CHAPTER ONE
1.0
INTRODUCTION
Ugba also called ukpaka is a popular food delicacy in
Nigeria especially among Ibo ethnic group. It is rich in protein
and is obtained by a solid state fermentation of the seed of
African oil bean tree (Pentaclethra macrophylla Benth). It is
essential food item from various traditional ceremonies where it
is mixed with slices of boiled stock fish (ugba and okpoloko). The
natural fermentation of the seed which at present is still done at
the house-hold level, renders the production nutritious, palatable
and non-toxic (Enujiugha, 2002).
Its production, like many African fermented foods depends,
entirely on mixed fermentation by microorganism from diverse
source.
Some of these seeds have been exploited as soup bases
such as Gbegiri from Vigna unguicuata (Akanbi, 1992). Others
are fermented and used as condiments and seasonings such as
okpei from Prosopis africana (Achi, 1992) and ogiri from Ricinus
cummunis (Odunka, 1989). Another of such seeds is the African
1
oil bean seed (Pentaclethra macrophylla Benth), a highly
nutritious leguminous crop seed abundant in the rain forest areas
of west and central Africa.
Pentaclethra macrophylla Benth is a large woody plant
abundant in the rain forest areas of west and central Africa. It’s
origin in Nigeria is believed to be around 1937 (Ladipo, 1984);
where it is found in the South Nigeria, (Mbajunwa et al, 1998).
“Ugba” Pentaclethra macrophylla Benth belongs to the
Family Leguminosae and sub-family microsoideae (Keay, 1989
and NFTA, 1995).
Ugba seeds are irregular and oval, they are flat, black and
hard pods. It is composed of oil, protein and small amounts of
carbohydrate (Obeta, 1982).
Production of Ugba is still on age old traditional family in
the rural area. The fermentation depends on random inoculation
of boiled slices of the oil bean seed by microorganism within he
immediate environment. Earlier report attribute the sources of
the micro-organism to the leaves used in packaging, human
2
handling, container and utensils used in processing (Obeta,
1993, Odunfa & Oyeyiola, 1985).
A number of researches have been carried out to know
what causes this fermentation, and was attributed to microorganisms that are probably introduced through the air, water,
and banana leaves. Organisms such as Micrococus roseus,
Micrococcus
luteus,
Leuconostoc
mesenteroides,
Staphylococcus aureus, Staphylococcus epidermidis, Bacillus
substilis, Bacillus megaterium and Bacillus circclans, E-Coli
Fermented African oil bean seed (ugba), has a high rate of
susceptibility to microbial spoilage and therefore has a very short
self life of 1-2 weeks (Enujiugha & Olajundoye, 2001).
Manifestation of food spoilage are many and vary typically
resulting in an off smell, colour, taste and texture.
Reports by Mbajunwa (1998) Obeta, (1983) and Nwagu et
al. (2010) indicates that micrococcus species do not play an
active role during microbial fermentation of Ugba.
However, further work on spoilage association of ugba by
Nwagu et al (2010) showed that the population of Micrococcus
3
sp increased with increase in keeping time of ugba. This
indicates the ability of micrococcus to thrive in the alkalophilic
environment while constituting as a spoilage organism of ugba.
This may be attributed to the ability of microorganism to produce
amylase, lipase, or protease able to utilize protein, carbohydrate
or lipid content of ugba as source of nutrition (Njoku et al., 1990).
Amylases are enzymes that breakdown starch or glycogen.
The amylases can be derived from several sources such as
plants, animals and microbes. The major advantage of using
microorganisms for production of amylase is in economical bulk
production capacity and microbes are also easy to manipulate to
obtain enzymes of desired characteristics
1.1
Aims and Objectives
This work is aimed at the following;
a.
isolating micrococcus from fermented ugba
b.
producing amylase from the micrococcus from ugba
4
CHAPTER TWO
2.0
LITERATURE REVIEW
2.1
FERMENTED FOODS
Many developing countries are still preparing traditional
fermented foods for human consumption (Campbell-Platt, 1987).
Fermented products remain of interest since they do not require
refrigeration during distribution and storage. The traditional
condiments have not attained commercial status due to the very
short shelf life, objectionable packaging materials, stickiness and
the characteristic putrid odor (Arogba et al., 1995).
Moreover, fermented condiments often have a stigma
attached to them they are often considered as food for the poor
(Ukwuoma, 2003; Duru 2005). The production of fermented
vegetable proteins for use as food condiments is craft based
(Achi, 2005). Remarkably, in many areas of Nigeria today, they
still made in traditional ways with success, depending upon
observance of good manufacturing practices and control of
environmental conditions during the manufacturing phase (Achi
2005).
