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Nigerian Food Journal 33 (2015) 54-60
www.elsevier.com/locate/nifoj
Controlled fermentation of cotton seeds (Gossypium hirsutum) for Owoh production using bacteria starter cultures
O.O. Ezekiela,n, A.A.O. Ogunsheb, D.E. Jegedea
aDepartment of Food Technology, University of Ibadan, Ibadan, Nigeria bDepartment of Microbiology, University of Ibadan, Ibadan, Nigeria
Available online 23 May 2015
Abstract
This work studied the potential of four bacteria as starter cultures for the fermentation of cotton seeds (Gossypium hirsutum) to produce Owoh condiment. Cotton seed was fermented using spontaneous traditional method and starter cultures. Single cultures of Bacillus subtilis, Bacillus licheniformis, Bacillus pumillus, and Staphylococcus spp. and their combinations with and without Staphylococcus spp. were used as starters for the fermentation at 35 °C for 3 days. Chemical components of fermented and unfermented samples of cotton seeds were analysed for titratable acidity, pH, gossypol and proximate using standard methods. Sensory evaluation of the fermented products was carried out for colour, aroma, taste, stickiness and overall acceptability. Data were analysed using Analysis of Variance (ANOVA) and means separated by Duncan's multiple range test. Crude protein, crude fat, crude fibre, ash, moisture content, dry matter, carbohydrate, pH, titratable acidity and gossypol ranged from 22.40% to 13.62%, 28.25% to 15.29%, 11.29% to 2.51%, 3.65% to 1.25%, 39.26% to 55.85%, 60.74% to 44.15%, 34.41% to 67.33%, pH 6.67 to 8.24, 0.36% to 0.02% and 0.004900% to 0.000233%, respectively. Sensory evaluation of the fermented products showed that Owoh produced by the spontaneous or traditional fermentation of cotton seed was not significantly different from Owoh produced with combinations of starters. Although most of the single starter culture did not produce acceptable products in the controlled fermentation of cotton seeds, they played some roles in the product quality. Overall, the use of combinations of isolated Bacillus species with and without Staphylococcus spp. produced Owoh of good quality.
© 2015 Association of Vice-Chancellors of Nigerian Universities. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Fermentation; Starter cultures; Cotton seed; Bacillus spp.; Staphylococous spp.
1. Introduction
Fermented foods form a substantial part of the diet of the people of Africa. These foods are prepared from maize, sorghum or millets or various mixtures of these cereals. Vegetable proteins are also fermented to produce condiments. Some of the oil seeds employed for fermentation include melon seed (Citrullus vulgaris), sesame seed (Sesamum undicum), African oil bean (Pentachletra macrophylla), African locust bean (Parkia biglobosa) and cotton seed (Gossypium hirsutum). These are
*Corresponding author.
E-mail address: funkeeze@gmail.com (O.O. Ezekiel). Peer review under responsibility of Association of Vice-Chancellors of Nigerian Universities.
fermented to produce condiments which are used as flavourings in soups and sauces. The fermented products usually have a strong but pleasing aroma in soups. Fermented vegetable proteins have a great potential as key protein sources. This emphasises the importance of soup flavourings in Nigeria. Fermentation of these vegetable proteins is usually in moist solid state and is by chance inoculated brought about by various species of micro-organisms. Members of the genus Bacillus and coagulase negative Staphylococcus have been reported to be involved in the fermentation processes such as in the fermentation of melon seeds (C. vulgaris) for 'ogiri' production (Odunfa, 1981b), the African oil-bean (P. macrophylla) for 'ugba' production (Obeta, 1983; Odunfa and Oyeyiola, 1985; Ejiofor et al., 1987), castor oil seeds (Ricinus communis) for 'Ogiri-igbo' (Odunfa, 1985), soybean (Glycine max) for 'dawadawa' (Ogbadu
http://dx.doi.org/10.1016/j.nifoj.2015.04.013
0189-7241/© 2015 Association of Vice-Chancellors of Nigerian Universities. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
and Okagbue, 1988) and African locust bean (P. biglobosa) for 'iru' production. Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus and coagulase negative Staphylococcus are also known to be involved in the fermentation of cotton seeds (G. hirsutum) for Owoh production. The traditional condiments have not attained commercial status due to their very short shelf-life, objectionable packaging materials and inconsistent product quality. The production of fermented vegetable proteins for use as food condiments is craft-based. Remarkably, in many areas of Nigeria today they are still made in traditional ways, with success depending upon the observance of good manufacturing practices and control of environmental conditions during the manufacturing phase. Starter cultures are not normally used in the fermentation process and therefore variations in the quality and stability and of the products are often observed and also the product's safety is not guaranteed. Despite extensive studies on Nigerian soup condiments, there has been little documented report on the production of Owoh. There is also a dearth of information on the nutritive potential of cotton seed in Nigeria. In the mid-western part of Nigeria, Owoh is a popular condiment prepared by fermenting cotton seed. The seed is not normally used in its natural state because it contains some anti-nutritional substances, in particular gossypol, which inhibits digestion. However, the presence of gossypol in the meal has impeded the acceptance of cotton seed flour and cotton seed protein for human food. Whole cotton seed contains up to 1.5-2.0% gossypol, all in the bound form, levels can reach as low as 0.4% in some species of commercial origin.
