ETHANOL TOLERANCE OF YEAST IN PALM WINE

ABTRACT

The investigation of the ethanol tolerant yeast in palm wine was carried out .isolation of yeast from fresh palm wine was examined with the use of Sabraud agar and glucose yeast agar. Yeast were isolated from palm wine and characterized and identified five randomly selected Palm wine were cultured by pour plate and spread plate method obtained results showed five primary yeast species with varying prevalent in palm wine samples. Five yeast were isolated Saccharomyce cerevisiae, Saccharomyce globbus, Saccharomyce uvarum and Saccharomyce latis. Sacchromyce cerevisiae was found in all samples.

TABLE OF CONTENTS

Title Page …………….………………………………………………………..………i

Certification………….……………………………………………………………….. ii   

Dedication ………….…………………………………………...……….……………iii 

Acknowledgment ….………………………………….……...………….……….…...iv 

Table of Contents …………………………………………………………………......vi

List of tables……………………………………………………………………...........vii

Abstract... …………………………………………………………………..……….…v 

1.0    Introduction...……………………………………………………………….........1

1.1    Background of study……………………………………………………………..1   

1.2    Objectives of the research……………………………...…………………………4

CHAPTER TWO      

2.0    Literature Review…………………………………………..……………………5

2.1    Yeast……………………………………………………………………………  6

2.2    Taxonomy of borne yeast………………………………………………………..6

2.3    Yeast strain………………………………………………………………………8

2.4    Characteristics of yeast………………………………………………………….12

2.5    Growth and nutrition of yeast……………………………………………...........13

2.6    Factors affecting yeast growth………………………………………………….13

2.7    Reproduction………………………………………………………………........14

2.8    Uses of yeast…………………………………………………………………….15

CHAPTER THREE

3.0   Materials and Methods…………………………………………………………..17

3.1   Source of materials……………………………………………………………….17

3.2   Sampling and sample preparations……………………………………………….17

3.3   Media preparations……………………………………………………………….17

3.4   Isolation of yeast from palm wine……………………………………..………....18

3.5   Tests from ethanol tolerance……………………………………………………...18

3.6    Characterization of yeast isolates………………………………………………...19

CHAPTER FOUR

4.0   Results……………………………………………………………………….……21

CHAPTER FIVE

5.0   Discussion…………………………………………………………………………30

5.1   Conclusion………………………………………………………………………...30

5.2   Recommendation………………………………………………………………….31

References

LIST OF TABLES

Table                                                  Title                                                            Page