5
As with any other fermentation process, the understanding
of the microbial ecology of vegetable fermentation requires the
knowledge of the fermentation substrates i.e. the seeds of the
various plants as well as the products obtained (Achi, 2005).
Apart from increasing the shelf-life, and a reduction in the
anti nutritional factors (Odunfa, 1985; Reddy and Prerson, 1999;
Bafimalaa et al., 1989; Achi and Okereka, 1999), fermentation
markedly improves the digestibility, nutritive value and flavours of
the raw seeds.
Although fermented food condiments have constituted a
significant proportion of the diet of many people, Nigerians have
exhibited on ambivalent attitude in terms of consumers’ tastes
and preferences for such foods (Achi, 2005). The introduction of
foreign high technology products especially processed ones
because of globalisation and liberalization of the ecology,
radically changed the Nigerian food culture into a mixed grill of
both foreign and local dishes (Ojo, 1991).
Information on the manufacturing and microbiology of
indigenous fermented legumes has been reported (Odunfa,
6
1985; Odunfa and Adewuyi, 1988; Achi, 1992; Sanni, 1993;
Sanni & Ogbonna 1998).
The use of these condiments, could be extended ingredient
included into most fabricated foods in order to further increase
their versatility and utility (Auiani and Bekebain, 1992; Achi,
1999). Fermented fluted pumpkin flavour has been incorporated
into
wearing
food
formulations (Achi, 1987; Banjo and
Akpapunam, 1987).
2.2
PENTACLETHRA MACROPHYLLA
Pentaclethra macrophylla is common in primary forest and
secondary forest and coastal savanna often in the vicinity of
creeks and rivers. It is most common at attitudes up to 500m;
although growth can be good at higher elevation where rainfall is
adequate and temperature are never cooler than 18 0C, it
requires a mean annual temperature of about 25 0C. It prefers
medium loamy, well drained soil. The natural distribution
suggests that it is adapted to relatively acid oil, and it tolerates
water logging. It belongs to the family leguminosae, subfamily
7
mimosoideae and recognized peasant farmers in the southeast
of Nigeria for its soil improvement properties. A related species
viz Pentaclethra macrophylla has been cultivated in Nigeria since
1937 (Lapido, 1984) and for many years in other West African
countries where its seed is relished as a food. Pentaclethra
macrophylla was not known to modulate until recently (Lapido et
al, 1993). With the diverse native uses of this species, and the
present research effort on it. Its utility would be further enhanced
for agro forestry growing and seedlings will achieve a height if
1.5m in the first year on good sites.
2.3
BOTANY
The tree has a characteristics low branding habit and an
open crown which allows substantial light under its canopy. This
characteristics accounts for the trees use in combination with
food crops on farms and particularly in home gardens in
southeast Nigeria. The bole produces a reddish brown (Keay,
1989); thin and partly with irregular places flaking off. Leaves
posses a stout angular petiole. The compound leaves are usually
8
about 20-45cm long and covered with rusty hairs giving a scurfy
effect particularly along the upper surface but this eventually falls
off. There are 10-12 pairs of stout pinnae, the middle pairs are 713cm long and also have rusty hairs along the central grove.
There are usually 12 – 15 pairs of opposite stalk less pinnules
(leaflets) each 12 – 15cm long and 5 – 10mm broad, with the
middle pairs longest. Leaflets often have a rounded tip but are
sometimes notched, the base is unequal.
Flowers are creamy yellow or pinkish-white and sweat
smelling, flowering commences at variable periods within West
Africa. The main flowering season is between March to April with
smaller flushes in June and November. Fruits are available at
most periods of the year because the large woody pods are
persistent. The pods are 40-50cm long and 5-10 wide. Fruits
splits open explosively with the valves curling up. This is the form
in which they appear on most trees usually pods contain
between 6-10 flat glossy brown seeds are up to7cm long. This is
the edible product and sources of the oil, hence the name “the oil
bean tree” (Aubre Ville, 1959).
9
2.4
ECOLOGY
Pentaclethra macrophylla is endemic to the humid and
some parts of the sub humid zones of West Africa. It does not
occur in the lands although growth can be good where rainfall is
adequate and temperatures are never cooler than 18oc (Oboh,
2007).
