ABSTRACT
The microorganisms involved in the fermentation of African oilbean seeds for Ugba production were investigated. This investigation revealed the presence of Bacillus, Staphylococcus, Micrococcus, Escherichia coli, Klebsiella and Proteus. It was obvious that no other microorganisms outside bacteria was involved in this fermentation Bacillus spp were seen to be the persistent bacteria throughout the fermentation period. The number of microorganisms increased from day 1 to day 3 and decreased from day 4 till the end of fermentation. Bacillus spp had the highest percentage occurrence while Micrococcus spp had the lowest. There was no presence of fungi. The study can be a key, leading to the development of starter culture for Ugba production.
TABLE OF CONTENTS
TITLE PAGE i
CERTIFICATION ii
DEDICATION iii
ACKNOWLEDGEMENT iv
TABLE OF CONTENTS v
LIST OF TABLES vii
LIST OF FIGURES viii
ABSTRACT ix
CHAPTER ONE
1.1 INTRODUCTION 1
1.2 AIMS
AND OBJECTIVES 3
1.3 LITERATURE
REVIEW 3
CHAPTER TWO
MATERIALS AND
METHODS 9
2.1 MATERIALS 9
2.2 METHODS 9
2.2.1 PREPARATION
OF OIL BEAN SEED FOR UGBA PRODUCTION 9
2.2.2 MICROBIOLOGICAL
ANALYSES 10
2.2.2.1 GRAM’S
STAINING 10
2.2.2.2 BIOCHEMICAL
TEST 11
CHAPTER THREE
3.1 RESULTS 14
CHAPTER FOUR
DISCUSSION, SUMMARY AND CONCLUSION
4.1 DISCUSSION 18
4.2 SUMMARY 19
4.3 CONCLUSION
20
4.4 RECOMMENDATION
20
REFERENCES
LIST OF TABLES
Table Title
Page
1: MICROBIAL
SUCCESSION ON “UGBA”
DURING
FERMENTATION 15
2:
BIOCHEMICAL CHARACTERIZATION AND IDENTIFICATION
OF
BACTERIA ISOLATES 17
LIST OF FIGURE
Figure Title
Page
1: Percentage occurrence
of each Isolate during the period of
fermentation
16
CHAPTER ONE
1.1 INTRODUCTION
Ugba
is the Igbo name for the Fermented African Oilbean Seeds (Pentaclelhra macrophlla, 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 Igbo’s
(Odunfa and Oyeyiola 1985). 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 (Njoku and Okemadu 1989). 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).
Ugba
is widely consumed in Eastern States of Nigeria with tapioca, stockfish and
garden eggs and leaves. It can also be
eaten with bitter kola (Garcinia Ikola)
or kola nuts (Cola acuminate and C. nitida).
When prepared with garden egg leaves are used to eat yam and cocoyam
(Okafor et al., 1991; Mbajunwa et al., 1998). It is an important and cheep source of
protein for people whose staple foods are deficient in proteins (Obeta 1983). The quantity of Ugba produced annually is not
known, since the seeds are collected by individuals and sold in the market to
Ugba producers.
Oil
bean seeds (Ugba) are obtained from a perennial legume tree, Pentaclethia macrophylla, Bentham
commonly called the oilbean tree. The
tree are planted along the sides of roads as shade trees and around communities
as cash crops. The fruit is a black hard
and woody pod measuring about 35-36cm long and 5-10cm broad. When mature, it splits open explosively to
release about eight flat, glossy, brown seeds measuring about 5-7cm in diameter
and weighing between 15-20g (Keay et al.,
1964; Odunfa, 1986a).
Ugba
the fermented African Food is of primary importance as cheap and easily
available source of plant protein in developing countries of the world and
Africa in particular. Fermented seeds
are not just palatable but serves as a delicacy amongst consuming regions where
it is consumed ganished with other vegetables or staple. Consumption of Ugba
seeds could pose as a means of addressing the prevailing Protein Energy
Malnutrition (PEM) in developing countries (Enujugha and Ayodele-Oni, 2003).
The
oilbean seeds contain 4-17% carbohydrate 44-4% oil which has been found out to
be rich in oleic acid (Nwokedi, 1975; Odoemelam, 2005) and linoleic acid
(Onwuliri, et al., 2004) also found
out that the saturated fatty acid lignoceric acid, occurred in high amounts
constituting about 10% of the total fatty acid concentration. Some workers said that the oil content could
be as low as 38% (Kar and Okechukwu, 1978).
They also reported that the oil contains about 75% saturated fatty acids
and 25% unsaturated fatty acids.
