CHARACTERIZATION AND STATER CULTURE POTENTIAL OF LACTIC ACID BACTERIA ISOLATED FROM KUNU ZAKI

ABSTRACT

Kunun zaki is a fermented non-alcoholic cereal beverage. It is a popular refreshing drink in northern Nigeria. The aim of this study is to determine the starter culture potentials of yeasts associated with the traditional fermentation of millet for kunun zaki production. The kunu- zaki were purchased from two different local house producers of kunu zaki in Umuahia, Abia State and was transported to the laboratory for microbiological analysis. The mixture was serially diluted in a distilled  water  and from the 10 fold dilutions, colony, colony forming units (Cfu) were determined using pour plate method. Plate counts were carried out using the following media, temperature and incubation periods, De Mann Rogosa- Sharpe (MRS) agar, 370C, 48hour  for LAB, Malt extract agar (MEA) supplemented with streptomycin (370C) 72hours for yeasts and nutrient agar (NA) 370C, 24-48h for total viable counts, incubation for LAB  was done under anaerobic conditions. After incubation and several biochemical and physiological test was carried out,  ten Lactobacillus species were revealed which include Lactabacillus fermentum, Lactabacillus plantarum, Lactabacillus salivarius, Lactabacillus amylolyticus, Lactabacillu sreuten, Lactabacillus paraplantarumi, Lactabacillus ikii, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus casei. The pH and microbial count of Kunu Zaki. pH level of the kunu zaki ranged from 6.3 to 2.69, the total bacteria count of the kunu zaki ranged from 7.229± 0.79 to 5.532±0.54, the coliform count showed ranged from 4.411±20.00 to 4±00, LAB ranged from 9.025±0.62 to 6.301±0.00 while the Yeast showed a range of 7.104±0.00 to 6.031±0.25. the Antibiotic resistance pattern of lactic acid bacteria isolated from kunu zaki this result it showed that the isolates where resistant to Chloramphenicol, Spafloacine, Amoxacillin, Gentamicin, Travid, Streptomycin, Augumentin and Erythromycin. The presence of coliform bacteria and Yeast in Kunun-zaki drinks as determined in this research will be of public health concern because teaming populace, especially students, relies on these drinks as cheaper alternative to the bottled soft drinks. Preservation method must therefore be applied during sales and distribution to make the product wholesome for consumption. The information obtained from this work should form baseline data on which preservation procedure should be adopted in order to retain the quality of this product.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

Lists of Tables                                                                                                                        vii

Abstract                                                                                                                                   viii

CHAPTER ONE

1.0  Introduction                                                                                                                1

1.1 Aim                                                                                                                                    4

1.2 Objectives                                                                                                                         4

CHAPTER TWO

2.0  Literature Review                                                                                                                   5

2.1 Production of Kunun-Zaki Using Starter Culture                                                            6

2.2 Quality Characteristics of Kunun Zaki Sold Within Bida Metropolis                                     7

2.3 Processing And Evaluation of Instant Kunun Zaki from                                                  10

Millet-Cowpea Malt And Millet-Soybean Malt

2.2       Historical Perspective of Fermentation                                                                      12

2.3       Starter Cultures                                                                                                           13

2.3.1    Bacteria                                                                                                                       14

2.4       Functions of Starter Cultures                                                                                      14

2.5       Characterization and Identification of Microorganisms In Fermented Foods        16

2.5.1    Phenotypic methods                                                                                                   16

2.5.2    Genotypic methods                                                                                                     17

CHAPTER THREE

3.0 Materials and Methods                                                                                                     19

3.1 Sample Collection                                                                                                            19

3.2 Media Used                                                                                                                       19

3.3 Sterilization                                                                                                                      19

3.4 Determination of pH                                                                                                         20

3.5  Microbiological Analysis                                                                                                20

3.5.1 Enumeration of Total Bacteria                                                                                      20

3. 5.2 Enumeration of Total Coliform                                                                                    20

3.5.3 Enumeration of Yeasts                                                                                                  21

3.5.4 Isolation of Lactic Acid Bacteria                                                                                   21

3.5.5 Isolation of Lactic Acid Bacteria                                                                                  21

3.5.6 Characterization of Lactic acid Bacteria                                                                       21

3.5.6.1 Morphological Characterization                                                                                 22

3.5.6.2 Biochemical Characterization                                                                                    22

3.5.6.3 Physiological Characterization                                                                                   23

3.5.7 Identification of Lactic acid bacteria                                                                             24

3.6 Haemolysis Test                                                                                                               25

3.6.1 Rate of Acid Production                                                                                                25

3.6.2  Antibiotic Resistance Pattern                                                                                       25

CHAPTER FOUR

4.0       Results                                                                                                                        27       

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation                                                          33       

5.1       Discussion                                                                                                                   33

5.2       Conclusion                                                                                                                  34

5.3       Recommendation                                                                                                       35        

References                                                                                            

LIST OF TABLES

Table                                                              Title                           Page

1      pH and microbial count of Kunu Zaki                                      28

