ISOLATION AND IDENTIFICATION OF PROBIOTIC LACTOBACILLUS SPECIES FROM AKAMU, A NIGERIA FERMENTED CEREAL

Abstract

Probiotic is a viable microbial dietary supplement that is beneficial for the host through its effects in the intestinal tract. Akamu is an important source of Lactobacilli. The “akamu” samples were collected aseptically from three different markets in Abia State. Pure culture of specific probiotic isolate from each sample was preformed and identified on the basis of their colonies morphology and some biochemical test such as catalase, oxidase, indole, citrate utilization and sugar fermentation test. Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii species were the bacteria isolate while in the bile salt test, the isolate Lactobacillus spp. was able to survive in 0.2, 0.3and 2.0% bile acid. The isolated Lactobacillus spp. was also able to multiply in above mentioned concentrations of bile acid. Therefore, it was concluded that the isolation of the microorganisms from the “akamu” samples confirmed that it could serve as a vehicle for the transmission of potentially pathogenic microorganism.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Declaration                                                                                                                             ii

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Abstract                                                                                                                                  ix

CHAPTER ONE: INTRODUCTION                                                                                 1

1.1 Background to the Study                                                                                                  1

1.2 statement of problem                                                                                                        3

1.3 objective of the study                                                                                                       3

CHAPTER TWO: REVIEW OF RELATED LITERATURE                                         4

2.1 Traditional Cereal-Based Foods                                                                                       4

2.2 Traditional Fermented Cereal-Based Foods In West Africa                                            6

2.3 Steam-cooked Granulated Products                                                                                 6

2.4 Importance of Fermented Cereal-Based Foods                                                                7

2.4.1 Healthy and Safe Products                                                                                            7

2.4.2 Probiotic and Prebiotic Potentials                                                                                 8

2.4.3 Nutritional and Health Benefits                                                                                     9

2.5 Selection Criteria of Dried Lactic Acid Bacteria Starter                                                  10

2.5.1 Cultures for Cereal Dough Fermentation                                                                      10

2.5.2 Ability to Realize Fast Acidification and Production of Antimicrobial Compounds 10

2.5.3 Ability to Dominate the Indigenous Microbiota                                                           10

2.5.4 Ability to Exert Probiotic Effects                                                                                  11

2.5.5 Ability to Improve the Nutritional Quality of the Fermented Food                                     11

2.5.6 Ability to Hydrolyse Starch                                                                                           12

2.5.7 Ability to Have Good Stability during Production and Storage                                     12

CHAPTER THREE: METHODOLOGY                                                                          14

3.1 Sample Collection                                                                                                            14

3.2 Media Used                                                                                                                       14

3.3 Isolation of Lactic Acid Bacteria                                                                                     14

3.4 Identification of Microbia Isolates                                                                                   14

3.4.1 Gram Staining                                                                                                               14

3.4.2 Motility Test                                                                                                                  15

3.4.3 Catalase Test                                                                                                                  15

3.4.4 Oxidase Test                                                                                                                  15

3.4.5 Indole Test                                                                                                                     16

3.4.6 Sugar Fermentation Test                                                                                               16

3.5 Bile Salt Tolerance Test                                                                                                   17

CHAPTER 4: RESULT                                                                                                       18

CHAPTER 5: DISCUSSION, CONCLUSION AND RECOMMENDATION                    20

5.1 Discussion                                                                                                                         20

5.2 Conclusion                                                                                                                        21

5.3 Recommendation                                                                                                             21

REFERENCE                                                                                                                         22

APPENDIX

LIST OF TABLE

Table 1: Result of bacteriological and biochemical analysis of isolate                 18

Table 2. Bile Salt Tolerance Test Result                                                               19

