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This study was aimed to characterize and determine the functional potentials of lactic acid bacteria isolated from fermenting tiger nut milk. The specific objectives were to isolate lactic acid bacteria from fermenting tiger nut milk, characterize and identify lactic acid bacteria using morphological, physiological and biochemical techniques and to ascertain the functional potentials of lactic acid bacteria isolated from fermenting tiger nut milk. The result of the analysis showed that tiger nuts are high in carbohydrate, fat and fibre content. . All the isolates were negative for Catalase test. Many isolates were negative for gas production from glucose and few isolates were positive. All the isolates indicated negative haemolytic test. The antimicrobial properties of the samples was determined by measuring the diameter of zone of inhibition, the maximum zone of inhibition was observed in case of (TMP2472) Lactobacillus pentosus with 14mm zone of inhibition while the minimum was observed in (TMP 051) Lactobacillus fermentum with 3mm zone of inhibition. It has been reported that lower temperature slows down chemical reactions, enzyme actions and microbial growth; it is therefore recommended that when tiger nut milk is not to be served immediately, it should be stored at low temperatures (i.e. in the refrigerator) so as to prevent the growth of spoilage organisms and maintain the quality of the milk.



Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of contents v

List of tables vii

List of figures viii

Abstract ix



1.0 Introduction 1

1.1 Aim and Objectives   4



2.0 Literature Review 5

2.1 Lactic Acid Bacteria (LAB) 5

2.2 Taxonomy of Lactic Acid Bacteria 5

2.3 Sources of Lactic Acid Bacteria 6

2.4 Classification of Lactic Acid Bacteria 6

2.5 Food fermentation 7

2.6 Benefits of Food Fermentation 9

2.7 Advantages of Food Fermentation 10

2.8 Importance of Lactic Acid Bacteria 10

2.9 Beneficial Role of Lactic Acid Bacteria 11

2.10 Importance of LAB and their Effect on Human Health 12

2.11 Lactic Acid Bacteria and Other Effects on the Immune System 13

2.12 Lactic acid fermentation 14

2.13 Importance of Tiger nuts 15



3.0 Materials and Methods 19

3.1 Sample Collection and Preparation 19

3.1.1 Tiger nut milk extraction 19

3.1.2 Glass wares and media preparation 20

3.1.3 pH determination 20

3.2 Proximate Analysis 21

3.3 Microbiological Analyses 23

3.3.1 Enumeration of total bacteria 23

3.3.2   Enumeration of Coliform 24

3.3.3 Enumeration of Yeast 25

3.3.4    Enumeration of Lactic acid bacteria 25

3.3.5   Isolation of Lactic Acid Bacteria 26

3.3.6. Characterization of Lactic Acid Bacteria 26 Morphological characterisation 27 Biochemical characterization 27 Physiological characterization 29

3.3.7  Identification of Lactic Acid Bacteria 30

3.4  Safety Properties of Lactic Acid Bacteria 30

3.4.1  Haemolytic test 30

3.4.2 Antibiotic resistance pattern    31

3.5    Determination of Functional Potentials 31

3.5.1   Tolerance to low pH 3.5 and 2.5 31

3.5.2   Tolerance to 0.3% Bile salt 31

3.5.3   Antimicrobial properties 32


4.0 Results 34


Discussion, Conclusion and Recommendations 45

5.1 Discussion 45

5.2 Conclusion 46

5.3 Recommendations 47

References 48




Table Title Page


1: Proximate Composition of Fermenting Tiger nut milk 33


2: pH and microbial load of tiger nut milk during fermentation 34


3: Morphological Characteristics of Lactic Acid Bacteria Isolated from Tiger

nut milk 36


4: Biochemical and physiological characteristics of Lactic Acid Bacteria

isolated from fermenting Tiger nut milk 37


5: Haemolytic activity of Lactic Acid Bacteria isolated from fermenting

Tiger nut milk 40


6: Result for antibiotic sensitivity 41


7: Antimicrobial properties of lactic acid bacteria 44















Figure Title Page


1: Tiger nut milk extraction 19

2: Tolerance to low pH 42

3: Tolerance to 0.3% bile salt








2.0                                            INTRODUCTION

At the turn of the 20th century the term “lactic acid bacteria” (LAB) was used to refer to “milk-souring organisms.” While similarities between milk-souring organisms and other bacteria producing lactic acid were soon observed. Lactic acid bacteria have traditionally been associated with food and feed fermentations, and are generally considered beneficial microorganisms, some strains even as health-promoting (probiotic) bacteria. However, some genera (Streptococcus, Lactococcus, Enterococcus, Carnobacterium) also contain species or strains that are recognized human or animal pathogens (Li et al., 2010).

Lactic acid bacteria (LAB) are a group of Gram positive, non-spore forming cocci or rod shaped, catalase negative and fastidious organisms, considered as generally recognised as safe (GRAS) organisms. They are a group of related bacteria that produce lactic acid as a result of carbohydrate fermentation. These microbes are broadly used in our day to day life in the production of fermented food products, such as yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.), sauerkraut (Leuconostoc spp.) and sausage. These organisms are heterotrophic and generally have complex nutritional requirements because they lack many biosynthetic capabilities (Campbell, 2011). Most species have multiple requirements for amino acids and vitamins. Because of this, lactic acid bacteria are generally abundant only in communities where these requirements can be provided. They are often associated with animal oral cavities and intestines (eg. Enterococcus faecalis), plant leaves (Lactobacillus, Leuconostoc) as well as decaying plant or animal matter such as rotting vegetables, fecal matter, compost, rhizosphere soils etc (Lengkez et al., 2009).

