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 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 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 micro-organisms 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 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;
- isolating micrococcus from fermented ugba
- producing amylase from the micrococcus from ugba
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).
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, 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 180C, it requires a mean annual temperature of about 250C. 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 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 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 7-13cm 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).
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).
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.
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 4.5-5.5cm 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.
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 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).
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, 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, 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 & 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).
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.
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).
TABLE 1: Common uses of Pentaclethra macrophylla in West Africa.
| Uses | Parts of plant | Countries |
| Food
Salt substitute Edible oils Fences and palings Charcoal Caroling bowls, etc Sees craft (beadings) Dye (mordant) Mild poison Medicine (Convulsion) Medicine (convulsion) Medicine (diarrhea) Medicine (itch) Medicine (lactogenicity) Medicine (wound treatment) ornamental |
Seed
Pod ashes Seeds Wood Wood Wood Seed (beadings) Pod ashes Bark and seed Pod Crushed seed Burnt leaf Leaf / steam bark Bark as liniment Bark decoction Bark as lotion
Whole tree |
Nigeria/ Ghana
Ghana Nigeria, Ghana, Togo, Cameroon Nigeria, Ghana, Togo, Cameroon, Cote d’ivoire
Nigeria, Ghana Nigeria Ghana Ghana Cameroon Ghana, Nigeria Ghana Ghana Ghana Ghana Ghana
Nigeria |
Abbiw (1990).
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 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 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 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.
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.
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 (105), 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.
- 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.
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 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.
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.
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 37oc 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 1210C 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 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 m mole glucose min-1mg-1 protein.
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.
Table 2: Biochemical test for the identification of Micrococcus
| Bacteria | Grain staining | Catalase (test) | Citrate test | Methylred test | Oxidase test | Cogulase test |
| Micrococcus | Positive(+) | Positive(+) | Negative(-) | Positive(+) | Negative(-) | Negative(-) |
4.2 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.
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.
Figure 4. Amylase production in medium containing bambara nut
Figure 5. Amylase production in medium containing Soluble Starch
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 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).
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.
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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.
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 1210C for 15min. The molten medium was allowed to cool at 450C before 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 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
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
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