ABSTRACT
Antimicrobial properties of probiotic bacteria from different sources were studied. Lactic Acid Bacteria were isolated from three different fermented food sources, Ogi, Ugba and Ogiri and were tested for their respective probiotic properties after which their microbial activities were tested against three pathogenic organisms, E. coli, Staphlylococus aureus and Candida spp. Results obtained showed the isolation of four Lactobacillus species, Lactobacillus plantarum, L.casei, L. fermentum and L. acidophilus with different occurrences levels in the fermented foods. Lactobactllus plantarum had 100% occurrence, L. casei (66.7%), L. fermentum and L. acidophilus had 33.3% each. The LAB isolates were confirmed to be probiotics following their successful but varied responses showing their ability to produce gas from glucose, salt and bile tolerance as well as growing in the presence of phenol. Test for antimicrobial ability of the probiotics show activities against the pathogens with mean inhibition diameters in the range of 10.67mm to 14.67mm against E. coli, 9.33mm to 10.67mm against Staphylococcus aureus and 12.33mm to 14.67mm against Candida albicans. Comparatively, Lactobacillus plantarum isolate from “ogiri” had the least antimicrobial effects. Also Candida albicans was the most susceptible to the probiotics while Staphylococcus aureus had the least resistance. It was observed that the LAB isolates were probiotics and demonstrated significant antimicrobial properties.
TABLE
OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of contents v
List of Tables vii
List of Figures viii
Abstract ix
CHAPTER ONE
1.0 Introduction 1
1.1
Description of some local food
condiments 4
1.1.1 Ogiri (Ricinus
communis) 4
1.1.2 Ugba (Pentaclethra
macrophylla, Benth) 5
1.1.3 Ogi (Cereal Starch L.) 5
1.2 Aims and objectives 6
CHAPTER
TWO
LITERATURE REVIEW
2.1 History and definition of probiotics 7
2.1.2 Classes of microorganisms
used as probiotics 8
2.1.3 Lactobacillus 10
2.1.4 Bifidobacterium 10
2.1.5 Bacillus 11
2.1.6 Enterococcus 12
2.1.7 Saccharomyces 13
2.1.8 Criteria for the selection
of probiotics 14
2.2 Applications and relevance of probiotics 17
2.2.1 Human health 17
2.2.2 Agriculture 18
2.2.3 Pharmaceutical industry 19
2.3 Lactic acid bacteria (lab) 19
2.3.1 Antimicrobial
peptide Bacteriocin produced by lactic acid bacteria 19
2.3.2 Bacteriocin
produced by Streptococcus spp. 21
2.3.3 Bacteriocins
produced by Enterococcus faecium 22
2.3.4 Bacteriocins
produced by Lactobacillus plantarum 23
2.4 Review of food sources used 24
2.4.1 Ogi (Cereal Starch L.) 24
2.4.2 Ogiri (Ricinus communis) 25
2.4.3 Ugba (Pentaclethramacrophylla, Benth) 27
CHAPTER THREE
MATERIALS AND METHODS
3.1 Source of materials 29
3.2 Media preparation 29
3.3 Isolation of lactic acid bacteria 29
3.4 Characteristics of lab isolates 30
3.5 Test for probiotic activity of LAB
isolates 35
3.5.1 Salt Tolerance Test 35
3.5.2 Phenol Tolerance Test 35
3.5.3 Bile Salt Tolerance Test 36
3.5.4 Temperature Tolerance 36
3.6 Antimicrobial activity test on probiotics 36
CHAPTER FOUR
4.0 Results 38
CHAPTER FIVE
DISCUSSION,
CONCLUSION AND RECOMMENDATION
5.1 Discussion 44
5.2 Conclusion 45
5.3 Recommendation` 45
REFERENCES
LIST
OF TABLES
Table Title Page
2.1: Microorganisms
used as probiotic cultures 9
2.2: Desirable properties
of probiotic bacteria 16
4.1: Antimicrobial
activity of LAB Probiotics from Ogi,
Ugba
and Ogiri 40
4.2: Test
Results for Probiotic 41
4.3: Occurrence
of LAB isolate in three formulated food
product 42
LIST
OF FIGURES
Figure Title Page
2.1: Process
flow chart for Ogi 25
2.2: Flow
chart for the production of Ogiri 26
2.3: Flow
chart for the production process of Ugba 28
CHAPTER
ONE
1.0 INTRODUCTION
The term probiotic
was defined as “a live microbial feed supplement which beneficially
affects the host animal by improving its microbial balance’’ (Aslam and Qazi, 2010).
Probiotic bacteria may produce various compounds, which are inhibitory to the
pathogen’s growth and these include organic acids (lactic and acetic acids),
bacteriocins, and reuterin. The organic acids not only lower the pH, thereby
affecting the growth of the pathogen, but they can also be toxic to the
microbes (Tambekar and Bhutada, 2010).
