Introduction

‘Owoh’ is a traditionally fermented high protein food that is solely produced from cotton seeds (Gossypium hirsutum) Its fermentation, like other soup condiments is still a family art (Sanni and Ogbuna,1991). Fermentation of ‘owoh’ is by chanced inoculation. This method exposes the seasoning agent to contamination by pathogens and results in lack of consistence in final product, hence reduces its acceptability.‘Owoh’ serves as a low cost substitute for animal protein, since it (‘owoh’) is very rich in amino acids and essential fatty acids (Enujuigha and Agbede, 2000). This is responsible for the formation of ammonia that accounts for the characteristic pungent odour and high pH of ‘owoh’ (Cadwell, 1995).

Bacillus sp had been implicated in the fermentation of condiments especially those mainly consumed in West Africa (Campbell-Platt, 1980). In production of ‘iru’, fermented locust bean (Parkia biglobosa), Odunfa (1985) and Oyeyiola (1988) identified Bacillus sp as most predominant organism. Obeta (1983) also reported the association of Bacillus sp with fermentation of ‘ugba’ a fermented product of Africa oil bean (Pentaclethra microphyla Benth) while Odunfa (1985) and Sanni et al.(1998) isolated Bacillus sp from ‘ogiri’, a fermented melon seeds (Citrullus vulgaris Schrad) and ‘owoh’, respectively.

Bacillus subtilis has been reported to be frequently encountered in the fermentation of oil-rich seeds (Campbell-Platt, 1980). This report made us investigated the advantage of single starter and the fermentative potentials of Bacillus subtilis isolated from other fermented oil-rich seeds.

In this work, interest was to develop a starter culture that produces ‘owoh’ with a better organoleptic property and without loss of acceptability among consumers.

Materials and Methods

Source of Cotton seeds

The cotton seeds used for this work were purchased from a market in Ado-Ekiti. They were collected in a new polythene bag and brought to the laboratory. The seeds were sorted to get rid of dirt and decomposing seeds. The seeds were boiled for one hour and dehulled. The cotyledons were washed and sorted again. The clean cotyledons were wrapped in aluminum foil and boiled for 2h. The water was drained off and allowed to cool before  ground in a mortar with a pestle

Source and Preparation of Inocula

The bacteria used for this work was collected from stock culture of Microbiology Department, University of Ado-Ekiti. The bacteria were isolated from condiments and characterized accordingly. The test organisms were Bacillus subtilis (BSWH), Bacillus subtilis (BSGR), Bacillus subtilis (BSRU ) Bacillus subtilis (BSSR), Bacillus lichenliformis (BLWH) and Bacillus pumilus (BPWH) the viability of each of the organism was ensured by resuscitating it in buffered peptone (Oxoid) broth and incubated at 37^oC for 18h. The starter cultures were standardized by the method of Bauer et al, (1966).

Twenty gram (20g) of mash was introduced into a100 ml conical flask, properly corked and sterilized at 121^oC for 15 min. After which two millilitre (2.00 ml) of standardized starter culture was introduced into the mash and mixed thoroughly with a surface sterilized spatula and incubated at 37^oC for five days.

Determination of Proximate Composition of ‘Owoh’

The dry matter of the sample was determined by weighing 2g in a crucible; dry in an oven at 105^oC.  The sample was removed, cooled in desiccator and weighed intermittently until a constant weight was obtained. The weight was expressed in g/100g of freshly fermented ‘owoh’.

The pH of the sample was determined by taking 2g of fermented sample into a clean mortar and homogenized to a pulp using a pestle .ten millilitre (10 ml) of distilled water was added to make fine slurry. The pH meter (ELE model) standardized using buffer at the pH of 4.00 and 9.00 was dipped into the samples to determine their pH values.

The standard method of AOAC (1990) was used to determine the ash and the lipid content of the sample. Dried sample was weighed into a platinum dish and ashed by heating at 500^oC in a muffle furnace until residue turned whitish gray. The ash content per unit weight was calculated and expressed as percentage (%). The lipid content of the samples was extracted quantitatively with petroleum ether (b.p. 40-60^oC) in a Soxhlet apparatus.

The indicator method of AOAC (1984) was adopted for the determination of titratable acidity. Twenty grams of sample was ground into pulp using a mortal and pestle. Then 100 ml of distilled water was added to make fine slurry. The slurry was filtered using Whatman No 1 filter paper .to 10 ml of filtrate in 100 ml conical flask was added 5 drops of 1% phenolphthalein indicator. Sample was titrated against 0.1N NaOH (in burette) until a pink colour developed. The result was expressed as normality (N) of the predominant citric acid.

The soluble protein was determined according to Lowry et al, (1951) while the crude fibre was determine according to AOAC (1984). Dried samples were extracted with petroleum ether; air dried and boiled in H₂SO₄ for 30 min. after cooling, insoluble matter (in water) was washed free of acid then boiled in alkali (NaOH solution). Residue was washed in 95% ethanol and then ether, before drying in oven at 100^oC.

