochemical characterization                                                                                   26

3.4.6.3  Physiological Characterization                                                                                   28

3.4.7    Identification of Lactic acid bacteria                                                                          29

3.5.2    Antibiotic resistance pattern                                                                                       30

3.6       Determination of Technological Properties                                                               30

3.6.3    Rate of Acid Production                                                                                             31

CHAPTER FOUR

4.0       Results                                                                                                                        32

CHAPTER FIVE

5.0       DISCUSSION, CONCLUSION AND RECOMMENDATIONS                 43

5.1       Discussion                                                                                                                   43

5.2       Conclusion                                                                                                                  45

5.3       Recommendations                                                                                                      46

References                                                                                                                  47

LIST OF TABLES

Table                  Title                                                                Page

1.                                        Identification of the LAB Isolates from the samples                     28

2.                                       Acidic pH tolerance test on the LAB species isolated                    29

3.                                       Bile salt Tolerance test on the Lab species isolated                        30

4.                                       Antimicrobial effect of the LAB  species metabolites on pathogens    31

CHAPTER ONE

1.0        INTRODUCTION

1.1       Background of the Study

Lactic acid bacteria (LAB) are the main group of microorganisms that are responsible for the production of fermented products with a great economic importance (Arici et al., 2014). Lactic acid bacteria are a group of gram-positive bacteria include the following genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus. Others include Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella species (Odoemelan, 2013). The general description of the bacteria included in the group is gram-positive, non-spore forming, they are either rod shaped (bacilli) or spherical (coccus) which produce lactic acid as the major end product during the fermentation of carbohydrates (Aslim et al., 2015).  They are microaerophillic, they outcompete other bacteria during natural fermentation. They can withstand increased acidity from organic acid production (e.g lactic acid). They are catalase negative and do not reduce nitrate. Lactic acid bacteria are amongst the most important groups of microorganisms used in the food industry (Leroy and de Vyust, 2014).

Lactic acid bacteria (LAB) have a long and known history about its use in fermented products: the fermentation process as in the production of antimicrobial substances including lactic acid and other organic acids and bacteriocins (Soccol et al., 2010). In addition to the mentioned benefits, LAB can also be characterized as potentially probiotic according to the beneficial effects on consumer health and safety use (Bibek, 2011). Among LAB, Lactobacillus species are usually described as possessing beneficial properties, as well as some Streptococcus, Leuconostoc, Pediococcus and Enterococcus (Fontana et al., 2013).

Lactic acid bacteria (LAB) have long been used as commercial starter culture in dairy industries, fermented beverages, meat and vegetable processing for their contribution to flavor and aroma development and spoilage retardation. They are food-grade microorganisms that are generally recognized as safe (Hammes et al., 2016). Lactic acid bacteria (LAB) have been used in the production of foods, especially fermented foods because not only are these components desirable for their effects on food taste, they can produce several compounds that contribute to smell, color, and texture of the foods but they also inhibit undesirable microflora. Hence, lactic acid bacteria and their products give fermented foods distinctive flavors, textures, and aromas while preventing spoilage, extending shelf-life and inhibiting pathogenic organisms (Bibek, 2011).  In addition, they can produce antimicrobial substances including bacteriocins that have the ability to inhibit pathogenic and food spoilage bacteria. Lactic acid bacteria are capable of inhibiting various microorganisms in a food environment and display crucial antimicrobial properties with respect to food preservation and safety (Achilleos and Berthier, 2013).

Lactic acid bacteria are grouped two; the first group are the homofermentative lactic acid bacteria such as Pediococcus, Streptococcus and Lactococcus species which produce lactic acid as the major or sole end-product of glucose fermentation .The second group are the heterofermentative lactic acid bacteria such as Weissella and Leuconostoc species which produce lactate, CO₂ and ethanol from glucose fermentation. The main LAB groups are gram-positive, catalase negative organisms and belong to genera Lactobacillus, Bifidobacterium, Lactococcus, Pediococcus and Leuconostoc (Leroy and de Vuyst, 2014). Lactic acid bacteria are able to produce acids, hydrogen peroxide and bacteriocins and possessed great potential as food bio-preservatives (Aslim et al., 2015).

LAB are commonly used in fermented food products and their importance is mainly associated with their physiological characteristics, such as their use of different substrates, their metabolic capabilities and their health promoting properties. They are characterised by their organoleptic characteristics, the determination of their products and their interference in the survival and detection of foodborne pathogens (Bas et al., 2014).

Lactic acid bacteria are very promising sources for novel products and applications, especially those that can satisfy the increasing consumer’s demands for natural products and functional foods. They can be used in the diet of humans and animals, with particular role in their health status. Despite recent advances, the study of LAB and their functional ingredients is still an emerging field of research that has yet to realize its full potential (Begley et al., 2016).

Tiger nut (Cyperus esculentus L.) is a tough erect fibrous-rooted perennial plant, 1 to 3 ft high, reproducing by seeds and by many deep, slender rhizomes, which form weak runners above the ground, and small tubers or nutlets at the tips of underground stems. This tuber is rich in energy content (starch, fat, sugar, and protein), minerals (mainly phosphorus and potassium), and vitamins E and C. There are mainly three varieties of tiger nut tubers namely: black, brown and yellow. Only the yellow and brown tubers are readily available in the Nigerian markets. The yellow variety is preferred to all other varieties because of its inherent properties like its bigger size, attractive colour and fleshier body. The yellow variety also yields more milk, contains lower fat and higher protein and less anti-nutritional factors especially polyphenols (Davidson et al., 2010).

The three major products of tiger nut tubers are tiger nut flour, tiger nut oil and tiger nut milk (Onwuakor et al., 2014). The easiest means of consuming tiger nut tubers is by chewing the raw tubers which has a slightly sweet and nutty flavour. Tiger nut-milk is known as tiger nut drink or tiger nut beverage or descriptively known as tiger nut-milk drink. Tiger nut drink, locally called ‘ayah’ is recommended for diabetics and people who are gluten intolerant (Bamishaiye and Bamishaiye, 2011).

Some researchers had isolated lactic acid bacteria (LAB) from the surface of tigernut tubers. In fact, LAB possess adhesional adaptation which gives it ability to survive different environments such as diverse food matrices (Hayek and Ibrahim, 2013). (Bi et al., 2016) in their study isolated LAB from the surface of tigernut tubers. Tiger nut being slightly acidic (pH 6.34) can support the growth of lactic acid bacteria. Previous researches reported that tiger nut is a substrate that can sustain microbial growth possibly due to the near neutral pH of tiger nut tubers which favours the growth of many microorganisms. Therefore, this research is aimed at characterization and identification of lactic acid bacteria isolated from fermented tiger nut milk.

1.2       Aims and Objectives

The aim of the study is concerned with the characterization of lactic acid bacteria isolated from fermented tiger nut milk. The objectives of the study are:

i.    To isolate lactic acid bacteria from fermented tiger nut milk

ii.   To access the identification of lactic acid bacteria isolated from fermented tiger nut milk.

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