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Improvement of the traditional processing and fermentation of African oil bean (Pentaclethra macrophylla Bentham) into a food snack – ‘ugba’
International Journal of Food Microbiology 59 (2000) 235–239 www.elsevier.nl / locate / ijfoodmicro
Short communication
Improvement of the traditional processing and fermentation of African oil bean (Pentaclethra macrophylla Bentham) into a food snack – ‘ugba’ a, b ,1 N.R Isu *, C.O. Ofuya a
Microbiology and Biotechnology Unit, School of Biological Sciences, College of Sciences, Abia State University, P.M.B. 2000 Uturu, Abia State, Nigeria b Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria Received 20 May 1999; received in revised form 6 April 2000; accepted 20 April 2000
Abstract Inocula for the improvement of the traditional production of ‘ugba’ were developed by attaching pure cultures of Bacillus subtilis responsible for the natural fermentation of the African oil bean seeds on cowpea granules. Changes in pH, amino-nitrogen and protease activity were used as fermentation indicators. In comparison with the natural fermentation, changes in these process variables were more pronounced. Results also showed that the production time could be significantly reduced. In addition, the overall product quality of ‘ugba’ produced by the developed inocula was good and highly acceptable. The cultures were stable and viable for over 6 months on the granules of cowpea. 2000 Elsevier Science B.V. All rights reserved. Keywords: Fermentation; Bacillus; Biological carriers; ‘Ugba’
1. Introduction ‘Ugba’, a popular, fermented and protein-rich food product with appealing organoleptic qualities is produced by an agelong, primitive and house-hold process of solid-state fermentation of the seeds of African oil bean tree (Pentaclethra macrophylla Bentham). The steps involved in production of ‘Ugba’ are shown in Fig. 1. Usually boiling for 12 h *Corresponding author. 1 This article is entirely dedicated to the memory of Dr. C.O. Ofuya who passed on while supervising part of this project.
is used and not autoclaving as indicated in the figure. The process takes five to ten days involving rudimentary utensils and produces just enough ‘ugba’ for immediate family consumption and occasionally for retail marketing. It serves as a snack or sidedish if fermented for 5–6 days or as soup condiment after 7–10 days of fermentation. It has been reported that ‘ugba’ is not only rich in essential amino-acids but also in carbohydrates, lipids and vitamins (Mba et al., 1974; Achinewhu, 1983; Achinewhu and Ryley, 1986). The traditional fermentation process is believed to be carried out by natural microorganisms arising from utensils, hand-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00318-4
236
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
2. Materials and methods
2.1. Isolation and identification of Bacillus subtilis
Fig. 1. Processing of African oil bean seed (Pentaclethra macrophylla Bentham).
ling and packaging materials (Obeta, 1983; Njoku et al., 1990). As a result, the process is unpredictable and often leads to ‘ugba’ of varied qualities. Previous studies however, showed that although many microorganisms are associated with the fermentation, only the Bacillus species are necessary for ‘ugba’ production (Njoku et al., 1990; Isu, 1995). ‘Ugba’ has the potential of serving as a low-cost, palatable and nutritious food item for over 40 million people who eat it (Mba et al., 1974) It is believed that improved processing and fermentation methods must be sought, that will not compromise the product safety and quality. It has also become evident that major improvements in the fermentation process will depend on developing bacterial cultures with commercially desirable fermentation traits and establishing conditions for their predominance of the process. This is necessary for a meaningful scale up. The objective of this study, therefore, was to develop stable inocula with desirable fermentation traits for an improved and facilitated production of ‘ugba’.
This was carried out according to Isu and Njoku (1997) as follows: Five packages of locally produced ‘ugba’ purchased from local producers were transported in sterile polyethylene bags to the laboratory and were prepared for analysis on arrival. Samples (5 g) from each package were aseptically placed in sterile stomacher bags (Seward, London, UK) and 45 ml of sterile 0.1% Peptone water (Oxoid) containing 1% Tween 80 were added. Homogenization was carried out by stomaching for 90 s in a colworth stomacher (Seward). After ten fold dilutions, 0.1 ml of the 10 26 dilution was inoculated on each of the dry, sterile surfaces of three tryptone soy agar (Oxoid) plates by the spread plate technique. Plates were incubated at 308C for 18 h and were observed and counted. Representatives of the predominant and less frequently occurring colony types were purified by repeated subculturing, Gram-stained and checked for purity. Standard microbiological techniques (Collins and Lyne, 1984) were used for preliminary identification of isolates. Gram positive, straight or curved rod like separate cells with endospores isolated on nutrient agar (Oxoid) were chosen. Further tests for the identification of the Bacillus subtilis were carried out using the classification scheme based on Sharpe (1979) and Harrigan and McCance (1976). Stock cultures of the purified Bacillus isolates were maintained on nutrient agar (Oxoid) slopes and stored at 48C for further use. Cultures were resuscitated by plating a loopful from the slopes on tryptone soy agar (TSA Oxoid) and incubating at 308C for 18 h. Five strains of Bacillus subtilis were used for further studies.