There is
increasing evidence that probiotics are beneficial in gastrointestinal
disturbances, such as diarrhoea, dysentery, typhoid etc. (Tambekar and Bhutada,
2010). It is important to underline when considering the effectiveness and
biological activity of probiotics, prebiotics or their combination (synbiotics)
that they are food products and not drugs.
Probiotic
inhibition of pathogenic microbes in the intestinal tract may involve a variety
of mechanisms, including competition for the use of nutrients, production of
antimicrobial compounds, or competition for specific adhesion sites
(competitive exclusion) (Gonzalez et al.,
2010).
Probiotics
may be alternative to antibiotics. Lactobacilli,
Bifidobacteria, Enterococci and Streptoccoci have been
used prophylactically to prevent travellers diarrhea caused by Enterotoxigenic E.
coli (Cunningham-Rundles et al., 2000) and used as therapeutic
agents against diarrheal diseases caused by Rotavirus and Clostridium difficle
(George and Cummings, 1999; Salih et
al., 2011).
Antimicrobials
have been used increasingly as a primary intervention for inhibition or inactivation
of pathogenic microorganisms in foods (Davidson and Zivanovic, 2003).
Generally, food antimicrobial agents are not used alone to control foodborne
pathogens, but are also tested as substitute to antibiotic.
Furthermore, in
many cases, their effects are mainly prophylactic in nature, rather than
therapeutic, that is preventive rather than curative (Suskovic et al.,
2001). Lactic acid bacteria were referred to as probiotics in scientific
literature by Lilley and Stillwell (1965). Lactic acid bacteria (LAB) are a
group of Gram positive, non-spore forming, Cocci or Rods which produce lactic
acid as major end product from fermentation of carbohydrates. Majority of
microorganisms used as probiotics belong to the LAB and Bifidobacteria.
Within the group
of LAB, Lactobacillus species are most commonly utilized group of
microorganisms for their potential beneficiary properties as probiotics. The
antagonistic activity of such bacteria is known to inhibit a large number of
enteric and urinary pathogenic bacteria (Hutt et al., 2006). Lactic acid
bacteria including Lactobacillus, Leuconostoc, Lactococcus, Pediococcus
and Bifidobacterium are found throughout the gastrointestinal
tract. Lactobacillus and Bifidobacterium species are prominent
members of the intestinal flora and are the commonly studied probiotics
bacteria.
They cause reduced
lactose intolerance, alleviation of some diarrhoea, lowered blood cholesterol,
increased immune response and prevention of cancer. The selection criteria or
probiotic LAB include: safety, viability/activity in delivery vehicles,
resistance to acid and bile, adherence to gut epithelial tissue ability to
colonise the gastro intestinal tract, production of antimicrobial substances,
ability to stimulate a host immune response and the ability to influence
metabolic activities such as vitamin production, cholesterol assimilation and
lactose activity (Savodago, et al., 2006).
Therefore an
attempt was made to isolate LAB from food samples and to evaluate their
potential as probiotics. Lactic acid bacteria (LAB) isolated from these foods
displayed probiotic properties such as hypolipidemic, hepatoproctective and
antibacterial and had been found to be effective in treating gastroenteritis in
man and animals (Aderiye, et al., 2007). The Lactic acid bacteria (LAB),
a component of several fermented foods including dairy products have long been
consumed by humans. Lactic acid bacteria are the focus of intensive research
for their essential role in most fermented foods.
These bacteria are
able to inhibit exogenous pathogens and exert many beneficial effects on human
health. Because lactic acid bacteria prohibit colonization by the invader and
control the intestinal pH through the release of acetic and lactic acids, these
bacteria could effectively prevent constipation and diarrhea caused by lactose
intolerance or pathogenic bacteria and also have a role in improving metabolism
and lowering cholesterol level in blood (Sindhu and Khetarpaul, 2001).
In recent years,
consumers’ demand for non-dairy-based probiotic products has increased, and
probiotics have been incorporated into drinks as well as marketed as
supplements in the form of tablets, capsules, and freeze–dried preparations
(Shah, 2001). It has been suggested that fruit juice could serve as a good
medium for cultivating probiotics (Mattila-Sandholm, et al., 2002). Vegetable juices processed by lactic acid
fermentation introduce a change in the beverage assortment (Karovicova et al., 2002).
These juices are produced mainly from cabbage, red
beet, carrot, celery and tomato. Raw material for juice production contains
substances with beneficial health effect. The fermentation techniques are often
a small scale and household basis, characterized by the use of simple
non-sterile equipment, chance or natural inoculums, unregulated conditions,
sensory fluctuations, poor durability and unattractive packing of the processed
products resulting in food of unpredictable quality (Olanrewaju et al.,
2009).