Fermentation is known to play a significant role in the gossypol level as well as the nutritional quality of the cotton seed. During fermentation, the anti-nutritional factors due to the gossypol in the cotton seed are reduced, which invariably makes the seeds edible without adversely affecting the consumers. Fermentation has also been used to improve the digestibility of some indigenous oilseeds.
The objectives of this work were to produce Owoh from cotton seeds (G. hirsutum) traditionally and under controlled fermentation condition using pure cultures of B. subtilis, B. licheniformis, B. pumilus and coagulase negative Staphylo-coccus spp. as starters; and evaluate the effects of these pure bacterial cultures on the chemical composition and sensory attributes of the fermented cotton seeds.
2. Materials and methods
2.1. Traditional fermentation of cotton seeds for Owoh production
Owoh was prepared in the traditional way using cotton seeds (Sanni and Ogbonna, 1991). The seeds were removed from the lint and boiled in water for 2 h until they became soft. After boiling, the water was carefully discarded and the seeds soaked in cold water overnight. The seed coat was later removed manually by pressing between the fingers to obtain the cotyledons. These were then tightly wrapped with banana leaves (Musa sapientum) in different packets and boiled again for 1 -2 h. The packets were removed after boiling and water was allowed to drain off
completely. The packets were then placed in calabash trays and covered with jute bags. Fermentation was allowed for 3 days. The fermented mash is Owoh, a dark-brown, semi-moist solid with a pungent smell.
2.2. Isolation of bacterial cultures
The isolation of the bacterial species for the controlled fermentation of cotton seeds (G. hirsutum) was carried out using a sample of Owoh from cotton seeds that was traditionally fermented. 10 ml of distilled water was discharged into six McCartney bottles and then sterilised in an autoclave at 121 1C for 15 min. The bottles were allowed to cool, after which a sterilised (flamed) wire loop was used to pick each of the samples into them to make suspensions. The bottles were gently shaken to dislodge the associated microorganism into the solution. Plate Count agar (PCA), De Man Rogosa Sharpe (MRS) Agar and Agar technical were prepared together and sterilised in an autoclave at 121 1C for 15 min. The PCA was used for plate count, MRS agra supplied the nutrients necessary for the growth of the bacterial species while agar technical assisted in the rapid gelling/solidification of the PCA. 1 ml of the suspensions of all the samples was measured with needle and syringe and dispensed onto sterile petridishes after which the cooled but not solidified sterile nutrient agar was then carefully poured onto the sterile petridishes and allowed to solidify. The petridishes were placed in the incubator at 35 7 2 °C for 24 h.
2.3. Purification of the bacterial isolates
Representative of each different bacterial colony types was randomly picked from the primary plates of each fermented sample and subcultured onto sterile plates by the streaking method. The isolated strains were subcultured by repeated streaking to obtain pure cultures. The bacterial isolates from the traditionally fermented samples of the cotton seeds (G. hirsutum), were kept in triplicates on PCA slants as working and stock cultures. Characterisation of the bacterial isolates: Taxonomic studies were carried out on the pure bacterial isolates from traditionally fermented samples of cotton seeds on the basis of their cultural, morphological, physiolological and biochemical characteristics.
