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This study was aimed at characterizing and determining the technological potentials of lactic acid bacteria isolated from fermented tiger nut milk. The result of the studies showed that tigernuts are high in carbohydrate, fat and fibre contents. All the isolates were negative for Catalase test. Some of the isolates were negative for gas production from glucose while few of the isolates were positive. All the isolates indicated negative haemolytic test. In the antibiotic sensitivity test, majority of the isolates tested were antibiotic sensitive while others were antibiotic resistance. Production of exopolysaccharides investigated in this study was considered a positive characteristic for the fermentation of tiger nut milk, except (TMT2464, TMT2472 and TMT2462). Amylase abilities by the lactic acid bacterial isolates from the tiger nut milk were all negative. The results showed fermented tiger nut milk contain 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 including yoghurt.


Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of contents v

List of figures vii

List of tables viii

Abstract ix



1.0 Introduction 1

1.1 Aims and Objectives 4



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


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 Proximate Analysis 18

3.4 Microbiological Analyses 22

3.4.1 Enumeration of total bacteria 22

3.4.2 Enumeration of Coliform 23

3.4.3 Enumeration of Yeast 23

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 Morphological characterisation 26   Biochemical characterization 26 Physiological Characterization 28

3.4.7 Identification of Lactic acid bacteria 29

3.5 Safety Properties of Lactic Acid Bacteria 29

3.5.1 Haemolytic test 29

3.5.2 Antibiotic resistance pattern    30

3.6 Determination of Technological Properties 30

3.6.1 Screening for Amylase Production 30

3.6.2 Screening for Esopolysaccaride Production 31

3.6.3 Rate of Acid Production 31



4.0 Results 32


5.0 Discussion, Conclusion and Recommendations 43

5.1 Discussion 43

5.2 Conclusion 45

5.3 Recommendations 46

References 47


Table 4.1: Proximate Composition of Fermented Tiger nut milk 35

Table 4.2: pH and microbial load of tiger nut milk during fermentation 36

Table 4.3: Morphological Characteristics of Lactic Acid Bacteria Isolated from

Fermented Tiger nut milk 37

Table 4.4: Biochemical and physiological characteristics of Lactic Acid Bacteria

isolated from fermented Tiger nut milk 38

Table 4.5: Haemolytic test results and antibiotic sensitivity of Lactic Acid Bacteria

isolated from fermented Tiger nut milk 39

Table 4.6: Exopolysaccharide and Amylase production of lactic acid bacteria isolated

from fermented tiger nut milk 40






Figure 4.1: Rate of acidification of MRS broth by selected lactic acid bacterial isolates

from fermented tiger nut milk 41










1.0                                                 INTRODUCTION


Lactic acid bacteria (LAB) are the main technological 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 technological potentials of lactic acid bacteria isolated from fermented tiger nut milk.

1.1 Aims and Objectives

The aim of the study is concerned with the characterization and technological potentials 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 determine the characterization of lactic acid bacteria isolated from fermented tiger nut milk using morphological, physiological and biochemical techniques.

iii. To access the technological potentials of lactic acid bacteria isolated from fermented tiger nut milk.



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