The annual mean temperature requirement is about 25oC
and rainfall between 1000-2000mm. After about 2 years growth
in the forest, trees become relatively fire resistance and re-sprout
readily when looped (Oboh, 2007).
2.5
Growth and Development
The bole is often gnarled and twisted and forked at a low
level and elephants often damage the base but trees with a
longer straight trunk are found. The crown has been described
as heavily branded and dense, but also as open and allowing
crops to grow well below the trees. Some species are leafless
during the rainy season, though the species is mostly evergreen
(Voorhoeve, 1965).
10
Pentaclethra macrophylla modulates and fixed atmospheric
nitrogen. The main flowering season in West Africa is March –
April, with small flushes in June and November; in Liberia trees
flower in February – April and fruit in September – December.
The flowers are strongly fragrant, very rich in nectar and much
visited by honeybees (Voorhoeve, 1965).
Pests and Diseases
No serious diseases or pests of Pentaclethra macrophylla
are known but many insects species and pathogen attack the
pods and seeds. The major insect pests are Cossus Cadam bae,
Sitophilus Spp, Spodoptera Exempta, and several giant silk
worms. Some of the insects skeletonize the green pods, some
bore onto the pod and seeds; others accelerate the pods,
causing lesions that allow fungal and bacterial pathogen to
mirade the seeds.
11
2.6
METHOD OF PRODUCTION
Production of Ugba involves boiling of the oil bean seed for
5 – 8 hours to ease removal of the hard shell (dehulling) of the
seeds. After removal of the shells, the cotyledons are washed
with water & sliced into sizes of 4 –5cm or more. The slices are
then washed and boiled for about 1 – 3 hours is then soaked in
water for about 10 – 12 hours (Mbata & Orji, 2005), (Obeta,
1982) reported that the seeds were boiled in water, for 16 – 18
hours and then they de-hulled easily. After dehulling the
cotyledon or mesocarp, they are cut into slices of about 4.55.5cm long and 3.0 – 4.0cm broad. It is then followed by boiling
for 30 minutes and left over night (16 hours) in water at room
temperature. This difference in boiling and soaking interval is not
significant as the over all which is aim to soften and reduce
bitterness was achieved with the two methods.
After soaking in water overnight or for 10 – 12 hours, the
slices are washed again and allowed to drain for 30 minutes in a
basket lined with banana leaves and allowed to dry a bit.
12
After draining the slices are wrapped with banana leaves
(Musa sapietum) this method was also employed in ogiri
fermentation just that plantain leaves are used). The wraps are
allowed to stay (ferment) for 3 – 4 days at room temperature.
After fermentation, ugba has a pH of above 7-8, it becomes light
brown, soft and shiny (Mbata & Orji, 2008).
Enujiugha & Akanbi (2005), reported that processing of the
seeds reduced the crude protein content, while oil increased.
Phosphorus and iron content of the seeds were reduced, while
calcium and magnesium content increased. Available reports
also indicate that thermal processing of the seeds raised nutrient
availability, digestibility and function ability (Enujiugha et al,
2003).
2.6.1 Microorganism involved in ugba fermentation:
Microorganisms isolated
from fermented
ugba were
Staphylococcus. epidermis, Micrococcus, luteus, Micrococcus
roseus, Leuconostoe mesenteriodes, Bacillus substilis, Bacillus
Lichenifornis and Bacillus megaterium. The organisms were
13
isolated from nutrient agar. In both laboratory and locally
prepared samples, Bacillus spp were more prominent even until
the
end
of
the
fermentation.
Staphylococcus,
E.
Coli,
Micrococcus protein also had high total viable counts. Ability of
the different isolates to ferment ugba was carried out. It was
confirmed that Bacillus spp softened the ugba slices as well as
produce the characteristic aroma of the fermented oil bean seed
(Obeta, 1982). Staphylococcus spp, Mcrococcus spp and
lactobacillus spp, produced no aroma while Bacillus megaterium
and Bacillus substilis has been used as started cultures for
laboratory fermentation of ugba, where the Bacillus spp
successfully
fermented
the
ugba,
and
produced
similar
characteristics as the locally prepared one. (Mbata & Orji, 2008).
The aforementioned result was also noted and reported in other
works (Njoku et al, 1990; Isu & Njoku, 1997).
The role of enzyme in fermentation is not yet known,
probably research would be carried out in the near future.
Enzyme system, especially the alpha-amylase aid in hydrolysis
of seed macromolecules (Enujiugha et al 2002).