Gram reactions and shape of cells were determined in 24 hold cultures of the pure bacterial isolates from samples of fermented cotton seeds (G. hirsutum) as described by Seeley and Van dermark (1972)
Determination of biochemical characteristics: Catalase, starch hydrolysis and carbohydrate fermentation tests were carried out according to the method described by Harrigan and McCane (1976). Gelatin liquefaction test was determined using the method of Edwards and Ewing (1972). Citrate utilisation test was carried out according to the method of Cruickshank et al. (1975). Hydrogen sulphide production test was carried out by stabbing the butt of Triple Sugar Iron Agar medium with the bacterial cultures and hydrogen sulphide production was detected by the blackening of the butt. Pulcherrimin
production test was carried out by culturing the bacterial isolates on Triple Sugar Iron (TSI) Agar and incubating at 35 7 2 °C for 48 h. The formation of a red pigment indicated pulcherrimin production. Oxygen Status test was conducted by culturing the bacterial isolates on plate count agar and incubating at 35 7 2 °C for 48 h under aerobic and anaerobic conditions, respectively.
Colonial morphology on triple sugar iron (TSI) agar: The cultural characteristics of the purified bacterial isolates were described as observed on culture media. Features examined included the elevation of colonies, pigmentation, surface structure, shape and light penetration of the colonies. Colonies developing on the plates were grouped based on their colonial morphologies (Prescott et al., 2002).
2.4. Controlled fermentation of cotton seeds for Owoh production
Owoh was prepared under controlled condition using cotton seeds (G. hirsutum). The seeds were removed from the lint and boiled under pressure for 1 h until they became soft. After boiling, the water was carefully discarded and the seeds soaked in fresh cold water overnight. The seed coat was later removed by pressing between the fingers to obtain the cotyledons. Thereafter, 36 g each of the dehulled cotton seeds was weighed and transferred into six sterilised 250 ml conical flasks. Sterilisation of the cotton seeds was carried out in an autoclave at 121 °C for 15 min minutes. The sterilised cotton seed was then allowed to cool in preparation for inoculation with the isolated bacterial cultures (B. subtilis, B. licheniformis, B. pumilus and Staphylococcus spp.). Inoculation of the cotton seeds was carried out singly with B. subtilis, B. licheniformis, B. pumilus and Staphylococcus spp.. Inoculation of the cotton seeds was also carried out in combinations with and without the Staphylococcus spp., 2 x 109 cfu/ml of the original dilution of the inoculums was used in inoculating in all the cases. Fermentation was thereafter carried out in a fermenter at 35 7 2 °C for 3 days.
2.5. Chemical analysis
The proximate analysis and determination of titratable acidity followed the official methods of analysis described by the Association of Official Analytical Chemists (Association of Official Analytical Chemists, 2005). Carbohydrate was determined by difference. The pH of the samples was determined using the Jenway 3505 pH meter, following standard procedures given in AOAC (2005). All analyses were carried out in triplicates.
2.6. Determination of gossypol content
The gossypol content was determined by the AOAC official method (2005) of Smith (1965). 2 g of the sample was weighed on a top pan Mettler Analytical Balance and transferred into a 100 ml volumetric flask. 10 ml of complexing agent, which is made up of 2:5 mixture of 3-amino-propanol with glacial acetic acid, was diluted to 100 ml with dimethyl
formamide. The mixture was heated in a boiling water bath for 30 min, cooled, and diluted to about 30 ml with 40:60(v/v) hexane-isopropyl alcohol. The solution was filtered and diluted to 50 ml with hexane-isopropyl alcohol. 1 ml of the filtrate was transferred into a 25 ml volumetric flask and two drops of 5 M HCl were added. A 5 ml aliquot of Ferric Chloric-bipyridyl reagent was added and mixed thoroughly, then allowed to stand for 5 min. 1 ml distilled water was added and the mixture was diluted to volume with (40/60) (v/v) hexane-isopropyl alcohol. A standard gossypol solution of range 0.01 mg/ml to 0.05 mg/ml was prepared from 0.10 mg/ml stock gossypol solution from Sigma Aldrich. These standard solutions were treated similarly like the extract above. The absorbances of coloured sample solutions and standard solutions were read on a Spectronic 21D Spectrophotometer at a wavelength of 620 nm against blank. This was carried out at 12 h intervals until fermentation ends.
