ABSTRACT
The effect of carbon sources on the growth of antimicrobial producing Bacillus species in Citrullus vulgaris (Ogiri) was studied in this research. A total of 11 antimicrobial producing strains were isolated of which 8 were identified through morphological and biochemical tests were of the genus Bacillus. The isolates were identified as B. subtilis, B. megaterium and B. firmus. Isolate K6 identified as B. subtilis produced the highest recorded zone of inhibition (22 mm) when all the isolates were grown in culture media seeded with the test organism S. aureus. The effect of different carbon sources (glucose, starch and sucrose) on the Bacillus isolates were investigated individually. It was observed that optimum growth was achieved when all the Bacillus isolates were grown on media having starch (5 and 10% concentration) and sucrose (5 and 10% concentration) respectively. Weak to moderate growths were observed in the presence of glucose with evident growth seen more at 10% concentration and as such was deduced to be less effective for growth of the Bacillus species isolated from Citrullus vulgaris. Although all isolates grew well in the presence of starch, isolates K2 and K5 identified as B. megaterium were not able to grow with starch as a carbon source but could grow in the presence of glucose and sucrose. This could be attributed to their inability to hydrolyze starch. Isolates K1. K3, K4, K6, K7 and K8 could all grow in the presence of all the tested carbon sources. From the experiment, it was observed that maximum growth was with starch and sucrose for 72 hours at 37oC.
TABLE OF CONTENTS
Title
page i
Certification ii
Dedication iii
Acknowledgments iv
Table
of contents v
List
of tables viii
Abstract ix
CHAPTER ONE
INTRODUCTION 1
1.1 Aim of the Study 3
1.2. Objectives of the Study 3
CHAPTER TWO
LITERATURE REVIEW 4
2.1 Fermentation 4
2.1.1 Importance of Fermentation 5
2.2 Fermented Foods 7
2.3 Traditionally Fermented Foods in Nigeria 8
2.4 Citrullus
vulgaris (Melon) 11
2.4.1 Description of the Seeds 11
2.4.2 Composition of the Seeds 11
2.4.3 Importance of Citrullus vulgaris 11
2.5 Ogiri 12
2.5.1 Production of Ogiri 12
2.5.2 Microbiology of Ogiri 13
2.6 Bacillaceae 14
2.6.1 History of Genus Bacillus 14
2.6.2 Genus Bacillus 15
2.6.3 Bacillus
as a Source of Antimicrobial Compounds 17
2.6.3.1 Ribosomal Peptide Antibiotics 18
2.6.3.2 Non-ribosomal Peptide Antibiotics 19
2.7 Effect of Different Carbon Sources on the
Growth of Antimicrobial Producing
Microorganisms 21
CHAPTER THREE
MATERIALS
AND METHODS 22
3.1 Sample
Collection 22
3.2 Isolation of Antimicrobial Producing
Organisms from Ogiri Samples 22
3.3 Characterization and Identification of
Antibiotic Producing Bacilli from Ogiri 23
3.3.1 Biochemical Analysis of the Isolates 23
3.3.1.1 Gram Staining 23
3.3.1.2 Catalase Test 24
3.3.1.3
Spore Formation Test 24
3.3.1.4 Methyl Red Vogues Proskauer (MR-VP) Test 25
3.4.1.5 Oxidase
Test 26
3.4.1.7 Citrate
Test 26
3.4.1.8 Starch Hydrolysis Test 26
3.4.1.9 Carbohydrate
Fermentation Test 27
3.5 Effect of Different Carbon Sources on the
Growth of Antimicrobial Producing Bacillus Species 27
CHAPTER
FOUR
RESULTS 28
CHAPTER FIVE
DISCUSSION AND
CONCLUSION 32
5.1 Discussion 32
5.2 Conclusion 32
References 35
LIST
OF TABLES
|
TABLE
|
TITLE
|
PAGE
|
|
2.1
|
Traditionally Fermented Foods in Nigeria
|
10
|
|
2.2
|
Antibiotics Produced by Some Bacillus species
|
20
|
|
4.1
|
Zone of Inhibition of
Antimicrobial Isolates from Ogiri samples
|
29
|
|
4.2
|
Characterization
and Identification of Antimicrobial Producing Bacillus Isolated from Citrullus vulgaris (Ogiri) samples
|
30
|
|
4.3
|
Effect
of Different Carbon Sources on Antimicrobial Bacillus species Isolated from Citrullus vulgaris (Ogiri) samples
|
31
|
CHAPTER ONE
INTRODUCTION
Microorganisms
are known to produce some of the most important medicines for various diseases.
