ABSTRACT
The enzymatic potentials of lactic acid bacteria and Saccharomyces cerevisiae from fresh and soured palm wine samples were determined from 50 different palm win samples collected from Ariam, Umudike and Umunneochi in Abia state. Lactobacillus planetarium and Leuconostic Mesenteroides were the dominated lactic bacteria and Saccharomyces cereuisiae and Saccharomyces uvarum were the dominated yeast species isolated from the palm wine sample the lactic acid bacteria and saccharomyces cerevisiae isolated were screened for amylase, cellulase, protease, pectinase and xylanase production using well plate method in order to determine their ability to produce these enzymes. Among 88 LAB isolated, 20 were able to exhibit amylase production, 15 produced protease, 10 produced pectinase, 7 produced cellulose and 5 produced xylanase with different zone of inhibition . The isolate LAB 35 was able to produce all the enzymes tested and it was selected and identified as Lactobacillus plantarium . In other hand, among the 50 S. cerevisiae isolated, 10 were able to exhibit amylase production, 5 produced cellulose, 8 produced protease, 7 produced pectinase and 2 produced xylanase with different halo zones. Amonge the isolates that were able to produce these enzymes S.cerevisiae 25 has the potential to produce all the enzymes tested (amylase, protease, cellulose, pectinase and xylanase).
TABLE OF CONTENTS
Cover
page
Title
page i
Declaration
ii
Certification iii
Dedication iv
Acknowledgement v
Table
of contents vi
List
of table xi
List
of figure xii
Abstract
xiii
CHAPTER
ONE : INTRODUCTION
1.1 Aims 2
1.2 Objectives 2
CHAPTER TWO:
LITERATURE REVIEW
2.1 Palm Wine 3
2.1.1 Palm Wine Production 3
2.1.2 Biochemical Constituents 4
2.1.3 Microbial Communities in Palm Wine 5
2.1.4 Benefits
of Palm Wine 6
2.2 Lactic Acid Bacteria 6
2.2.1
Condition that Favors the Growth of Lactic Acid Bacteria. 7
2.2.2 The activities of Lactic Acid Bacteria in
Palm Wine 8
2.3 Yeast.
9
2.3.1 Conditions
Necessary for the Growth of Yeast 9
2.3.2 Uses
of Yeast 11
2.3.2.1 Brewing 11
2.3.2.2 Alcoholic Beverages 11
2.3.2.3 Beer Production 11
2.3.2.4 Production of wine 12
2.3.2.5 Baking 12
2.3.2.6 Food
Spoilage 12
2.3.2.7 Bioremediation 13
2.3.3 Saccharomyces cerevisiae 13
2.3.3.1 Factors
that Affects the Growth of Saccharomyces
cerevisiae. 14
2.3.3.2 The Activities of Saccharomyces cerevisiae in Palm Wine 14
2.4 Enzyme 15
2.4.1 Types of Amylase 15
2.4.1.1
α-Amylase 15
2.4.1.2
β – Amylase 15
2.4.1.3. γ – Amylase 16
2.4.2 Sources of amylase 16
2.4.3
Microorganisms capable of producing amylase; 16
2.4.4 Industrial Applications of α-Amylase 17
2.4.4.1
Bakery Industry 17
2.4.4.2
Detergent Industry 17
2.4.4.3 Desizing of Textiles 18
2.4.4.4 Paper Industry 18
2.4.4.5
Fuel Alcohol Production 18
2.5 Xylanase 19
2.5.1 Application
of xylanase 19
2.5.2 Xylanase sources 20
2.6 Cellulose 20
2.6.1 Microorganisms having cellulolytic
abilities. 21
2.6.2 Application
of Cellulases in Various Industries 21
2.6.2.1 Pulp and Paper Industry 21
2.6.2.2
Textile Industry 21
2.6.2.3
Bioethanol Industry 22
2.6.2.4 Wine and Brewery Industry 22
2.6.2.5
Food Processing Industry 22
2.6.2.6 Animal Feed Industry 22
2.6.2.7 Agricultural Industries 23
2.6.2.8
Detergent Industry 23
2.6.2.9
Waste Management 23
CHAPTER
THREE: MATERIALS AND METHOD
3.1 Sources and collection of samples 24
3.2 Sterilization
of Materials 24
3.3 Normal Saline Preparation 24
3.4
Media Preparation 24
3.5 Isolation
of yeasts (saccharomyces) 24
3.6
Identification of yeast (saccharomyces) 24
3.7
Isolation of Lactic Acid Bacteria 25
3.8 Sub-culturing 25
3.9 Characterization
and Identification of bacterial isolates 26
3.9.1
Gram staining techniques 26
3.9.2 Motility test 26
3.9.3 Catalase test: 26
3.9.4 Coagulase
test 26
3.9.5 Methyl
red test: 27
3.9.6
Voges-proskaeur test: 27
3.9.7 Indole test: 27
3.9.8
Citrate test: 28
3.9.9 Oxidase
test: 28
3.10 Sugar
utilization test 28
3.11 Qualitative
Screening for Extracellular Enzyme Producing Isolate by plate assay 28
3.11.1 Production of Xylanase enzyme 28
3.11.2 Production of Amylases enzyme 28
3.11.3 Production of Cellulases enzyme 29