2.7. Sensory evaluation
A reference sample and seven coded samples of cotton seeds fermented traditionally and under controlled condition were given out to 30 panellists in a ventilated and well-lit room to ensure proper sensory evaluation, and grading of the samples by the panellists was done using the 9-point hedonic scale. Preference test was conducted on the data collected from the questionnaire using the multiple comparison test (Larmond, 1977). All data collected were analysed using the Analysis of Variance with means separated using Duncan's Multiple Range test.
3. Results and discussion
3.1. Bacteria isolation
B. subtilis, B. pumilus, Bacillus licheniformis and Staphylo-coccus spp. were isolated from Owoh condiment. The Bacilli were all Gram-positive rods when viewed under the microscope while the Staphylococcus spp. was found Gram-positive cocci. All were catalase positive, indicating their ability to produce a catalase enzyme that is able to break down hydrogen peroxide into oxygen and water. The bacterial isolates were also able to utilise citrate as their only carbon source in their metabolic activities. Only B. subtilis and B. licheniformis were able to hydrolyse starch. B subtilis was able to liquefy gelatin while B licheniformis was also able to hydrolyse at a slow rate. B. subtilis produced a black pigment on Triple Iron Agar, indicating its ability to produce Hydrogen sulphide gas. All the bacterial isolates were facultative, indicating their ability to grow under aerobic and anaerobic conditions. B. licheniformis was able to produce pulcherrimin (red pigment) on Triple Sugar Iron Agar and this shows the distinct characteristic of B. licheniformis to produce pulcherrimin on any agar containing sufficient iron (Table 1).
Table 1
Biochemical and colonial morphological tests of bacteria isolated from "Owoh" condiment.
Biochemical and colonial morphological tests Isolate A
Isolate B
Isolate C
Isolate D
Gram's status
Catalase
Citrate
Starch hydrolysis
Gelatin liquefaction
Hydrogen sulphide
Pulcherrimin production
Oxygen status
Xylose
Sucrose
Fructose
Galactose
Dextrose
Maltose
Colonial morphology on triple sugar iron (TSI) Agar Suggested identity
+ rods +
+ + + +
Facultative anaerobe
Yellow mucoid pool, light-red, black base of pool Bacillus subtilis
+ rods +
Slow Strong
Facultative anaerobe Acid
Acid and gas Acid
Acid and gas Acid and gas Acid and gas
Yellow pool, actively spreading, red, hair-like outgrowth
Bacillus licheniformis
+ rods +
Facultative anaerobe Acid and gas Acid and gas Acid and gas Acid and gas Acid and gas Acid and gas Raised, mucoid, cream coloured Bacillus pumilus
+ cocci +
Nil test Nil test Nil test Nil test
Facultative anaerobe
Nil test
Nil test
Nil test
Nil test
Nil test
Nil test
Nil test
Staphylococcus spp.
3.2. Effects of fermentation on chemical composition of cotton seed
Table 2 presents the effect of fermentation on the chemical composition of cotton seed. There was significant difference in the crude protein content among the traditionally fermented cotton seeds, unfermented cotton seeds and cotton seeds fermented under controlled conditions. A reduction in the crude protein content after fermentation was observed, decreasing from 22.40% in the unfermented cotton seeds to 13.62% in cotton seeds fermented using a combination of all the four bacterial cultures. This decrease in the crude protein content could be due to several factors, such as the loss of ammonia during fermentation, or the hydrolysis of the protein in the seed, which subsequently provides excellent sources of nitrogen for microbial growth (Sanni and Ogbonna, 1991).
There was significant difference in crude fat contents between the traditionally fermented cotton seeds and unfer-mented cotton seeds as well as cotton seeds fermented under controlled conditions. There was a noticeable decrease in crude fat content in the fermented cotton seeds when compared with the unfermented cotton seeds. This reduction in value was from 28.25% in the unfermented cotton seeds to 15.29% in the fermented cotton seeds using combinations of all the four bacterial cultures in inoculating. This decrease in the crude fat content in the fermented samples could be attributed to their utilisation by the associated microorganisms during fermentation (Sanni and Ogbonna, 1992).