They are the source of many life saving drugs and also effective antibiotics
against bacterial and fungal infections (Thakur et al., 2007). After the discovery of penicillin in 1928,
antibiotics have been recognized as the only means of effective control of
microorganisms. Since then, there has been continuous search for more effective
antibiotics (Kuta, 2008). In spite of tremendous success of secondary metabolite
research for antibiotics, the numbers of antibiotics are currently approaching
a saturation curve with an apparent limit of application in the near future.
Along with the usage of new antibiotics as therapeutics, there is emerging menace
of drug resistance among microorganisms worldwide. The increase in antibiotic
resistance has been attributed to inappropriate usage and inadequacies on the
part of the manufacturers, thereby steady decline of effective antibiotics
(Blomberg, 2008). Due to the above said facts, there is increasing demand for
new lead molecules as antimicrobials and has enforced to search for novel
organisms with new metabolites in so far untouched habitats (El-Banna and
Qaddoumi, 2016).
Bacillus
genus is an aerobic Gram-positive spore former rod commonly found in soil and
groundwater. These microorganisms are metabolically chemoorganotrophs being
dependent on organic compounds as sources of carbon and energy. The genus of Bacillus, one of the most abundant
bacterial strains found in soil, is able to produce two dozens of antibiotic
compounds with various chemical properties, among which peptide derivatives are
more studied (Stein, 2005). Furthermore, other groups of antibiotics, like
phospholipid derivatives (i.e., Bacilysocin) were also found as antibacterial
agents produced by this earthborn genus (Tamehiro et al., 2002). Polypeptide antibiotics which constitute the Bacillus bacteria have been gaining
importance as a result of studies. The Bacillus
species that produce antibiotics are B.
subtilis, B. polymyxa, B. brevis, B. licheniformis, B. circulans, B. cereus
and the antibiotics produced that are used for medical treatments are
bacitracin, gramicidin S, polymyxin and tryotricidin (Yilmaz et al., 2006). Most of the species from
the genus Bacillus are considered as
safe microorganisms and they possess remarkable abilities to synthesize many
substances that have been successfully used in agriculture and for industrial
purposes. The secondary metabolites produced by several species and strains of
the genus Bacillus have been found to
show antibacterial or antifungal activity against different phytopathogens
(Beric et al., 2012).
Melon
seeds (Citrullus vulgaris) are
presently fermented using traditional processes of uncontrolled solid state
fermentation in Nigeria, where the product (ogiri egusi) is used as a condiment
for flavouring soups and stews (Achi, 2005). Ogiri refers to a fermented oily
paste that is used as soup condiments for its strong smell. It is a product
prepared by traditional method of uncontrolled solid state fermentation of
melon seeds (Citrullus vulgaris),
involving the use of natural inoculation or chance fermentation. This
fermentation process is known to enhance the palatability, increases protein
value, vitamin content and mineral levels of such condiments. It increases
variety in the diet, improves nutritional value, reduces anti-nutritional
compounds and in some cases, it improves functional properties. Members of the
genus Bacillus have been reported to
be involved in the fermentation processes such as in the production of ‘ogiri’,
‘ugba’, ‘ogiri-igbo’, ‘dawadawa’ and ‘iru’ and Bacillus subtilis have been identified as the main bacterium
involved in the fermentation of melon seeds for the production of ogiri (Ibeabuchi
et al., 2014).
The
production of secondary metabolites from the genus Bacillus can be influenced by optimization of the nutritional
requirements and cultural conditions. These conditions play an important role
in the production of these secondary metabolites (Khattab et al., 2016). The ability of microorganisms to form these bioactive
compounds is not a fixed property but can be greatly increased or decreased
under different conditions of nutrition and cultivation media. Hence media
composition plays a vital role in the efficiency and economics of the ultimate
process. Therefore, designing an appropriate fermentation medium is of critical
importance in the production of secondary metabolites. Changes in the nature
and type of carbon sources have been reported to affect antibiotic biosynthesis
in microorganisms (Bundale et al.,
2015).
1.1 Aim of the Study
This
study was aimed at evaluating the effects of carbon sources on the growth of
antimicrobial producing Bacillus
species in Citrullus vulgaris.
1.2. Objectives of the Study
The
specific objectives of the study included;
1. To
isolate, characterize and identify Bacillus
species from Citrullus vulgaris
2. To
identify antimicrobial producing Bacillus isolates from Citrullus vulgaris
3. To
determine the effect of different carbon sources on the growth of antimicrobial
producing Bacillus isolates from Citrullus vulgaris.
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