1                                              Characterization of yeast isolate                              21

2                                       Frequency of occurrence of yeast in fresh palm wine    24

3                     Tolerance of yeast isolates for ethanol produced during palm wine 26

4                    Changes in acohol content of fermenting palm wine                         28

CHAPTER ONE

1.0  INTRODUCTION

1.1 BACKGROUND OF STUDY

The tolerance mechanism in yeast is not very well understood. This may result from the complex inhibitory mechanisms of ethanol, the lack of a universally accepted definition and method to measure ethanol tolerance and its complex polygenic characteristic. Recently, there has been some progress in understanding ethanol tolerance. Plasma membrane phospholipids have been shown to play an important role in the ethanol tolerance mechanism increases in membrane unsaturated fatty acids result in increased yeast ethanol tolerance. Supplementation of growth media with combinations of unsaturated fatty acid, vitamins and proteins also enhance ethanol tolerance. Physiological factors such as mode of substrate feeding, intracellular ethanol accumulation, temperature and osmotic pressure all contribute to the ethanol tolerance of yeast. The complex nature of ethanol toxicity suggests that a number of different genes are likely to be involved in the ethanol tolerance mechanism. Genetic approaches such as spheroplast or protoplast fusion, hybridization and continuous culture selection have been used to obtain ethanol tolerance yeasts; isolation and characterization of such strains may provide a better understanding of ethanol tolerance (1987 published by Elsevier Inc.). Nowadays, manufacturers have begun to show unprecedented interest in innovations by introducing benefits from re-engineering and genetic manipulation. These innovations were ushered in because of certain constraints intrinsic to the organism being used for the respective manufacturing process. These include the need to improve yeast resistance to ethanol, mutagens, temperature, carbon dioxide as well as eliminating production of other compounds which may contaminate the production of other compounds which may contaminate the production of other compounds which may contaminate the product and of course improve both yield and product recovery (Aunstrup,2007).In order to obtain strain showing more suitable properties genetic manipulation method have been used (Dubey,2009).However due to the euploid, diploid or polyploid nature of most strains of yeast used diploid or polyploidy nature of most strains of yeast used in ethanol fermentation, traditional crossing techniques have not been very useful. This made the use of other technologies such as classical mutagenesis, protoplast fusion and transformation necessary (Arnord and jose,2008).However classical mutagenesis which involves the production of mutants by the exposure of microbial strains to mutagenic chemicals or ultraviolet rays to induce changes in their genomes leading to the production of improved strains would be the main focused of this research work seven days (168 hours ) ethanol tolerance saccharomyces specie s isolated from palm wine sap would be subjected to mutagenic effect of acridine orange (a basic fluorescent dye that selectively binds to genetic material and can differentiate between deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),such mutants may, however show undesirable secondary mutation which can influence the behavior of cultures during fermentation. Sacchromyce appear microscopically as globose or ovoidal cells with multilateral budding and possibly psedohyphae .The yeast form one to four ascospors ,which are smooth and ellipsoidal. Colonies appear smooth, usually flat and occasionally raised and opaque (martini and martini, 1998). The two primary species found in wines, Sacchromyce bayanus and Sacchromyce cerevisiae (anamorph: candida robusta),ferment glucose, sucrose, maltose, raffinose,and ethanol but not nitrate. Sacchromyce can not utilize five carbon sugars ( pentoses) (Kudo et al,1988). On a dry weight basis Sacchromyce  contains 3%to5% phosphate,2.5% potassium,0.3%to 0.4% magnesium,0.5% sulfure, and trace amount of calcium, chlorine, copper, iron, zinc, and manganese(monk, 1994; walker,1998).Other minerals needed by Sacchromyces during fermentations have a variety of function but are used primarily as enzymes activators. Beside minerals,yeasts require various vitamins such as thiamin, riboflavin, pantothenic acid,pyridoxine,nicotinamide, biotin,and inositol depending on species and specific growing conditions (Monk, 1994;Ough et al.,1989).In general, practically all strains of Sacchromyces require biotin and pantothenic acid while some also need inostilol and thiamin for synthesis of nucleic acid, protein and fatty acids (Walker, 1998).

Palm wine is a traditional wine extracted from palm trees Elaeis guineesis and Raphia hookeri.palm wine is consumed because of its probiotic content (Ezeronye 2004;lourens-hattinigh and Wiljeon 2001; Heller 2001). Palm wine is consumed throughout the tropics and appears as a whitish liquid produced by natural fermentation of the sap of Elieas guineensis and Raphia hookeri (Uzogara et al, 1990; Uzochukwu et al, 1991). The unfermented sap is clean, sweet, colourless syrup containg about 10-12% sugar which is mainly sucrose (Bassir, 1962; okafor,1975a).Upon fermentation by the natural microbial flora,the sugar level decreases rapidly as it is converted to alcohol and other products (Obire, 2005) whereas, the sap becomes milky-white due to the increase microbial  suspension resulting from the prolific growth of the fermenting organism (okafor,1975). Palm wine is characterized by an effervescence of gas resulting fro the fermentation of the sucrose content, by the fermentation organisms( Bassir, 1962). Previous study of microbiology of  Elaies guineensis and Raphia hookie  have incriminated several bacterial and yeast flora to be involved in the fermentation process (Fapa-runsi and Bassir, 1972; okafaor, 1972; okafor, 1975; Eze and ogan, 1987; Amanchukuru  et al., 1989; Ejiofor,1994; Orimaiye, 1997; nester et al., 2004).These organism have also been reported to originate from several sources, which include tapping equipment, containers, the environment, etc (Faparunsi and Bassir 1972; Eapen, 1979).

1.2 OBJECTIVE OF RESEARCH

The main objective of this study is to identify high ethanol tolerant yeast in palm wine. The objectives specifically include the following:

1.     To isolate, characterize and identify yeasts from palm wine.

2.     To test the ability of the different yeasts to tolerate during growth.

3. To identify the particular yeasts that can withstand high ethanol concentration.

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