14
2.6.2
Ugba Fermentation
Ugba Fermentation is therefore a mixed wild fermentation
with Bacillus, Micrococcus and Lactobacillus as the predominant
organism. Bacillus spp are the organisms, which actually ferment
the oil bean seeds. The oil bean seeds are largely composed of
oil, protein and carbohydrate. The ability to break down these
major components of the seed is an important characteristic of
organisms able to ferment the seeds. Most organism isolated
from
fermented
ugba
possessed
this
characteristics.
Micrococcus spp & Bacillus spp are known to breakdown oil and
proteus is proteolytic (Frazier, 1976).
It is worthy of note that bacillus spp are important source of
amylase and proteases (Forgarty, 1971; Forgarty & Griffin,
1973).
The bacteria involved in the oil been 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,
15
bacillus spp are known to produce resistance spores (Mbata &
Orji, 2008) which may survive the prolonged heating.
2.6.3 Ugba Defects
Though of high nutritive value, fermented ugba is reported
to posses of new defects.
Nwanjo et al, 2005 & Onwuiri et al, 2004 studied that
presence
of
anti-nutritional
factor
in
the
Pentaclethra
Macrophylla seed; the phytochemical screening of the seed are
revealed that the seed possesses some anti-nutritional factors,
such as pancine, cyanide, oxelate, saponin. Eniviugha & Akinbi
(2005) also reported that phytic acid phylate – P, and tannins
were present in unfermented African oil bean seed. These anti
nutritional factors can invariably bind to some of the protein and
make it unavailable (Ladeji et al, 1995).
2.6.4 Shelf life and Preservation of Ugba
This major problem of shelf-life is often associated with the
uncontrolled wild fermentation and poor packaging of ugba,
16
which often allows maggots to develop on the product as a result
of eggs laid by fires that gain entry into the wrapped product.
According to Ogbulie et al in 1993, the development of off flavour
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 & 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 quinines, which
spontaneously, polymerize to form a brown pigment (Enujiugha
and Akanbi, 2005). Fermented ugba has restricted availability
and exploitation because of its high rate of deterioration, as it is
known to loose acceptability beyond two weeks of ambient
storage (Enujiugha, 2000). The locally fermented ugba spoils
within
three
to
four
days
of
fermentation
under
room
temperature, the spoilage is associated with increased softness
(Enujiugha, 2000), colour change, off-flavour, sliminess (Mbata &
17
Orji, 2008) and production of pungent ammonia odor (Ogbulie et
al, 1993).
This major problem of ugba having a poor shelf life is often
associated with the uncontrolled wild fermentation and poor
packaging which often allows maggots develop on the product
as a result of eggs laid by flies that gain entry into the wrapped
product. The off flavour reported by Ogbulie et al in 1993 is due
to increase in ammonia nitrogen during shortage as a result to
increased hydrolytic activities of microbial enzymes. An increase
in pH was observed during spoilage, which was explained by
increased hydrolytic activities of microbial enzymes (Mbata &
Orji, 2008). Spoilage is also observed in change of colour from
light to dark brown. This off colour is believed to be a result of
both enzymatic and non enzymatic browning, polyphenol
oxidase a major enzyme contained in oil bean seed cotyledon,
catalyzes the oxidation of phenolic substances of quinines, which
spontaneously, oxidation of phenolic substances of quinines,
which spontaneously, polymerize to form a brown pigment
(Enujiugha and Akanbi, 2005).
18
2.6.5 Uses/ Application of Ugba
Pentaclethra macrophylla (Ugba), is planted or retained
along the edges of home gardens and farms mainly for its seed
from which edible oil can be extracted. Throughout the forest
zone of West Africa, the seeds are eaten boiled or roasted. They
are also fermented to yield a snack or condiment with a meaty
taste, very popular in southeastern Nigerian where it is called
“Ugba” (Oboh, 2007).
The empty dry pods are used as fuel for cooking, farmers
protect this species on farms because, its open crown does not
severely affect crop growth and because some trees are leafless
during the growing season. The leaves also contribute to soil
fertility (Oboh, 2007). Pentaclethra macrophylla wood, called
“Mubala” or “Ovala” is suitable as fuel, wood and for charcoal
making. As few trees develop a straight trunk of harvestable
size, timber of large sizes is only occasionally available. The
wood is hard and difficult to work, but suitable for poles, railway
sleepers and general carpentry.
19
Silkworm moths Nuduarella oyemensis called “minsangula”
and imbrasia obscura called “minsendi” feed on the leaves. Bees
forage the flowers for honey (Latham, 2004).