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND TO THE STUDY

Akamu is a name given to a popular fermented cereal porridge made from crops, maize, sorghum, and millet. “Akamu” is a nutritive diet that is mostly eaten at infancy as a weaning food. But adults also enjoy this delicacy (Ezendianefo and Dimejesi, 2014). According to Adeyola et al., (2018) maize slurry (Akamu) is a product of fermentation of maize (corn), millet or sorghum. Maize slurry (Akamu) can be processed into a whitish or yellow-like custard depending on the variety of maize used for the fermentation. Akamu is classified among the breakfast foods (Obire and Amadi, 2015). Akamu is a porridge prepared from fermented maize. It is a popular breakfast cereal and infant weaning food among the Igbo speaking people of Nigeria. Akamu is similar to ogi, a lactic acid fermented food made from maize, sorghum or millet which may the fortified with legumes (Banigo and Muller, 2011). According  to  Odunfa, 2015 akamu is a locally prepared food from fermented maize, sorghum or millet in Nigeria. It serves as a weaning food for infants. The preparation of akamu involves soaking of corn in water for 1 to 3 days followed by wet milling and sieving. The filtrate is fermented for 2-3 days to produce white starchy sediment. Akamu is prepared by soaking clean maize grains in water for 2-3 days. The grains are washed and ground to a paste. The paste is sieved to smooth slurry which is allowed to settle and the supernatant decanted. The slurry is mixed with hot water with stirring until it forms a get which serves as food (Ighedioh, Ogbeni, and Adole, 2016). Production of “akamu” is carried out mainly by local producers, and there is risk of high microbial contamination which often makes the food products undesirable due to the presence of organisms that cause food spoilage, food poisoning or food intoxication in the food product (Awada et al., 2015). It is the first native food given to babies at weaning. Preparation of Akamu involves the steeping of the maize grains for two days and wet milling thereafter. Water is added to the mash and is sieved through a clean cloth. The filtrate is allowed to sediment for a day and dewatered with a clean cloth sac (Obire, and Amadi, 2015).

Fermentation process serves as a mean of providing a source of nourishment for large rural populations Fermentation enhances the nutrient content of foods through the synthesis of proteins, vitamin, and essential amino acids (Zhang, et al., 2010). A wide spectrum of micro organism is involved during fermentation process. But a few types usually determine the quality of end products (Abegaz, 2007). Therefore, isolation, characterization and identification of the microorganisms involved in fermentation of cereal with a prospective selection of starter cultures would be important to support the technical process and to obtain a predictable end-product with a desired quality. The major microorganisms implicated in fermentation of maize products include lactic acid bacteria (LAB) such as Lactobacillus fermentum, Lactobacillus reuteri, Lactobacillus rhamnosu, Lactobacillus casei, Lactobacillus plantalum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus alimemtarius, Lactococcus lactis, Entorococcus faecium, Leuconostoc mesentoroids and Pediococcus spp as well as yeasts (Tamang, 2016)

The Lactobacillus genus consists of a genetically and physiologically diverse group of rod-shaped, Gram-positive, non-spore forming, nonpigmenetd (Hasan and Frank, 2010), catalase negative and microaerophilic to strictly anaerobic lactic acid bacteria (LAB) that have widespread use in fermented food production and are considered as generally recognized as safe (GRAS) organisms and can be safely used for medical and veterinary applications (Fuller, 2018).

Lactic acid bacteria (LAB) are non pathogenic organism widely distributed in nature. LAB have an important role in the preservation of foods and fermented products and are designated as GRAS (Generally regarded as safe). The genus Lactobacillus is the largest group among the Lactobacteriaceae, and contains over 100 species (Canchaya et al., 2006). They are characterized as Gram-positive rods, anaerobic but aero tolerant, non-sporulating and catalase negative (De Vuyst and Leroy, 2007).

Probiotics are live microorganisms, which when administered in adequate doses, confer health benefit on the host. However, it is recommended that any probiotic strain used in food matrix should generally be regarded as safe (GRAS) devoid of potentially transferable antibiotic resistant traits (FAO/WHO. 2012).

1.2 statement of problem

Traditional fermentation depends on inoculation of earlier fermented products, however, commercial starter cultures are currently available to ensure constancy and reliability of processes and products. Traditional fermented products are mostly processed in a non-sterile environment which creates a selection of microorganisms that produce the desired end product however there is an increased risk of spoilage and unsafe products as a result of uncontrolled fermentation.

It is common knowledge in Nigeria that some persons including children and adult fall ill after the consumption of Akamu. Therefore microbial load and types of microorganisms with emphasis on Lactobacillus species in fermented and dewatered maize slurry (Akamu), and their isolate and characterize of the Lactobacillus species and the antibiotic susceptibility of the Lactobacillus species isolated because most bacteria are potential pathogens.

1.3 Objective of the study

The main objective of the study is to isolate and identify the probiotics Lactobacillus species from akamu, a Nigeria fermented cereal. The specific objectives are to;

1.     To isolate and identify lactobacillus spp. from akamu

2.     Determination of probiotic potential.

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