ISOLATION AND IDENTIFICATION OF MICROORGANISMS FROM PALM WINE SOLD IN UMUDIKE, ABIA STATE

ABSTRACT

Microbiological analysis was conducted on fresh and fermented palm wine in Umudike, Abia state. Fresh and fermented palm wine samples were collected from a palm wine tapper immediately after tapping and from different palm wine bars respectively, using tightly capped sterile containers and was taken to a microbiology laboratory for analysis. Spread plate and streak plate method was used for the isolation of microorganisms. The microorganisms isolated were identified based on their cultural, morphological and bio-chemical characteristics. The microorganisms include Bacillus spp, Streptococcus spp, Lactobacillus spp, Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Acetobacter spp, Candida spp, and Saccharomyces spp. The mean average microbial counts of the sample were 2.7 × 104cfu/ml, 3.1 × 104cfu/ml, 0.3 × 104cfu/ml, and 3.2× 104cfu/ml for heterotrophic count, fungal isolates, coliform bacteria count and isolates on De Man, Regosa and Sharpe agar respectively. Saccharomyces spp occurred in all the samples (100.0% occurrence) while Bacillus spp, Klebsiella spp and Escherichia coli had the least occurrence (20.0%). In the assessment of the survival of isolates in relation to time (0-96hrs) Klebsiella pneumoniae, Staphylococcus aureus were eliminated from the sample after 24hours while Streptococcus spp, Lactobacillus spp, Escherichia coli and Candida spp were eliminated after 48hours. Bacillus spp, Acetobacter spp and Saccharomyces spp survived beyond 72 hours of fermentation. The presence of Bacillus spp, Staphylococcus aureus, Klebsiella pneumoniae, and Candida spp indicated unhygienic handling of the beverage. It is recommended that appropriate agencies should ensure that proper personal and environmental hygiene is enforced to prevent outbreak of disease.

TABLE OF CONTENTS

Title page i

Certification ii

Dedication iii

Acknowledgements iv

Table of Contents v

List of Tables vii

Abstract viii

CHAPTER ONE 1

INTRODUCTION 1

Background of the study 1

General objective 2

Specific objectives 3

CHAPTER TWO 4

LITERATURE REVIEW 4

2.1 Production of palm wine 4

2.2 Biochemical constituents of palm wine 5

2.2.1 Sugar identified and their concentration in palm wine. 6

2.2.2 pH and organic acid concentration in palm wine 7

2.2.3 Ethanol concentration in palm wine 8

2.2.4 Minerals and trace elements present in palm wine 9

2.2.5 Odorant of palm wine 9

2.3 Microbial communities in palm wine 10

2.4 Fermentation of palm wine 12

2.5 Uses of palm wine 13

CHAPTER THREE 14

MATERIALS AND METHODS 14

3.1 Study Area 14

3.2 Sample collection 14

3.3 Preparation of media 14

3.4 Serial dilution 15

3.5 Isolation of Microorganisms 15

3.6 Characterization and identification of isolates 15

3.6.1 Fungi 15

3.6.2 Bacteria 16

3.6.2.1 Gram staining 16

3.6.2.2 Spore staining 16

3.6.2.3 Catalase test 17

3.6.2.4 Coagulase test 17

3.6.2.5 Oxidase test 17

3.6.2.6 Citrate test 17

3.6.2.7 Indole test 18

3.6.2.8 Carbohydrate fermentation test 18

CHAPTER FOUR 19

Results 19

CHAPTER FIVE 25

DISCUSSION, CONCLUSION AND SUMMARY 25

5.1 Discussion 25

5.2 Conclusion 26

5.3 Recommendation 26

REFERENCES 27

LIST OF TABLES

Table                          Title                Page

1.          Microbial counts of microorganisms isolated from palm wine samples                    20

2.          Identification of bacterial isolates from palm wine samples.                                   21

3.           Identification of fungal isolates from palm wine samples.                                             22

4.          Occurrence of each isolate in the different samples collected from five locations      23

5.           Percentage of occurrence of the isolates from palm wine.

   (fresh and fermented) in relation to time.                                                            24

CHAPTER ONE

INTRODUCTION

1.1 Background Of The Study

Palm wine is a whitish, effervescent alcoholic beverage produced by the spontaneous fermentation of palm sap of palm trees (Elaeis guineensis, Raphia vinifera and Borassus flaelliffer) by indigenous microbes which consist of fungi and bacteria (Ezeronye, 2004; Okereke and Okereke, 2008). This fermentation proceeds until the vinegar is produced and as this takes place, the microbial quality fluctuates (Obire, 2005). However, among the various fermentation stages of palm wine, people consume more of the twenty four (24) hours palm wine and this is due to the alcoholic content as well as the nutritive quality of the beverage, which is due to the activities of the microbes present in palm wine.

Natural palm sap is clean sweet, colourless syrup containing about 10 – 20% of sugar that exudes from the trunk of the various species of palm tree during tapping operations (Elijah et al, 2010).

It is cloudy whitish beverage with a sweet alcoholic taste and has a very short life of only one day. The wine is an excellent substrate from a growing palm. It is collected by tapping the palm (Chandrasekhar et al., 2012).

In Africa, the sap is most often taken from wild date palms such Phoenix sylvestis and caryota urens, from oil palms such as Elaeis guineensis or from Raphia, kithul or nipa palms (Bassir, 1962).

In various traditional African societies, palm wine plays significant role in customary practices (Amoa-Awu et al., 2006). The sap of the palm tree (Elaeis guineesis) serves as a rich substrate for various microorganisms to grow. In various African countries and beyond, the sap of the palm tree is tapped and allowed to undergo spontaneous fermentation, which allows the proliferation of yeast species to convert the sweet substrate into an alcoholic beverage (Amoa–Awu et al., 2006).

The microorganisms in the overnight palm wine have the effect on the palm wine and this effect is due to the increase in their population as well as their activities (Chandrasekhar et al., 2012).

Palm wine differs from conventional beers and table wines produced in the modern brewery and winery in three ways. First, the media fermented for such beers are usually grains and for wines, grapes and fruit juice are used in wine. The basic principle is however, is the same; a sugar solution is fermented, essentially by yeast (Saccharomyces spp). Secondly, whereas there is control of fermentation during the production of modern beers and wine, fermentation of palm wine is not controlled. Thirdly, the European beer and wines are usually clarified by removing microbial cells and other suspended material. Palm wine on the other hand is consumed without such clarification. In essence, the basic differences between the true wines and the palm wine are a matter of technological differences between wine making techniques and palm wine production methods. The method of producing palm wine are likely to continue to change with enhanced technological advancement of the consuming countries (Benjakul et al., 2009).

1.2 GENERAL OBJECTIVE

The main aim of this study is to isolate and identify the microorganisms associated with palm wine sold and produced in Umudike.

1.3 SPECIFIC OBJECTIVE

1. To isolate the bacteria and fungal species present in the palm wine sold and produced in Umudike.

2. To identify the bacteria and fungal isolates from the palm wine.

3. To ascertain the frequency of occurrence of the isolates.

4. To compare the occurrence of the isolates from palm wine sold in Umudike and those produced in Umudike.

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