There was also a significant difference in the crude fibre content between the traditionally fermented cotton seeds, unfer-mented cotton seeds as well as those fermented under controlled conditions. A substantial decrease in the fibre content was noticed. This reduction in value was from 11.29% in the unfermented cotton seeds to 2.51% in the fermented cotton seeds using all the four bacterial cultures for inoculation. According to
Ejiofor et al. (1987), the fermentative activity of the associated microorganisms could have led to the breakdown of the fibre content of cotton seed with eventual utilisation by the microbes.
A decrease in the ash content was observed in the fermented cotton seeds, with the reduction ranging from 3.65% in the unfermented cotton seeds to 1.25% in cotton seeds fermented using combinations of all the four bacterial cultures for inoculation. The decrease in ash content could be related to the decrease in the crude fibre content (Ejiofor et al., 1987).
There was no significant difference in the moisture content and dry matter between cotton seeds fermented by inoculating with single starter cultures of B. subtilis and B. licheniformis. However, there was a great increase in the moisture content of fermented cotton seeds. The increase in the moisture content ranged from 39.26% to 55.85%% in the unfermented cotton seeds and those fermented when the combinations of all the four bacteria were used as starters, respectively. The increase in moisture content observed in the fermented seeds is due to the moist solid nature of the fermentation and the hydrolytic decomposition of the fermenting substrate. Also, there was an observed decrease in the dry matter of the fermented cotton seeds.
There was significant difference in the carbohydrate contents between the fermented and unfermented cotton seeds. However, a noticeable increase in the carbohydrate content was observed in the fermented cotton seeds. This increase in value ranged from 6.44% to 15.72% in the unfermented cotton seeds and fermented cotton seeds using a combination of all the bacterial cultures in inoculating, respectively. This could be because the microorganisms did not hydrolyse carbohydrate in their fermentative activity.
The pH ranged from 6.67% to 8.24% in the unfermented cotton seeds and fermented cotton seeds using all the four bacterial cultures in combinations, respectively. An increase in pH tending towards alkalinity was observed in the fermented cotton seeds. This increase in pH resulted from the production
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of amines and ammonia by the hydrolytic activity of the microorganisms This view agrees with that of Odunfa, (1981a, and b) on related fermented vegetable proteins . There was no significant difference in pH value between traditionally fermented cotton seeds and those fermented by inoculating with single starter cultures of Staphylococcus spp and B. lichenifor-mis There was no significant difference in titratable acidity between traditionally fermented cotton seeds and cotton seeds fermented with all the four bacterial cultures in combinations under controlled conditions A decrease in the titratable acidity was noticed in the fermented cotton seeds This reduction in value was from 0 36% in the unfermented cotton seeds to 0 02% in fermented cotton seeds when combination of all the starters was used in inoculating This decrease in titratable acidity could be due to the increase in pH value observed after fermentation
There was no significant difference in the free gossypol level between cotton seeds fermented under controlled conditions when inoculation was done with all the four bacterial cultures in combinations and when inoculation was done with only the combination of the three Bacilli spp There was also no significant difference between fermented cotton seeds using combinations of the three Bacilli spp and those fermented with single starter culture of B. pumilus With the use of starter cultures singly, no significant difference was observed in the level of free gossypol among the samples . However, there was a significant decrease in the free gossypol level in the fermented cotton seeds when compared with unfermented samples Free gossypol is an anti-nutritional and a toxic substance secreted in the pigment glands of cotton seeds The free gossypol level of cotton seeds ranged from 0 0049% in the traditionally fermented cotton seeds to 0 0002% in cotton seeds fermented under controlled conditions when inoculation was done with the combination of all the four bacterial cultures This reduction in the free gossypol of cotton seeds for Owoh production met the acceptable limits of free gossypol level in edible cotton seed products specified by the FDA and FAO/WHO The United States Food & Drug Administration (USFDA, 2014) and the Protein Advisory Group of the United Nations Food and Agriculture and World Health Organization (FAO/WHO/UNICEF, 1972) have set maximum guidelines of 450 ppm (0.045%) and 600 ppm (0 .06%), respectively, for free gossypol in edible cotton seed products (Lusas and Jividen, 1987)
3.3. Sensory evaluation of fermented cotton seeds samples
The results of the sensory evaluation carried out by the 30 panellists are as shown in Table 3 The results indicated that Owoh produced traditionally was rated the best among all the others in terms of the colour and aroma and this was not significantly different from cotton seeds fermented using single starter culture of B. pumilus as well as when combinations of all the four starters were used for inoculation Also, cotton seeds fermented under controlled conditions when inoculated with single starter of B. pumilus were most preferred in terms of the taste and stickiness and this was not significantly different from cotton seeds fermented spontaneously or
Table 3
Sensory evaluation of fermented cotton seeds samples.