Ugba
production is locally done through a mixed wild bacteria. Fermentation of sliced boiled and soaked
African oil bean seeds. Unprocessed African oil bean seeds are bitter and
having anti-nutritional factors amongst which are pancine cyaride, oxalates,
saponin, phytic acid, phytate and tannine (Ownuliri et al., 2004).
The
major problem with the fermented oil bean seed Ugba is the restricted
availability due to its very short shelf life.
Under room temperature, fermented Ugba spoils within three to four
days. Spoilage is identified with
increase softness, colour change, off flavor and sliminess and production of
punget ammonia odour.
1.2 Aims and Objectives
This
work is aimed at finding out the microorganisms involved in the fermentation of
African oil been seeds (Ugba).
- Production
methodology
- Favourable
conditions
- Fermentation
conditions
1.3 Literature Review
Various
microbiological studies conducted on Ugba by Obota (1983); Odunfa and Oyeyiola (1985),
Ogueke and Aririatu (2004) showed that food pathogens such as Clostridum perfringens, C. botulinum, Salmonella
sp. Shigella sp and Vibrio sp. have not been isolated from
Ugba. However, such bacteria as Esherichia coli and Staphylococcus aureus have been isolated. These are bacteria capable of causing food
infections/poisoning. But since the
preparation of the delicacy or addition as condiment to soup involves heating,
they will be eliminated during the process.
Azubune and Isu (2006) studied the fungal contamination of the
fermenting product. They isolated Aspergillus
flavus A. niger, Penicillium chrysogenum and Fusarium sp. This poses a
serious health risk as these are moulds that produce mycotoxins in foods. This calls for the observation of Good
Manufacturing Practice (GMP) during the production. However, application of starter cultures and
immobilized cells in the fermentation process will eliminate these
possibilities of contamination with unwanted organisms.
Njoku
and Okemadu (1989) detected a-amylase,
proteolytic and lipolytic enzymes from the start of Ugba fermentation. These enzymes attained their maximum levels
at 24 – 36h. They suggested that this could be assumed to be period of maximum
microbial activity. The initial enzyme
activity detected could be, due to the activity of natural microflora of the
oilbean which developed particularly during the soaking the cooked beans. They
therefore suggested that it could well be that fermentation began much earlier
during the soaking of the sliced beans.
Enujugha et al., (2002) have
demonstrated that the raw seeds contain both a-amylase and lipase. They observed that the specific activity of
the purified a-amylase from the raw and fermented seeds were 0.037ml-1 min-1
and 0.88ml-1min-1respectively. They also
claimed that these enzymes complement the bacterial enzymes during
fermentation. However, since the seeds were boiled for several hours before
fermentation they could not have contributed to the fermentation as the boiling
must have inactivated them. Enujugha et al., (2004) in his study observed
that the activity of lipase isolated from the seeds were inhibited up to 36% by
NACI. However, they found that presence of Ca2+ increased the
activity of enzyme by 64%.
Mbata
and Orji (2008) that spontaneous activities of microorganisms present lead to
short shelf-life of product. Colour
change is throughout to be as a result of the presence of polypyhenol oxidase
(a major enzyme contained in oil bean seed) that catalyzes the oxidation of phenolic
substances to quinine which is polymerized to form a brown pigment.
Apart
from increasing the shelf-life, and a reduction in the anti nutritional factors
(Odunfa, 1986b) fermentation markedly improves the digestibility, nutritive
value and flavours of raw seeds.
Although
fermented food condiments have constituted significant proportion of the diets
of many people, Nigerians have exhibited on ambivalent attitude in terms of consumer’s
tastes and preferences for such foods (Achi, 2006). The introduction of foreign high technology
products especially processed ones because of globalization and liberalization
of the ecology, radically changed the Nigerian food culture into a mixed grill
of both foreign and local dishes (Ojo, 1991).
The
bacteria involved in the oil bean fermentation are randomly introduced through
the air, water utensil, banana leaves or by handling during the preparation
stages. It is believed that their initial step of boiling or long period would
kill most of the natural microbial flora of the seeds. However, bacillus spp
are known to produce resistance spores (Mbata and Orji, 2008) which may survive
the prolonged heating.
According
to Ogbulie et al., 1993, the
development of off flavor is due to the increase in ammonia nitrogen during
storage as a result of increased hydrolytic activities of proteolytic enzymes.
The pH of Ugba is reported to increase during spoilage and the increased in pH
(Mbata and Orji, 2008). The acceptable
colour of Ugba is light brown; dark brown colour is believed to be a result of
both enzymatic and non enzymatic browing.
Poolyphenol oxidase major enzyme contained in oil bean seed cotyledon
catalyzes the oxidation of phenolic substances to quinine, which polymenized to
form brown pigment (Enujiugha and Akambi, 2008).