Sensory Evaluation of Owoh

A randomly selected nine-man panel conducted sensory evaluations. The panel consists of students and staff members of the University of Ado-Ekiti. A scorecard was six-point hedonic scale (with excellent, very good, good, poor, and very poor representing 6, 5, 4, 3, 2 and 1 respectively) was supplied to each of the panelists. Attributes scored include appearance, colour texture, odour, and general acceptability. The identity of each of the samples was concealed from the panelists as suggested by Moskowisz (1999)

The results were subjected to statistical analysis using analyses of variance (ANOVA). The level of significance was determined at 0.05 level of probability.

Results

The mean value of triplicate determination of proximate composition analyses of ‘owoh’ fermented by different Bacillus species was presented in Table 1. The protein content of the samples varied from 14.03 in sample BSPLO to 33.79g/100g in sample BLO. ‘Owoh’ produced by single starter culture of Bacillus species had higher amount of crude fibre than the sample with the combination of the starter cultures. The amount of free nitrogen extract (NFE) was lower in pure starter cultures.

Table 1: Proximate composition of ‘owoh’ fermented by different Bacillus species (g/100g dry matter).

Table 2: Proximate composition of ‘owoh’ produced by Bacillus subtilis strains isolated from different condiments (g/100g dry matter).

Legend

BSRU = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘iru’

BSSR = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘soy-iru’

BSWH = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘owoh’

BSGR= ‘Owoh’ fermented by Bacillus subtilis isolated from ‘ogiri’

Control = Naturally fermented ‘owoh’

Table 3: Sensory evaluation of ‘owoh’ fermented by Bacillus subtilis from different soup condiment.

Legend

BSRU = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘iru’

BSSR = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘soy-iru’

BSWH = ‘Owoh’ fermented by Bacillus subtilis isolated from ‘owoh’

BSGR= ‘Owoh’ fermented by Bacillus subtilis isolated from ‘ogiri’

Control = Naturally fermented ‘owoh’

The dry matter of ‘owoh’ fermented by Bacillus subtilis isolated from different condiment ranged from 61.65 to 71.53g/100g. Sample BSRU had the highest value (71.52g/100g). Amount of soluble protein, ash, and crude fibre was highest in BSRU with values 45.15, 1.52 and 6.90g/100g respectively. Naturally fermented ‘owoh’ had the highest amount of NFE (28.68g/100g). The titratable acidity of the product ranged from 0.052 to 0.086. The least value (0.052) was recorded in sample BSRU.

The acceptability of ‘owoh’ produced by different strains of Bacillus subtilis varies. Sample SRBS had the highest value in general acceptability with sample GRBS.

Discussion

‘Owoh’ is produced from an oil seed,it is very rich in amino acids and essential fatty acids(Enujuigha and Agbede, 2000). ‘Owoh’ is nutritionally better than raw cotton seeds (Enujuigha et al.,2002). Bacillus subtilis was found to be the best for the fermentation of ‘owoh’. This report is in agreement with the work of Oyeyiola and Odunfa (1985)

Bacillus subtilis had been reported to produce antibiotics that act against other bacteria (Campbell-Platt, 1980). The combination if the starter cultures produced low quality of ‘owoh’. This may be as a result of competition for the available nutrients or antagonistic interaction between the organisms that consequently reduced their efficiency (Woodburn, 1992).

The pH of fermented ‘owoh’ skates between 5.10 and 7.30. This agrees with the findings of Whitaker (1978) who reported that the pH of fermenting oil seeds tends to alkalinity as against the starchy substrates that increase acidity with fermentation period. Gilbert (1973) reported that the genus Bacillus utilizes different carbon sources. It was also noted that the amount of nitrogen free extracts (carbohydrates) of the fermented cotton seed compare to the value reported for the raw seeds by Faulkner (1974).

Sample BSRU produced ‘owoh’ with highest amount of protein. Aderibigbe and Odunfa (1988) reported that Bacillus subtilis produces protease that degrades the protein in the Parkial biglobossa during the production of ‘iru’. This process resulted in steady increase in the amino acids content of the final product.

High measure of dietary fibre (5.61 to 6.90g/100g) of fermented ‘owoh’ is in close agreement with 6.50% reported by Sanni et al., (1998) the high crude fibre has been reported to aid digestibility and prevents constipation and appendicitis Ibitoye, (2005).

The result of the sensory evaluation test of ‘owoh’ fermented by Bacillus subtilis strains is shown in Table 3. Based on texture, the control sample was rated best with the score of 3.50 while in term of odour BSRU, BSSR, and control were rated same with 3.63 points the general acceptability presents sample BSSR to be the  best. According to Sarkar and Tamang (1994) this product judgment was the best way of rating and selecting the highest quality amongst the total products. There was no significant difference in the attributes examined (at p>0.05)

It may be concluded that single starter culture of Bacillus subtilis produced a better ‘owoh’ than other species. The combination of the organisms does not produce a nutritionally better ‘owoh’.

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