ABSTRACT
Microbial load of African fermented oil bean seed ( pentalethra macrophylla bentham) was studied. A total of four locally fermented samples of oil bean seed wrapped with traditional leaves called ‘ororompo leave’ were used. The four locally fermented samples were randomly purchase from different markets in Imo State which include Eke Onunwa, Orji market, Relief market, Orie Ekwe, and then taken aseptically to the laboratory for preparation and microbiological analysis, the sample Eke Onunwa, Orji market, Relief market. The microorganisms identified were identified as Bacillus sp, Mucor sp, Aspergillus sp, Proteus sp, Staphylococcus sp, Escherichia Coli, Micrococcus sp and Candida sp. There was no remarkable change in the organisms, except for the increase in number and it was observed that as time increases the colour and texture of the ugba changes from soft, light brown to dark brown, Very soft and very slimy the last day of fermentation.
CHAPTER ONE
- INTRODUCTION/LITERATURE REVIEW
1.1 INTRODUCTION
Ugba is a fermented product from African oil bean seed Pentaclethra macrophylla Benth, a woody plant predominant in the rain forest areas of West and Central Africa belonging to the family Leguminosaea, sub-family Mimosoidae (Keay, 1989). Ugba 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 serve as a delicacy amongst consuming regions where it is consumed garnished with other vegetables or staples. Consumption of ugba seeds could pose as a means of addressing the prevailing Protein Energy Malnutrition (PEM) in developing countries (Enujiugha and Akanbi, 2008).
Preparation of ugba is by mixed fermentation carried out spontaneously by a number of microorganisms. Microorganisms isolated from fermented ugba include Micrococcus sp., Lactobacilli, Staphyloccus sp., Leuconostoc mesenteroides, Proteus and E. coli (Isu and Njoku, 1997; Mbata and Orji, 2008; Obeta, 1983). 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 increased softness (Enujiugha et al., 2008), color change, off flavor and sliminess (Mbata and Orji, 2008) and production of pungent ammonical odor (Ogbulie et al., 1993).
Extending the shelf life of ugba has been a case of interest to many researchers. Reports on strategies to extend the shelf life of fermented ugba include treatment with varying concentrations of sodium chloride (Ogbulie et al., l993), preservation in high density polyethylene sachets and aluminum foil wraps (Ogbulie et al., 1998), canning within tomato puree, brine solution or refined groundnut oil (Enujiugha and Akanbi, 2005) and use of starter cultures to shorten period of fermentation (Mbata and Orji, 2008). The deterrent in the methods reported so far include the cost of implementation, no remarkable difference in shelf life extension and lack of ease in method application making it difficult or impossible for local commercial dealers to implement such methods.
A key selling point to using fermented oil bean seed ugba to address Protein Energy Malnutrition (PEM) issues is the ease of process adoption by local producers. Advocating for commercialization of this process though commended may inadvertently lead to a hike in product prize making this rich source of protein unavailable to the impoverished that are direly in need of the nutrients, which this can provide. A concise knowledge of the spoilage associations of fermented oil bean seed will enable the identification of the chief sources of spoilage and therefore help to identify simpler techniques to address spoilage problems.
1.2 AIM AND OBJECTIVES
Aim
This study is aimed to at determining the microbial load of African fermented oil bean seed (Pentaclethra macrophylla bentham).
Objectives
The objectives are stated thus;
- Determination of microbial load on fermented African oil bean (Pentaclethra macrophylla bentham)
- Isolation of microorganisms associated with fermented African oil bean (Pentaclethra macrophylla bentham)
- Characterization of microorganisms associated with fermented African oil bean (Pentaclethra macrophylla bentham)
1.3 LITERATURE REVIEW
Ugba is a fermented product from oil bean seeds (pentaclethra macrophylla benthham) a woody plant predominant in the rain forest areas of west and central African belonging to the family Luguminosae. Ugba is a primary important as cheap s easily available source of plant protein developing countries of the world and African in particular.
Fermented seed are not just palatable but serves as a delicacy amongst consuming religious where it is consumed garnished with other vegetables or staples consumption of ugba seeds could pose as a means of addressing the prevailing protein energy malnutrition in developing countries (Enujigba and Akanbi, 2008).
Processing of the seeds entails boiling, wrapping in banana leaves and fermentation. Processing ugba seeds drastically reduces the level of the anti-nutritional compound mentioned while increasing iron, calcium, potassium thiamine and riboflavin levels (Enujiugba and Ayodele Oni, 2003).