Sample Codes Colour Taste Aroma Stickiness Overall acceptability
QXR 7.47a 7.03a 7.70a 6.80a 7.40a
QXV 6.90a 6.63a 7.60a 6.93a 7.07a
QXL 6.17b 5.57b 6.43b 5.63b 5.90b
QXZ 4.67c 4.17d 3.90d 3.77c 4.17c
QXP 5.73b 4.90c 5.70c 5.33b 5.37b
QXW 7.03a 7.23a 7.40a 7.37a 7.20a
QXK 4.10c 3.57e 3.77d 4.13c 3.77c
Samples in the same column with the same letter(s) are not significantly different from each other at P < 0.05
QXR, Owoh produced from traditionally fermented cotton seeds; QXV, Owoh produced from fermented cotton seeds using combination of all the four bacterial cultures for inoculation; QXL, Owoh produced from fermented cotton seeds using combination of the three Bacilli for inoculation; QXZ, Owoh produced from fermented cotton seeds using single starter culture of B. subtilis for inoculation; QXP, Owoh produced from fermented cotton seeds using single starter culture of B. licheniformis for inoculation; QXW, Owoh produced from fermented cotton seeds using single starter culture of B. pumilus for inoculation; QXK, Owoh produced from fermented cotton seeds using single starter culture of Staphylococcus spp. for inoculation.
traditionally as well as those fermented when combinations of all the four starter cultures were utilised. Owoh produced from fermentation of cotton seeds using the combinations of the three Bacilli spp. was rated moderately in terms of the colour, taste, aroma and stickiness. Owoh produced from fermented cotton seeds using single starter culture of B. licheniformis was also rated moderately in terms of the colour and stickiness, and was not significantly different from Owoh produced from the fermentation of cotton seeds using the three Bacilli spp. as starters. Also, Owoh produced using single starter of B. subtilis and Staphylococcus spp. were rated the least in terms of the colour. Although Owoh produced traditionally was considered the best in terms of the overall acceptability it was not significantly different from samples fermented singly with B. pumilus and with the combination of all the four bacteria.
4. Conclusion
The role played by fermentation on the use of cotton seeds in the production of Owoh offers a good processing method for utilisation of cotton seeds. Although, single starter cultures did not produce acceptable products in the controlled fermentation of cotton seeds, they played some roles in the product quality. However, the use of combination of B. subtilis, B. licheniformis, B. pumilus with and without Staphylococcus spp. produced Owoh of good quality in terms of both sensory and chemical characteristics. The Owoh produced from those combinations was not significantly different from the traditionally fermented one in terms of the sensory attributes.
There was significant reduction in gossypol level with the use of combined starters in comparison with spontaneous or traditional fermentation. It is of great importance that high priority be given to the reduction of free gossypol level in cotton seeds, since free gossypol has been a major impedance or hindrance to the optimum utilisation of cotton seeds, especially in human food as well as in animal feed. It is therefore recommended that a combination of B. subtilis, B. licheniformis, Bacillus pumillus with and without Staphylococcus spp. be used as starters for the fermentation of
cotton seed at 35 °C for 3 days during the production of Owoh condiment.
Acknowledgement
The authors wish to acknowledge the financial support of the University of Ibadan Senate Research Grant given to Ezekiel O.O. to carry out this research work. (Grant No. SRG/ FT/2010/5A).
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