2      Morphological characteristics of lactic acid bacteria isolated from kunu zaki             29

3      Biochemical and physiological characteristics of lactic acid bacteria isolated from Kunu zaki           30

4      Haemolystic test for lactic acid bacteria isolated from kunu zaki                31

5      Antibiotic resistance pattern of lactic acid bacteria isolated from kunuzaki       32      

CHAPTER ONE

1.0       INTRODUCTION

Kunu which is also known as kunu-zaki is the traditional Hausa name of a non-alcoholic beverage which is widely consumed in Nigeria. It’s a major source of calories and supplied large proportion of energy in form of carbohydrates. Kunu is taken as an alternative to beverage and is mostly consumed throughout Nigeria, mostly in the North (Ikpoh et al., 2013). Kunu drink is relatively cheap due to the availability of its constituent materials. It is made from cereal grains such as maize (Zea mays), Millet (Pennisetum typhoideum) and Sorghum (Sorghum vulgare) (Gaffa et al., 2002). Garlic, pepper and ginger are some other ingredients which are added to enhance its flavor, while honey or sugar is also added to serve as sweetener. Fura, Burukutu, Buwo, Burakosko, Akamu, Danwake, Bulum, Pito to mention but a few are other traditional foods which are often fabricated from cereals in the northern part of Nigeria (Mamudu et al., 2013).  Acha, guinea corn, maize and millet contain 70 to 77% of carbohydrate with little fat and protein and about 12% moisture contents in the whole grain (Olusola et al., 2000). The variety of drinks made from sorghum is a milky light-brown colour, whilst that is made from millet and maize is whitish in colour. Carbohydrate, proteins, minerals and fat can be obtained from cereals but carbohydrates are the prominent diet produced from cereals, while vitamins and proteins are less produced from cereals (Adebayo et al., 2010).

Minerals such as manganese, iron, zinc and copper can be obtained from the intake of kunu (Cheesbrough, 2004). In Nigeria, particularly in the North, kunu is widely consumed by young and old as thirst quencher and also for refreshment at home, office or in the market place. Owning to the low protein contents, kunu is often supplemented (Innocent et al., 2011).

One of the most commonly used supplements in Nigeria is milk. Kunu can be taken with bread or snacks. Some people prefer to take kunu drinks as an alternative to soft drinks which many believe to be associated with diabetes. Gaffa et al., (2002) reported the microbiological quantity, consumption rate and nutrient contents of kunu. The high demand for kunu drinks is believe to be due to the presence of carbohydrates which serve as source of energy, protein (although of low content) and vitamins usually vitamins B (Otaru et al., 2013). When kunu beverage is exposed to air without refrigeration for two to three days, its nutritive value diminishes and this lead to the spoilage of the kunu product owning to enzyme actions, poor handling and frequent fermentation process of the carbohydrate contents (Amusa and Ashaye, 2009). According to healthdiaries.com, millet contains a polynutrients called lignin, which has cancer fighting properties and is beneficial in the treatment of heart disease and also reduce the risks associated with diabetes. Ginger content in kunu helps to lowers cholesterol level and prevents the formation of blood cloths. Chronic inflammatory diseases such as rheumatoid arthritis can also be prevented through the intake of kunu (Essien et al., 2011).

Fermentation involves the use of microorganisms and enzymes to produce foods with distinct quality attributes, quite different from the original agricultural raw material. The process depends on the biological activity of microorganisms to produce a range of metabolites which suppress the growth and survival of undesirable microflora in foodstuffs (Ross et al., 2002). It is one of the oldest and most economical methods of producing and preserving food, and provides a natural way to reduce the volume of the material to be transported; destroys undesirable components, enriches the nutritive value and appearance of the food, decreases the energy required for cooking and results in a safer product (Kassa and Hailay, 2014).

Fermentation may be a useful strategy for reducing bacterial contamination of food. The number of harmful microorganisms (Staphylococci, Coliform bacteria "E.coli" and Salmonella) in sorghum significantly decreased with the increase of fermentation period and could also reduce the prevalence of diarrheal diseases (Kassa et al., 2014).

According to Egounlety et al, (2002), fermentation is a low-cost and the most economical technique of production and preservation of foods. It helps to preserve perishable foods and to improve their nutritional and organoleptic qualities. As of 1995, fermented food represented between one quarter and one third of food consumed in Central Europe (Essien et al, 2011). In addition, fermented foods are of particular importance in ensuring adequate intake of protein and/or calories in the diet. Food fermentation, and especially lactic acid fermentation, is an important technology in Africa, indigenous and adaptable to the culture of the people.  There are many cereal based fermented foods in Africa, such as ogi and mahew in Benin, kenkey in Ghana, injera in Ethiopia, poto-poto in Congo, ogi and kunu-zaaki in Nigeria, uji and togwa in Tanzania, kisra in Sudan (Blandino et al., 2003). The desirable changes of taste, flavor, acidity, digestibility, and texture in these gruels are contributed by fermentation. The cereals most commonly fermented are maize, sorghum, millet, tef and occasionally rice and wheat (Egounlety et al., 2002).

The use of starter cultures has been suggested by Kimaryo et al. (2000), as an appropriate approach for the control and optimization of the fermentation process in order to alleviate the problems relevant of variations in organoleptic quality and microbiological stability observed in African indigenous fermented foods. The development of starter cultures is however one of the pre-requisites for the establishment of small scale industrial production of fermented foods in Africa (Ross et al., 2002).

1.1       AIM

The aim of this work is to determine the starter culture potentials of yeasts associated with the traditional fermentation of millet for kunun zaki production.

1.2       OBJECTIVES

·       To determine the physicochemical properties of Kunu zaki

·       To enumerate microorganisms associated with Kunu zaki.

·       To isolate and characterize Lactic acid bacteria from kunu zaki.

·       To evaluate the suitability of selected Lactic acid bacterial isolates as starter culture.

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