Phylogenetically, lactic acid bacteria belong to the Clostridium branch of Gram positive bacteria. They are non-sporing, aero tolerant anaerobes that lack catalase and respiratory chain, with a DNA base composition of less than 53 mol% G+C. According to their morphology LAB are divided to rods and cocci and according to the mode of glucose fermentation to homofermentative and heterofermentative. The homofermentative LAB convert carbohydrates to lactic acid as the only or major end-product, while the heterofermentative produce lactic acid and additional products such as ethanol, acetic acid and carbon dioxide (Kivanç et al., 2009). Thus, the main metabolism of LAB is the degradation of different carbohydrates and related compounds by producing primarily lactic acid and energy. Although many genera of bacteria produce lactic acid as primary or secondary fermentation products, typical lactic acid bacteria are those of the Lactobacillales order, including the following genera: Lactobacillus, Carnobacterium, Lactococcus, Streptococcus, Enterococcus, Vagococcus, Leuconostoc, Oenococcus, Pediococcus, Tetragonococcus, Aerococcus and Weissella (Leben and Daft, 2015).

Many strains of lactic acid bacteria are among the most important groups of microorganisms used in the food and feed industries, although some of the genus Pediococcus cause deterioration of foods, which results in their spoilage. Lactic acid bacteria have been used in food preservation and for the modification of the organoleptic characteristics of foods, for example flavors and texture (Bukola et al., 2008). Various strains of lactic acid bacteria can be found in dairy products (yoghurt, cheese), fermented meats (salami), fermented vegetables (olives, sauerkraut), sourdough bread, etc. The European Food Safety Authority (EFSA) has stated that several LAB strains can be considered to have “Qualified Presumption of Safety” QPS-status (Kebede, 2008).

Lactic acid bacteria are used in the food industry for several reasons. Their growth lowers both the carbohydrate content of the foods that they ferment, and the pH due to lactic acid production. It is this acidification process which is one of the most desirable side-effects of their growth. The pH may drop to as low as 4.0, low enough to inhibit the growth of most other microorganisms including the most common human pathogens, thus, allowing these foods with prolonged shelf life (Benson, 2012). The acidity also changes the texture of the foods due to precipitation of some proteins, and the biochemical conversions involved in growth enhances the flavor. The fermentation and growth of the bacteria is self-limiting due to the sensitivity of lactic acid bacteria to such acidic pH. Besides their lactic acid forming capacity, lactic acid bacteria also have the ability to contribute to other product characteristics like flavor, texture and nutrition (Kavitha and Pradeep, 2010).

Mankind has exploited lactic acid bacteria for thousands of years for the production of fermented foods because of their ability to produce desirable changes in taste, flavor and texture. Different antimicrobial molecules such as lactic acid, acetic acid, hydrogen peroxide and bacteriocins produced by these bacteria are widely known to inhibit foodborne pathogens and food spoilage micro-organism, thereby extending the shelf-life and enhancing the safety of the food products (Azadnia and Nazer, 2009). The LAB in the fermented foods not only contributes to the taste, aroma and texture but also lower the product’s pH, effectively promoting its quality and safety. Lactic acid bacteria (LAB) are widespread microorganisms which can be found in any environment rich mainly in carbohydrates, such as plants, fermented foods and the mucosal surfaces of humans, terrestrial and marine animals (Arwiyanto et al., 2010). In the human and animal bodies, lactic acid bacteria are part of the normal microbiota or microflora, the ecosystem that naturally inhabits the gastrointestinal and genitourinary tracts, which is comprised by a large number of different bacterial species with a diverse amount of strains (Karnwal, 2009; Aslam and Javed-Qazi, 2010).

Tiger nut (Cyperus esculentus) is an edible perennial grass-like plant. In Nigeria, tiger-nut is known as ‘Aya’ in Hausa, ‘Ofio’ in Yoruba and ‘Akiawusa’ in Igbo. Three varieties (black, brown and yellow) are cultivated in the country and among these, only two varieties; (yellow and brown) are readily available in the markets. The Yellow variety also yields more milk upon extraction, contains lower fat and more protein and possesses less anti-nutritional factors especially polyphenols (Okafor and Nwachukwu, 2013). Tiger-nut milk popularly known in the Northern part of Nigeria as ‘Kunun aya’ is one of the indigenous, locally fermented, non-alcoholic beverage drinks that is widely consumed for its thirst-quenching and nutritive properties (Okafor and Nwachukwu, 2013; Ayo and Okaka, 2008).

The study of functional and beneficial properties of lactic acid bacteria has being considered a trend in the food industry in the last decades. Among different positive aspects that these bacteria can provide and deliver to consumers, the characterization of their probiotic properties is of particular interest from nutritional and medical aspects. The characterization, functional potentials and health benefits of lactic acid bacteria may include balancing intestinal flora, modulation of the immune system, production of bioactive substances, pathogen exclusion, and reduction of the risk of intestinal infectious diseases, cardio vascular disease, obesity, and cancer (Frazier and Westhoff, 2009; Bibek, 2011). Among lactic acid bacteria, Lactobacillus species have a great attention for their functional potential effects in human health, and are probably the most important bacteria in the food industry because they are widely used as starter cultures to play significant roles in the production of fermented foods (Farhath et al., 2011;  Akoma et al., 2016).

1.1 Aim and Objectives  

The aim of this work is to characterize and determine the functional potentials of lactic acid bacteria isolated from fermenting tiger nut milk.

The objectives for this study are to:

i. Isolate lactic acid bacteria from fermenting tiger nut milk

ii. Determine the characteristics and identify lactic acid bacteria using morphological, physiological and biochemical techniques.

iii. Determine the functional potentials of lactic acid bacteria isolated from fermenting tiger nut milk.



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