ABSTRACT
Amylolytic activity of Lactic acid bacteria isolated from Nunu (a dairy product) was studied. Lactic acid bacteria were isolated from Nunu obtained from three sources and identified accordingly. Amylolytic activity of the LAB isolate were evaluated under varying conditions of temperature, pH and incubation time for optimization purposes. Results obtained show that three species of Lactic acid bacteria were isolated from the Nunu with varying levels of occurrences including Lactobacillus fermentum (80%), Lactobacillus pentosus (60%), and Pediococcus acidilatici (60%). All these isolates demonstrated amylolytic activities creating halo zones on starch containing medium with diameters of 5.03mm, 4.33mm and 3.8mm for Lactobacillus fermentum, Lactobacillus pentosus and Pediococcus acidilatici respectively. Also the optimum growth conditions for enzyme (amylase) activity of the three LAB isolates shows pH 7.0 with amylase activity of 5.0mm halo zone size, pH 6.0 with amylase activity of 4.57mm halo zone size and pH 7.0 with amylase activity of 4.0mm halo zone size for the same isolates respectively. Optimum incubation temperatures of 50°C with amylase activity of 4.75, 60°C with amylase activity of 4.57 and 60°C with amylase activity of 4.43 also for the same isolates respectively. The optimum incubation time was 72hrs for Lactobacillus fermentum, 72hrs for Lactobacillus pentosus and 96hrs for Pediococcus acidilatici at which the respective amylase activities were 4.9mm, 4.57mm and 4.33mm halo zone sizes respectively. It was observed that there were variations in the type and occurrence of LAB in Nunu while their optimal growth activities varied slightly but significantly.
TABLE
OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Charts viii
List of Tables ix
Abstract x
CHAPTER
ONE
1.0 Introduction 1
1.1 Aims And Objectives 2
CHAPTER
TWO
2.0 Literature Review 4
2.1 History 4
2.2 Human Evolution 5
2.3 Alpha-amylase 6
2.4 Beta-amylase 7
2.5 Gamma-amylase 7
2.6 Uses 7
2.6.1 Fermentation 7
2.6.2 Flour Additive 8
2.6.3 Molecular Biology 9
2.6.4 Medical Uses 9
2.6.5 Other Uses 9
2.7 Hyperamylasemia 10
2.8 Microorganisms and Amylase 10
2.9 Production of Amylase 12
CHAPTER
THREE
3.0 Materials and Methods 14
3.1 Materials/Equipment 14
3.2 Sources of Materials (Sample Collection) 14
3.3 Sample/Media Preparation 14
3.4 Isolation of Lactic Acid Bacteria from Nunu 15
3.4.1 Isolation of Lactic Acid Bacteria 16
3.4.2 Characterization of Lactic Acid Bacteria Isolates 17
3.4.2.1 Colony Characteristics 17
3.4.2.2 Microscopic Characteristics 18
3.4.3 Biochemical Tests 18
3.4.3.1 Catalase Test 18
3.4.3.2 Citrate Test 19
3.4.3.3 Motility Test 19
3.4.3.4 Oxidase Test 19
3.4.3.5. Voges Proskauer (V.P) Test 20
3.4.4.1 Growth
at Different Temperatures 20
3.4.4.2 Salt Tolerance Test 20
3.4.4.3 Carbohydrate Fermentation Test 20
3.5 Determination of Amylase Activity 21
3.6.0 Optimization of Growth Factors for Amylase Activity 21
3.6.1 Optimization of Temperature for Amylase
Production 21
3.6.2 Optimization of pH for Amylase Production 22
3.6.3 Optimization of Incubation Time 22
CHAPTER
FOUR
4.0 Results 24
CHAPTER
FIVE
5.0 Discussion 30
5.1 Conclusion 31
5.2 Recommendation 31
References 32
LIST OF FIGURES
Figure Title Page
4.1 Occurrence of LAB isolates in Nunu. 24
4.2 Amylase
activity of LAB isolates from Nunu 25
4.3 Effect
of pH on amylase activity of LAB isolates of Nunu. 26
4.4 Effect
of incubation temperature on amylase activity. 27
4.5 Effect
of incubation time on amylase activity 28
LIST OF
TABLES
Table Title Page
4.1 Morphological,
Physiological and Biochemical Characteristics of
Isolated Genera Of Lactic Acid Bacteria 29
CHAPTER
ONE
1.0
INTRODUCTION
Amylases are enzymes that breakdown starch
(carbohydrates) and are reputed to be among the most important enzymes with
great significance in present day biotechnology and industrial microbiology
(Ekunsami, 2004). Although amylases can be derived from many different sources,
such as plants, animals and microorganisms, Sindhu et al., (1997) observed that microbial amylases generally meet
demands of industries involved in starch products due to their high degree of
thermostability.
The production of amylases by
microorganisms is a normal characteristics in which the enzyme is produced
extracellularly for breaking down of insoluble carbohydrate thereby absorbing
the end product (sugars) into their cells (Ekunsami, 2004). There are two major
classes of amylases identified in microorganism namely alpha amylases and
glucoamylases. With the advent of new frontiers in biotechnology, the spectrum
of amylase application has widened in many fields including clinicals, medicals
and analyticals, food, textiles, brewing and distilleries.
While many microbes produce amylase
enzymes, Reddy et al, (1998) observed
that the most commonly used microorganisms for industrial production of amylase
are Bacillus species. High purity amylases are required in various fields that
require the enzyme application. Gupta et
al., (2003) observed that the many applications of amylase enzyme is the
driving force behind the current research interest in the enzyme production at
optimum, safe and convenient manner. Essentially, the use of microorganisms for
Amylase production stems from the relative advantages which include the fact
that microbes grow rapidly which in turn quicken enzyme production time. Also
they are easy to handle, cost effective and require less space than other
methods. More importantly, microorganisms are easy to manipulate to obtain
optimum enzyme production. They can be tailored through genetic engineering,
for stain improvement, mutation and other desired changes (Konsoula and
Liakopoulou-kyriakides, 2007).
Nunu is a natural milk from animals mostly
cattle. It is a normal secretion from the mammary gland of females which is
secreted during gestation in the udder (i.e. breast) and used during lactation
to feed their young. According to Adebolu, (2007), natural milk is sterile from
the animal body but soon receives an influx of microorganisms once out of the
body. This is attributed to its rich nutritional composition which makes it
fertile for microbial habitation and proliferation. Milk contamination can come
from varied sources including air, soil, the handles and psychrotrophic
microorganisms as external contaminants. Majorie and Kathlean, (2006) observed
that many different species of microorganisms are found in Nunu some of which
are potential pathogens but lactic acid bacteria represents a substantial
percentage of the microflora of raw milk (Adebolu, 2007). This makes Nunu,
natural milk a good source of lactic acid bacteria for use in this project
work.
1.1
AIMS AND OBJECTIVES
The aim of this project is to isolate
lactic acid bacteria from Nunu (raw milk) and evaluate their amylase production
activity.
The objectives of this project work include the
following
a.) To isolate and identify lactic acid bacteria from Nunu
(raw milk).
b.) To determine their amylase production and activities
of isolates
c.) To determine the optimal conditions for amylase
activity including pH, temperature, and incubation time.
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