Micrococcus
is generally thought to be a saprotrophyte or commensal organism, though it can
be an opportunistic pathogen, particularly in hosts with compromised immune
system such as HIV patients. It can be difficult to identify micrococcus as the
case of an infection, since the organism is normally present in skin microflora
and the genus is seldom linked to disease.
In rare cases death of immune compromised patients has occurred from
pulmonary infections caused by micrococcus micrococci may be involved in other
infections, including recurrent bacteremia, septic shock, septic arthritis,
endocarditis, meningitis and cavitating pneumonia in immune suppressed patients
(Smith, 1999).
Micrococci have been isolated from human
skin, animal, diary product, bear and spoilt foods like Ugba and also found in
many other places in the environment micrococcus luteus on human skin
transforms compounds in sweat into compounds with an unpleasant odour. Micrococci can grow well in environment
with little water, high salt concentration they grow optimally at 37oC
and can easily grow on inorganic nitrogen agar or simon’s citrate agar.
Bacillus licheniformis were seen as
light green raised colories with rough surfaces on blood agar.
Mbajunwa
et al., (1998) fermented Ugba turned
softer with time due to the presence of certain bacteria species. Softness during fermentation was attributed
to Bacillus species especially Bacillus subtilis.
Obeta
(1983); Mbata and Orji (2008) opened that activity of Pseudomonas chlororaphis was
also responsible for Ugba softening.
Enujiugha
and Akanbi (2008) isolated and used Pseudomonas
Fluorescence as starter culture for Ugba production, but the reported that
its activity during production was insignificant. It is also worthy to note that Bacillus sp.
have been implicated in all the reported cases of Ugba fermentation, either
when fermented spontaneously or inoculated specifically as starter cultures.
Mbajunwa
et al., (1998) explained that Ugba
fermentation by B. subtalas was
accompanied by extensive production of whitish mucilaginous strands (gums)
which caused the sticking together of Ugba strands.
Ogueke
and Aririatu (2004) – They identified the main fermenting microorganisms as
proteolytic Bacillus sp. Obeta (1983)
which include B. subtilis, B.
licheniformis and B. circulans. B.
megaterium, B. macerans. Their
numbers increased tremendously from 103 at the end of the fermentation 72
hours.
Moreover,
other bacteria identified in the fermenting slices include coagulase negative, Staphylococcus sp. Micococcus sp. Their numbers decreased after 72 hours of
fermentation, Leuconostoc mesenteroids; Lactobacillus
plantarum and E. coli.
Lipase
are composed in industrial applications and additives in food processing
(Flavour modification) fine chemicals (synthesis) detergents (hydrolysis of
fats) waste water treatment (decomposition and removal of oil substances)
diagnostics, cosmetics (removal of lipids) pharmaceuticals (digestion of oil
and fats in foods). Leather (removal of lipids from animals skins) and medical
(blood triglyceride assey) (Sarkar et
al., 1993)
Lipase
are produced by many microorganism and higher eukaryotes. Enzyme-producing microorganisms include
bacteria, fungi, yeast and actinomycetes. Especially lipase from microorganism
(Sarkar et al., 1993).
Ejiofor
et al., (1987) Oyeyiola (1981). They isolated the yeasts Candida tropicalis and Geotrichum
candidum during fermentation. It can
be assumed that the Bacillus sp are
the main fermenting organisms. They were
found to persist until the end of the fermentation and their numbers increased
throughout the period of fermentation while the numbers of others decreased
after 24 hours of fermentation.
Isu
and Abu (2000). They observed that the availability of the cells in association
remained stable at 94.5% for 6 months at 30oC and up to 10months at
4oC while the viability of the spores in association remained at 96%
for up to 10months at both 4 and 30oC. They also observed that the
indicators of fermentation were more pronounced than in the natural
fermentation and fermentation was completed within 48 hours.
Mohamadu
et al., (2008) opened the increase of
ash content in the fermented product could be attributed to the increased
metabolic activities of the fermenting microorganisms. That some of the biosynthetic mechanisms,
especially those involving Bacillus species are capable of synthesizing
divalent metals.
However,
Kabuo et al., (2007) studied various
flavor and aroma components present in the beans fermented with pure cultures
of microorganisms isolated from Ugba.
The sample fermented with B.
substilis and B. licheniformis were
found to produce the best Ugba with its typical aroma and flavor. The compounds identified were ethyle
state (3.60%) ethyloleate (4.70%) ethyl linoleate (14.14%) ethyl phenol (6.94%)
and ethyl benzoate (18.40%) ethyl carbonate (5.557%) methyl pentanone (1.67%) and
ethyl octanoate (4.72%).
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