Pentaclethra macrophylla is used in Africa in traditional human
and veterinary medicine. The ripe fruits are applied externally to
heal wounds. Extracts of the leaf, stem, bark, seed and fruit pulp
have anti-inflammatory and anti-helminthic activity, and are used
to treat gonorrhea and convulsions, and also used as analgesic.
The root bark is used as a laxative, as an enema against
dysentery and as a liniment against itch. In Cameroon, an
infusion of the bark is used as an abortifacient.
Pentaclethra macrophylla is occasionally planted along
roads. It plays a role in various traditional ceremonies (Ladipo &
Boland, 1995).
20
TABLE 1: Common uses of Pentaclethra macrophylla in West
Africa.
Uses
Parts of plant
Countries
Food
Seed
Nigeria/ Ghana
Salt substitute
Pod ashes
Ghana
Edible oils
Seeds
Fences and palings
Wood
Charcoal
Wood
Caroling bowls, etc
Wood
Nigeria, Ghana, Togo,
Cameroon
Nigeria, Ghana, Togo,
Cameroon, Cote d’ivoire
Nigeria, Ghana
Sees craft (beadings)
Seed (beadings)
Nigeria
Dye (mordant)
Pod ashes
Ghana
Mild poison
Bark and seed
Ghana
Medicine (Convulsion)
Pod
Cameroon
Medicine
(convulsion) Crushed seed
Ghana, Nigeria
Medicine (convulsion)
Burnt leaf
Ghana
Medicine (diarrhea)
Leaf / steam bark
Ghana
Medicine (itch)
Bark as liniment
Ghana
Medicine (lactogenicity) Bark decoction
Ghana
Medicine (wound
Bark as lotion
Ghana
Whole tree
Nigeria
treatment)
ornamental
Abbiw (1990).
21
2.7
MICROCOCCUS SPECIES IN UGBA
Micrococcus occurs in a wide range of environment
including water, dust and oil. Micrococci are grant +ve spherical
cell ranging from about 0.5-3 micrometers in diameter and
typically appears in tetrads. Micrococcus has a substantial cell
wall, which may comprise as much as 500% of the cell mass.
The genome of micrococcus is rich in quinine and cytosine (x),
typically exhibiting 65 75% GC content.
Micrococci often carry plasmids (ranging from 1 –100MDa
in size) that provide the organism with useful traits. Some
species of micrococcus such as M. luteus (yellow) and
Micrococcus roseus (red) produce yellow or pink colonies, when
grown on mannitol salt agar. Isolates of Micrococcus luteus have
been found to over produce riboflavin when grown on toxic
organic pollutants like pyrimidine. Hybridization studies indicates
that species within the genus micrococcus are not closely
related, showing as little as 50% sequence homology. This
suggests that some micrococcus species, may on the basic of
22
ribosomal RNA analysis, eventually be reclassified to other
microbial genera (Doddamar milt and Ninnekar. H. 2001).
2.8. Environmental Isolation of Micrococcus
Micrococci have been isolated from human skin, animal,
diary product, beer 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, or high salt concentration (Green
blaf et al (2004) ) they grow optimally at 37oc and can easily
grow on inorganic nitrogen agar or Simon’s citrate agar.
2.8.1 Pathogenesis
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
23
normally present in skin microflora and the genus is seldom
linked to disease. In rare cases death of immuno 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 immuno
suppressed patients (Smith et al., 1999).
2.8.2 Industrial Uses of Micrococci
Micrococci,
like
many
other
representatives
of
the
Actinobateria can be catabolically versatile, with the ability to
utilize a wide range of unusual substrates, such as pyridime
herbicides,
chlorinated
biodegradation
of
many
other
environmental pollutants (Zhuangw et al; 2003).
Amylase are enzyme that breakdown starch or glycogen.
Amylases can be derived from several sources such as plants,
animals
and
microbes.
The
major
advantage
of
using
microorganisms for production of amylases is in economical bulk
24
production capacity and microbes are also easy to manipulate to
obtain enzymes of desired characteristics.
Amylases stand out as a class of enzymes, which are of
useful applications to the food, brewing, textile, and mainly
employed for starch liquefaction to reduce their viscosity,
production of maltose, oligosaccharide mixtures, high fructose
syrup and maltotetraose syrup.
25
CHAPTER THREE
3.0
MATERIALS AND METHOD
3.1
Equipment
Weighing
Balance,
petri
dishes,
autoclave,
incubator,
microscope, wire loops, slides and digital microscope eyepiece.