According to (Odoemalam, 2005) the oil bean seeds contain 4-17% carbohydrates, 44-47% oil, which has been found to be rich in oleic acid. The oil bean seed also contains linoleic acid. (Onwuliri et al., 2004) also found out that the saturated fatty acid lingnoceirc acid occurred in high amounts constituting about 10% of the total fatty acid concentration of the oil. The major problem with the fermented oil bean seeds, ugba is the restricted availability due to its very short sheelf-life. Under room temperature ugba spoils within 3-4days. Spilage is identified with increase softness (Enujiugba et al., 2008) colour change. Off and sliminess (Mbata and Orji, 2008).
Extending the shelf life of ugba hasd been a great interest to many researchers reports on strategies to extend the shelf life of fermented ugba including treatment with varing concentrations of sodium chloride, preservation in high density polyethylene sachets and aluminum foil wraps. Canning within tomato pure, brine solution of refined groundnut oil (Enujigba and Akanbi, 2005) and use of starter cultures to shorten period of fermentation (Mbata and Orji, 2008) the deterrent in the methods reported so far include the cost of implementation, no remarkable difference in shelf life extension and lack of ease in method application making application difficult or impossible for local commercial dealers to implement such methods (Nwagu et al., 2010).
1.3.1 UGBA DISTRIBUTION
Ugba is a fermented food produced from the fermentation of African oil bean seeds (Pentaclethra Macrophylla Bentham). The seeds are obtained from a large tree all over the southern part of Nigeria especially in the east. The seeds are oval, flat, tough, and brown in color (Achinewhu, 1986).the seeds are distributed by dispersal method. Currently, Ugba is gaining wide acceptance being consumed by people of different ethnic group and socio-economic classes in Nigeria and in west Africa sub-region (Ogbulie et al., 1993).
- UGBA PRODUCTION
Traditionally, Ugba is produce first, boiling the seed to remove the hard shells for 5-8 hours. It was cooled and the cotyledons were washed and sliced into sizes. The sliced cotyledons were washed and boiled, after it has cooled own, it was soaked in water for about 1-2 hours. After that, the cotyledons were washed and allowed to drain for1 hour in a basket lined with leaves. The cotyledons were wrapped with ‘’ororompo” leaves and allowed to incubate for 2 hours at room temperature before eating. This method was described by Njoku and Okemmadu (1989) as fermentation.
FLOW CHART OF UGBA PRODUCTION BY LOCAL PRODUCERS
Dry seeds
Boil for bout 5-8 hours to remove the hard shells
Slice and wash the cotyledons
Boil and soak in water for 1-2 hours
Wash and allow to drain for 1 hour
Wrap the cotyledons with orormpo leaves
Ugba.
- COMPOSITION AND NUTRITIONAL VALUE OF UGBA
Ugba, which is a protinacious food,produced by a 3-day fermentation of the leguminous seeds of African oil bean. Ugba has been reported to be very nutritious with a crude protein content that includes twenty essential amino acid with the important amino acids like thiamin and riboflavin (1974). He further showed that ugba, though low in its content of sulphur containing amino acid is rich in lysine while the carbohydrates and lipid contents were about 14.8% and 58% Respectively.
Ugba is rich nutritionally and hence a major source of plant protein,widely consumed traditionally in southern Nigeria (Kolawole and okonkwo, 1984). The demand may therefore exceed its supply. With the global economic crisis and its attendant food scarcity, ugba has the potential of serving as a low cost palatable and nutritious food item to over 40 million people who eat it(Mba et al., 1974).
Elsewhere, other works have referred to ugba as a good source of low cost protein (Achinewhu, 1982; Ogbuie, 1998; Njoku and Okemmadu, 1989). Generally, the nutritional values of ugba have greatly contributed to the number of people that consume it throughout the world.
However, ugba is available because of its short self-life (perishable nature) mainly due to the activities of micro-organisms (Obeta, 1983, Njoku et al., 1990) as its high nutrient level supports the growth of micro-organisms shortly after fermentation. Because of chance of inoculation and consequently variation in processing, ugba of variable qualities it obtains (Mba et al., 1974).