3.11.4
Screening of Isolates for the Pectinase
Activity 29
3.11.5 Screening of Proteolytic Activity 30
CHAPTER FOUR: RESULT
4.1 Discussion
40
CHAPTER FIVE: CONCLUSION
5.1 Conclusion 42
References 43
LIST OF
TABLES
Number Title Page
Table 4.1: Identification and Characterization of
Lab Isolates From
Different Palm Wine Samples 33
Table 4.2: Percentage
occurrence of lactic acid bacteria isolated from different
Palm Wine Samples 34
Table 4.3:
Isolation And Identification of
Yeast From Different Palm
Wine Samples 35
Table 4.4; Percentage occurrence of yeast isolated from
different palm
wine samples
35
Table 4.5 : Total Viable Plate Count In CFU/Ml From
Different Palm Wine Samples 36
Table 4.6: Enzyme producing isolates and their inhibition
zone area (mm) 38
Table 4.7: Enzyme producing isolates and their
inhibition zone in mm 39
CHAPTER ONE
INTRODUCTION
Palm
wine is an alcoholic beverage resulting from the spontaneous fermentation of
the sap of the palm, tree by indigenous microbes which consists of yeast (Saccharomyces cerevisiae) and bacteria
(mainly lactic Acid bacteria LAB and Acetic Acid bacteria AAB). (Onwuka, 2011;
Okpara et al., 2013).It is the fermented sap of certain
varieties of the tropical plant of the palmae family which includes (Elaeis guineensis, Raphia vinifera and Borassusf
laellitter).Fresh palm wine is sweet, clear, colourless juice containing
bout 10-20% of sugar, small amount of protein, and minerals (Opara et al., 2013). According to Onyka et al., (2009), it contains
nutritionally important components including amino acids, proteins, Vitamins
and Sugars. Which makes the wine a veritable medium for the growth of a
consortium of microorganisms whose growth in turn change the physiological
conditions of the wine giving rise to competition and successions of organisms.
In traditional African societies, the palm wine plays a significant role in the
customary practices (AmoaAwua et al., 2006).
Over ten million people consume palm wine in West Africa (Onwuka 2011).
Traditionally, it is believed that when taken by nursing mothers palm wine
stimulates lactations and also dras diuretic effects. Palm wine has also been
used to enhance potency due to yeast cell concentration. (Amoa- Awu et al., 2006).
Despite
all these good qualities of palm wine it is highly perishable sap due to
fermentation which starts soon after the sap is collected through a process
called tapping and within an hour or two it becomes reasonably high in alcohol
(cup to 4%) if palm wine is allowed to continue to ferment for more than 24hrs,
it starts to turn into vinegar. This makes it unacceptable to consumers. (Awu et al.,2006).Fermentation in palm wine
is possible because it constitutes a good growth medium for numerous microorganisms
especially for yeast, lactic acid and Acetic acid bacteria (Bechemet al., 2007). Saccharomyces cereuisae constitutes about 70% of the total yeast of
palm wine and enzymatic activities of these microbes are believed to be
responsible for the conversion of sugar in palm sap to alcohol and after a
shout time while bacteria induces the conversion of alcohol into vinegar
(Onwuka, 2011).
According
to Abolhasan et al., (2007) it was
due to the enzymatic of activities for the palm wine microbes saccharomyces cerevisiae, lactic acid
bacteria and Acetic acid bacteria that leads to the souring of the palm wine
after 24 hours of tapping. They do these by oxidizing the ethanol content of
the palm wine to acetic acid by catalytic reactions of alcohol dehydrogenase
and aldehyde dehydrogenase which are located on the periplasmic side of their
cytoplasmic membrane. These enzymes are important in the industrial production
of Acetic acid (Ameh et al., 2011).
1.1 AIMS
The
aim of this research is to determine the enzymatic potentials of lactic acid
bacteria and saccharomyces cerevisae
from fresh and soured palm wine.
1.2 OBJECTIVES
To
isolate the bacteria and yeast species present in the palm wine
To
test the enzymatic activities of the lactic acid bacteria and yeast isolates
form the palm wine.
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