3.1.1
Media
Nutrient agar, MacConkey agar, Bambara-but starch medium,
Soluble starch, Basal medium.
3.1.2
Reagents
Distilled water, Peptone water, crystal violet, safranin, 3,5-
Dinitrosalicyclic acid and sodium hydroxide.
Their composition and method of preparation are presented
both in this chapter and the appendix.
3.2
Sample Collection
Fresh ugba samples used were purchased from a market
in Awka, the capital city of Anambra State.
26
3.3
Isolation of Micrococcus from Ugba
Exactly 1g of ugba was aseptically introduced in a sterile
mortar and pounded with a pestle. The crushed ugba were
introduced into a 3ml of distilled water, later it was made up of
10ml. Serial dilutions was carried out for 6 times i.e. up to (10 5),
with a wire loop, sample was collected from the last dilute into a
petri dish containing nutrient agar. The nutrient agar plates were
there after incubated at room temperature (30+20C) for 24 hours.
After which it was gram stained, and viewed under the
microscope with an objective of 1600 magnification. A picture
was taken using a digital microscope eyepiece attached to the
microscope. It showed cocci cells indicating the presence of
micrococcus.
3.4
Identification Of Isolates
The isolates were identified using their colonial appearance
and on the recommendation of Kogan (2001). The following test
were carried out; Grain staining, catalase test, citrate test,
oxidase test and coagulase test.
27
3.5
Gram Staining
The method used was described by carpenter (1977) and
Thomas (1973). Smears of the isolates are prepared and heat
fixed on clean grease free slides. The smears are stained for one
minute with crystal violet. This was washed out with gentle
running tap water followed by flooding the slides with dilute
Gram’s Iodine solution. This was washed off with water and the
smear decolorized with 95% alcohol till the blue colour are more
dripped out (about 30 seconds) the smears are then counter
stained with Saffranin solution for about 10 seconds. Finally, the
slides were washed with tap water, air dried and observed under
oil immersion x100 objective.
3.6
Biochemical Tests
3.6.1 Catalase Test
The method employed here was that described by Speck
(1976).
Drop of 3% of hydrogen peroxide was placed on a clean
slide using a sterile wire loop. A reasonable colony of the test
28
microorganism (micrococcus) is collected and placed on the top
of the hydrogen peroxide. The reaction is observed immediately.
Gas production indicated by the production of gas bubbles
confirmed the presence of catalase. Micrococcus sp is catalase
positive.
3.6.2 Citrate Test
This test is based on the ability of an organism to use
citrate as its only source of carbon. The organism is cultured in
medium containing sodium citrate ammonium salt and an
indicator (Bromo-thymol blue) which changes to blue if the
organism is positive. The medium used for this test was the
Simon’s citrate agar.
Method:
Using a sterile wire loop, a colony of the Micrococcus
sp is collected and inoculated into Simon’s citrate agar slant.
This was incubated at 370C for 24 h, after which it was observed.
A change in colour from green to blue after about 24 hours of
incubation indicated positive result.
29
3.6.3 Oxidase Test
A piece of filter paper was placed on a glass slide. The filter
paper was soaked with freshly prepared oxidase reagent and the
test colony was picked with a sterile glass rod and streaked
across the filter paper. A development of blue-purple colour
within a few seconds indicated positive result
3.6.4 Coagulase Test
Two drops of human plasma were placed on three different
spot on a slide the first was inoculated with the test organism.
The second was inoculated with Staphylococcus aureus as
control (positive). The slide was rocked for one (1) minute after
which the test microorganism spot was compared with the
positive and negative controls. A clumping of the plasma
indicated presence of coagulase organism.
30
3.6.5 Methyl Red Test
This
test
is
used
to
detect
whether
the
isolate
(micrococcus) could produce and maintain sufficiently a stable
acid
product
from
glucose
fermentation
of
the
Enterobacteriaceae (Baker, 1976). This test is carried out as
described by Kirk et al (1975).
Tubes are buffered glucose – peptone broth are lightly
inoculated with the isolate. The tubes are incubated at 37 oc for
not less than 48hours. About 5 drops of the methyl red reagent is
added into 5ml on the addition of the reagent show a positive
test. Methyl red test indicator consist of 0.1g methyl red and
300ml of 95% ethyl alcohol.