1.3.4 MICRO-ORGANISMS ASSOCIATED WITH FERMENTATION
The activities of micro-organisms in the fermentation of African oil bean seeds starts with the soaking of the seeds. Some of the micro-organisms are inhibited while others tend to proliferate. The dominant micro-organisms are Lactobacillus Spp, Micrococcus Spp, BacillusSspp, and Staphylococcus Spp (Isu et al., 1995). They further reported that Bacillus Spp. were most persisted from the onset to the of fermentation and identifed the species of Corynebacterium, Staphylococcus, Bacillus, Citrobacter and Alcaligenes have been isolated during the processing of the African oil been seed (Njoku et al., 1989). Bacillus Spp play significant roles in modifying the substrate biochemically, nutritionally and organoleptically (Obeta 1983, Njoku et al., 1990). No yeast but mold was isolated. Apart from the work of Obeta (1983), the microbiology of the fermentation had not been studied in detail. For example, the source of the fermenting microorganisms is not known, but it has been speculated that they may have originated from several sources such as the steeping water, wrapping leaves and utensils used in processing.
However, the objective of this study is therefore to determine the organisms responsible for the fermented African oil been seeds to ugba. (Pentaclethra Macrophylla Bentham).
1.3.5 CHANGES THAT OCCUR DURING FERMENTATION
Various biochemical changes occur during the fermentation (Obeta, 1983) found that pH increased from 6.5 at 0hour to 9.0 at 48hours and declined to 7.1 at 72hours. The rise in pH has been attributed to the abundant production of ammonia during the fermentation due to protein hydrolysis. The increase in pH would encourage the growth of bacillus sp. which have been found to grow well at pH 7.0 to 8.0 (Odunfa and Oyegiola 1985). The drop in pH to 7.1 at 72hours could be attributed to the fact that B.subtitlas and B. lincheniforms use ammonia as nitrogen source (Odunfa 1986). However, Odunfa and Oyeyiola (1985), Njoku and Okemadu (1989), Ogueke and Aririatu (2004) found that the pH rose throughout fermentation from 5.0 – 5.7 at 0hour to 7.9 – 8.7 after 3-5 days of fermentation.
The temperature of fermentation was observed to increase from about 30.8 to 34.5 – 38.50C within the first 24-36 hours of fermentation and decreases gradually afterward to 30-32-350C at the end of fermentation (Odunfa and Oyeyiola, 1985; Njoku and Okemadu, 1989) thus ugba fermentation is exothermic.
This initial increase in temperature has been attributed to the intense metabolic activities of microorganisms (period of maximum microbial activity) and represent the most active and important period of the fermentation. This is because enzyme studies (Njoku and Okemadu, 1989) have revealed that a – amylase, proteolytic and lipolytic enzyme activities attained their maximum levels at 24-36 hours of fermentation. Thus it could be the enzyme already produced rather than the presence of the microorganisms that continued the fermentation later. Moisture content was also found to increase throughout the period of fermentation (52-56.90% to 71.20-73%) (Odunfa and Oyeyiola 1985, Njoku and Okemadu 1989, Ogueke and Aririatu 2004). The increase in moisture is believed to be due to the hydrolytic activities on the microorganisms. However, the high moisture level has been suggested to predispose the product to rapid spoilage.
Njoku and Okemadu (1989) detected a – amylase, proteolytic and lipolytic enzymes from the start of ugba fermentation. These enzymes attained their maximum levels at 24-36hours. They suggested that this could be assumed to be the period of maximum microbial activity. The initial enzymes activity detected could be due to activity of the natural micro flora of the oil bean which developed particularly during the soaking of the cooked bean. Njoku and Okemadu (1989) therefore suggest that, it could be that fermentation began much earlier during the soaking of the slice beans. Some workers (Enujiugha, 2002, 2004) have demonstrated that the raw seed contained both a – amylase and lipase. They observed that the specific activity of the purified a – amylase enzymes complement the bacteria enzymes during fermentation they could not have contributed to the fermentation as the boiling must have motivated them.
The proteinase enzyme is considered the most important enzyme in ugba fermentation (Njoku and Okemadu, (1989) detected as seven fold increase in the level of amino nitrogen Enujiugba (2003). Only a two fold increase in reducing sugar was found while the activity of lipase was minimal compared to the other two enzyme (Njoku and Okemadu, 1989). This agrees with the report of (Achinewihu, 1986) that fermentation has to effect on the fatty acid content of pentaclethra macrophylla bentham. The minimal activity of the lipase could be attributed to the effect of Nacl that is usually added during fermentation (Enyiugha et al., 2004). In their study of the lipase activity in dormant seeds of African oil been seeds observed that the activity of lipase isolated from the seeds were inhibited up to 36% by Nacl.******