With increasing industrialization and urbanization,
efforts are presently geared towards the development of large-scale factory
production facilities for these foods where the quality of the finished product
will be assured (Agarry et al., 2010). This process enhances the
palatability, increases protein value, vitamin content and mineral levels of
such foods. It also improves food preservation, food safety, enhances flavour
and acceptability.
It increases variety in the diet, improves
nutritional value, reduces anti-nutritional compounds and in some cases, it
improves functional properties (Achi, 2005; Obute, et al., 2007). Although fermented food condiments have constituted
significant proportion of the diet of many people, Nigerians have exhibited
preference in terms of consumer tastes and preferences for such foods (Achi,
2005).
1.1 DESCRIPTION OF SOME LOCAL FOOD CONDIMENTS
1.1.1 Ogiri (Ricinus communis)
Ogiri generally refers to as an oily paste made
from oil seeds in West Africa. They are also used as soup condiments with
strong smell. It is a product of fermentation of melon seeds (Ricinus communis)
(Achi, 2005) consumed by the Ijebu and Ondo tribes in the forest zone of South
Western Nigeria. Ogiri egusi is a food flavouring condiment prepared by
traditional methods of uncontrolled solid state fermentation of melon seeds
involving the use of chance fermentation.
In ogiri preparation, melon seeds are boiled until
they are very soft and mashed. The mashed melon seeds are then wrapped tightly
in banana leaves and left to ferment for five to seven days. Thereafter, the
fermented mashed melon is placed in earthen well pot and covered with jute bags
which provide low oxygen tension (Odunfa, 1985; Omafuvbe, 2004). The fermenting
mashed melon is still wrapped in leaves, placed on a wire mesh, smoked over
charcoal heat at a distance for about two hour and pulverized before it can be
used in cooking (Odunfa,1985; Achi, 2005). Ogiri can also be made from castor
oil (Ricinus communis) seeds.
1.1.2 Ugba
(Pentaclethra macrophylla, Benth)
Ugba is the Igbo name for the fermented
African oil bean seeds (Pentaclethra macrophylla, Benth). It is called
Ukana by the Efiks in Southern Nigeria. It is consumed by an estimated 15
million people in Eastern Nigeria, majority of whom are Igbos (Odunfa and Oyeyiola, 1985; Ogueke et
al., 2010). It is a traditional food generally prepared in homes as a small
family business. The method of production varies from one producer to another
resulting in a non-uniform product. The beans that have been fermented for more
than three days are taken as a delicacy. Well fermented beans are added to soup
as flavouring (Odunfa and Oyeyiola, 1985; Ogueke et
al., 2010). It is widely consumed in eastern states of Nigeria with
tapioca, stock fish and garden eggs and leaves. It can also be eaten with
bitter kola (Garcinia kola) or kola nuts (Cola acuminate and C.
nitida) and when prepared with garden egg leaves are used to eat yam and
cocoyam. It is an important and cheap source of protein for people whose staple
foods are deficient in proteins (Obeta, 1983; Ogueke et al., 2010).
Methods for ugba preparation vary from one
community to the other. In this method described by Obeta (1983); (Ogueke et
al., 2010), the seeds are boiled in water for 16-18 h to remove the tough
testa. The cotyledons are then sliced, boiled again for 30 min and left
overnight in water at room temperature. The sliced cotyledons are then washed
in water and packaged in leaves of banana and allowed to ferment for 48 to 72
hours by chance inoculated microorganism.
1.1.3 Ogi (Cereal
Starch L.)
Ogi is a fermented cereal pudding from Nigeria,
typically made from maize, sorghum, or millet .
Traditionally, the grains are soaked in water for up to three days, before wet milling and
sieving to remove husks. The filtered cereal is then allowed to ferment for up
to three days until sour. It is then boiled into a pap, or cooked
to make a creamy pudding. It may be eaten with Moin Moin or Akara (Wikepedia, 2017).
A number
of studies have been carried out to improve the nutritive value of ogi. Fortification of sorghum-ogi can be achieved locally by
addition of sugar, milk, chocolate in order to enhance the sour taste. Afolayan et al. (2010) observed sorghum-ogi generally as one of the
malnutrition food among infant and this led many researchers attempt to
enrichment its nutritional value with plant sources such as soy bean, soy
flour, okra, cashew nut, cowpea and pawpaw (Ajanaku et al., 2010; Owuamanam et al., 2011; Egounlety et al., 2002).
1.2 AIM AND
OBJECTIVES
The aim
of this study is to determine the antimicrobial properties of probiotic
bacteria from different sources (Ogiri, Ugba and Ogi). The specific objectives
of this study were:
1) To isolate, characterize, identify and select
potentially probiotic strains of lactic acid bacteria from Ogi, Ugba and Ogiri.
2) To determine the antimicrobial activity of
the LAB isolates against specific pathogenic bacteria.
3)
To statistically
analyze the levels of variations in the antimicrobial activity of the different
LABS against the different bacteria pathogens.
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