3.7
Culture Conditions and Amylase Production
Culture media (150ml) in 500ml conical flasks were
sterilized in an autoclave at 15psi and 121 0C for 15 minutes. The
flask was cooled at room temperature. The flasks containing the
culture media were inoculated with 3ml of suspension containing
700µg of isolated Micrococcus sp biomass in saline. The flask
31
was left on the bench with occasional shaking. Exactly 5ml of the
sample was collected for analysis of biomass (OD600nm). The
amylase activity of the culture filtrate was estimated at 6 hourly
intervals for 96h.
3.8
Amylase assay
Amylase activity was assayed as described by Wood
and Bhat (1988) using a reaction mixture (2ml) comprising 1ml of
enzyme solution and 1ml of soluble starch (10gl-1) in 0.1M
acetate buffer pH 7.0. The mixtures were incubated for 10min at
300C. Total reducing sugars were determined by dinitrosalicylic
(DNS) method (Miller, 1959). A total of DNS was added to the
reaction mixture, the mixture was properly covered and boiled for
10mins. Boiled mixture was prompt cooled, diluted with 10ml of
distilled water and read off at 540nm with a spectrophotmeter.
One unit of amylase activity was defined as the amount of
enzyme which released
 mole glucose min
-1
mg-1 protein.
32
CHAPTER FOUR
4.0
RESULTS
4.1
Isolation of Micrococcus sp
After isolation of the colonies, Micrococcus sp was identified by
viewing under the microscope and using other biochemical tests. The
organism appeared yellow on nutrient agar as shown in Figure 1 and
was Gram positive as illustrated in Figure 2, with clusters of the cells
observed as cocci arranged in tetrads and clusters of tetrads.
Fig 1. Colonies of Micrococcus sp isolated from fermented oil
bean seed.
Fig 2. Gram positive cocci on gram stain, x100 magnification, oil
immersion.
33
Table 2: Biochemical test for the identification of Micrococcus
Bacteria
Micrococcus
4.2
Grain
Catalase
staining
(test)
Positive(+)
Positive(+)
Citrate test
Methylred
Oxidase test
test
Negative(-)
Positive(+)
Cogulase
test
Negative(-)
Negative(-)
Growth of Micrococcus sp in different types of media
Micrococcus sp isolated from ugba was used for amylase
production in three different types of media. The basal medium
contained soluble starch, gelatin and olive oil while the bambara
medium contained bambara nut starch instead of the afore listed
and the third media contained only soluble starch. Micrococcus
grew in the all the media and highest growth was observed after
76h. Growth in soluble starch medium was highest, followed by
bambara and then basal medium as shown in Figure 3.
34
35
4.3
Amylase production
Highest amylase activity was observed after 46h cultivation
of Micrococcus sp grown in basal medium and bambara nut
medium. From figure 4, it is obvious that enzyme activity was
higher in bambara nut medium (35 U/ml) than in basal medium
(19 U/ml). Highest activity of 30.8 U/ml was obtained after 56h
cultivation in soluble starch medium.
36
37
Figure 4. Amylase production in medium containing bambara nut
38
Figure 5. Amylase production in medium containing Soluble
Starch
39
CHAPTER FIVE
5.0
DISCUSSION, CONCLUSION AND RECOMMENDATION
5.1
Discussion
The microorganism isolated from the fermented African Oil
bean seed (Ugba) in this project tally with those reported in
earlier works by Obeta (1983).
Micrococcus sp appeared as smooth yellowish colonies in
nutrient agar as shown in Figure 1. Micrococcus was Gram
positive (Figure 2), catalase positive and positive to methyl red
test. However, it was negative to the other biochemical test like
oxidase test, coagulase test and citrate test.
Obeta in 1982 tried to use pure cultures of the above
isolate to ferment ugba, he reported that Bacillus spp softened
the slices of ugba, as well as produced the characteristics aroma
of the commercially fermented oil bean. However, the role of
Micrococcus sp ugba was not well understood (Mbajunwa et al.,
1998). Though its role in ugba fermentation remains unclear,
Micrococcus sp is a constant microbial association of ugba
40
according to previous reports (Njoku et al., 1990). African oil
bean seeds are largely composed of oil, protein and relatively
small amount of carbohydrate, the ability to breakdown these
components of the seed is an important characteristics of
organism able to ferment and persist till the seed spoils. This
work shows that Micrococcus specie produces amylase when
growing on different types of media. This probably explains its
ability to persist in ugba as part of its spoilage association
(Nwagu et al., 2010). Bacteria involved in the fermentation are
probably introduced through the air, water, utensil, banana
leaves or by handling during preparatory stages (Mbata and Orji,
2008). The ability to produce enzymes able to hydrolyze the
nutrient components of ugba ensures the establishment of an
organism as the spoilage association of ugba. This explains why
micrococcus spp and bacillus spp, which are known to
breakdown amylases and oil, were prominent throughout the
experiment (Mbajunwa et al. 1998).
41
5.2
Conclusion
Micrococcus isolated from ugba using nutrient broth was
confirmed be a good extracellular amylase producer which may
partly explain its ability to thrive in ugba during fermentation. For
production of amylase from Micrococcus sp isolated from ugba,
Bambara-nut starch is best recommended for a higher yield than
using other medium like basal medium and soluble starch
medium.
5.3
Recommendation
Amylase and its products are used in numerous industrial
processes; further studies may be needed to determine whether
the amylase from the Micrococcus from ugba posses unique
properties which can be applied in industrial processes.
42
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of prosopis Africana seeds for Production of Okpiye.
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(Pentadethra Macrophylla Benth” Journal of Food Science.
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Aderigbe E.Y., (1988), Studies on fermented oil bean seed.
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Adelusi E.Y. and Olowookere S.C. (1985) Determination of
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Adewuayi and Odunfa, (1988). Information on the Manufacturing
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microbiology 24:2-8.
Akanbi, L. S.(1992). Important uses of African oil bean seed
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43
Aiyer, P.U, (2005). Amylases and their applications Afr.J
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Enujiugha V.N. and Akanbi C.T. (2005), “Compositional changes
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(7): 27-39.
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McGraw- Hill
44
Garbult J. (1997). Essential of food microbiology. Amold
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45
Mbajunwa (1998), Obeta (1983), and Nwagu et al (2010), Role of
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46
APPENDIX I
Reagents
Distilled water, crystal violet, saffranin, 3,5 -Dinitrosalitcyclic acid,
potassium, Methyl red, sodium chloride
Media
Nutrient broth, MacConkey agar, Basal medium, Bambara-nut
starch medium, soluble starch medium.
Equipment and apparatus
Weighing balance, petri dishes, autoclave, test tubes, Bunsen
burner, incubator, measuring cylinder, microscope, wire loop,
refrigerator, slides.
47
APPENDIX II
MEDIA USED
NUTRIENT AGAR
This is a general medium that supports the growth of most
microbial species. It was used to isolate micrococcus from Ugba
sample.
Composition
g/litre
Lab-Lemco powder
1g
Yeast extracts
2g
Peptone
5g
Sodium chloride
5g
Agar
15g
PH
7.4g
Preparation:
The
medium
was
prepared
as
directed
by
the
manufacturer. 2.8g of the medium are dissolved in 100ml of
distilled water. The suspension was first dissolve completely by
heating and then sterilized by autoclaving at 121 0C for 15min.
The molten medium was allowed to cool at 450C before
48
dispending into sterile petri dishes in which it was allowed to get.
The slant was also prepared by dispensing into sterile bijou
bottles or test tubes and then allowed to solidity in slanting
position.
MacConkey Agar:
This is a differential medium, use to differentiate lactose
fermenters from non-lactose fermenters.
Composition
g/litre
Peptone
20g
Lactose
10g
Bile salt
5g
Neutral red
0.015g
Agar
12g
PH
7.6g
Preparation:
The powder medium was prepared according to the
manufacturing directors. 2.5g of the medium were dissolved in
100ml of distilled water. It was dissolved completely by heating
49
them sterilized at 1210C for 15 min in the autoclave. The molten
media was allowed to cool to 350C and then poured into sterile
petri dishes. The medium was then allowed to solidify in the petri
dishes.
Bambara Medium
Used for amylase production
Composition
g/litre
Lab-lemco powder
1.0g/l
Yeast extract
2.0g/l
Peptone
5.0g/l
Sodium chloride
5.0g/l
KCL
15g/l
Bambara groundnut starch
10g/l
Basal Medium
Used for enzyme test
Composition
g/litre
Lab-lemco powder
1.0g/l
50
Yeast extract
2.0g/l
Peptone
5.0g/l
Sodium chloride
5.0g/l
KCL
15g/l
Soluble starch
5.0g/l
Gelatin
2.5g/l
Olive oil
2.5g/l
Soluble starch
Used for enzyme test
Composition
g/litre
Lab-lemco powder
1.0g/l
Yeast extract
2.0g/l
Peptone
5.0g/l
Sodium chloride
5.0g/l
KCL
15g/l
Soluble Starch
10g/l
51