2.2. Biological carriers The biological carriers used in the study were sterile granules of cowpea (Phaeseolus vulgaris) and that of maize (Zea mays) white variety. They were produced and sterilized according to Isu (1995) as follows: The seeds (100 g of each) were autoclaved at 1218C for 15 min; dried at 808C for 24 h and dry-milled using a warring blender (Moulinex, UK). Particles (500–700 mm) were obtained by sieving
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
with laboratory test sieve (Endocotts Ltd., UK). The different fractions were collected separately, dispensed into 1 g portions in bijoux bottles and sterilized at 808C for 8 h in a hot air oven (Gallenkamp, UK).
2.3. Immobilized inocula Five strains of Bacillus subtilis were employed. Suspensions of the cultures grown on TSA were made in 0.05 M potassium phosphate buffer (pH. 7.0) and standardized using a spectrophotometer (Gallenkamp SPT-260, UK). Using sterile Pasteur pipettes, 0.05 ml of each culture containing about 5 3 10 7 bacterial cells were aseptically added to 1.0 g of the sterile cowpea or maize granules in bijoux bottles. They were thoroughly mixed and aseptically dried at 298C618C using sterile air from a monopoint dryer (Bra un Ag., Type 4547, Germany). The dry attached cells were stored at 48C and used as immobilised inocula for ‘ugba’ production. The viability of the immobilised cells was assessed monthly for 6 months by resuspending 0.1 g in 9.9 ml sterile deionized water, mixing and determining colony forming units per gram (CFU / g) by spread plating onto TSA (Oxoid) and incubating at 308C for 48 h. Cowpea granules which showed the highest CFU were used for further studies.
2.4. Processing and fermentation of African oil bean seeds African oil bean seeds obtained from a local market in Umuahia, Abia State, Nigeria, were processed by a modified method of the traditional process summarized in Fig. 1. Different starter cultures, natural inocula freshly grown vegetative cells (on TSA) as well as immobilised cells were each used to inoculate sterile African oil bean slices in high density polyethene bags. Fifty grams of sterile oil bean slices were inoculated with ca. 10 6 cells or 0.1 g of the immobilised cells and allowed to ferment at 308C for 6 days. Fermentation was monitored using changes in pH, amino nitrogen and protease activity of the substrate as indicators. Sterile oil bean slices inoculated with 0.1 g of sterile cowpea granules served as control.
237
2.4.1. pH determination The pH of the fermenting substrate was determined by the method of Ngo et al. (1982) using a pH meter (Pye Unicam, UK). 2.4.2. Amino nitrogen determination The method of Pham and Del-Rosario (1983) was employed in determining amino nitrogen. 2.4.3. Protease activity determination The protease activities of the different starter cultures were determined by the modified casein digestion method described by Kunitz (1947). 2.4.4. Evaluation of product quality The sensory qualities of ‘ugba’ as snack were evaluated by a taste panel comprising persons familiar with the product, using a 9-point hedonic scale. The products were assessed for flavour, texture, colour and general acceptability, using characters previously described (Njoku et al., 1990). In the scoring, 9 5 excellent / best while 1 5 not good / worst.
3. Results and discussion The dominant microorganism was Bacillus subtilis, but Bacillus coagulans, Bacillus pumilus and Bacillus megaterium were also observed. As seen from Tables 1 and 2 immobilisation showed better survival of Bacillus subtilis on cowpea than on maize. The effect of the different starter cultures on the process variables of the fermentation of African oil bean seeds to ‘ugba’ showed that the attached immobilised cells fermented the substrate in 48 h as against 96 h by the natural inoculum and 72 h by the freshly grown cells in broth culture. Fermentation by the immobilised cells were characterized by a more pronounced increase in pH, from 5.9 to 8.0 in 48 h, accompanied by a corresponding increase in protease activity from 4.560.85 mg nitrogen per minute (mg N / min) to 27.6561.50 mg N / min (results not shown). This character has been reported to be important for an efficient production process for ‘ugba’ (Achinewhu and Ryley, 1986). During the same period, the change in pH of the substrate fermenting by the natural inoculum was from 5.9 to 6.5 while protease activity increased from 4.5060.80
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
238
Table 1 Viable counts of five strains of Bacillus subtilis attached to cowpea granules and stored at 308C a Storage period (months)
B
C
D
E
F
0 1 2 3 4 5 6
4.2 (0.2) 3 10 6 4.0 (0.4) 3 10 6 4.0 (0.0) 3 10 6 4.1 (0.1) 3 10 6 4.0 (0.5) 3 10 6 3.8 (0.8) 3 10 6 3.9 (0.1) 3 10 6
5.5 (0.5) 3 10 6 5.5 (0.8) 3 10 6 5.2 (0.0) 3 10 6 5.2 (0.2) 3 10 6 5.0 (0.5) 3 10 6 5.0 (0.8) 3 10 6 5.0 (0.2) 3 10 6
5.0 (0.1) 3 10 6 5.0 (0.5) 3 10 6 5.0 (0.8) 3 10 6 5.0 (0.5) 3 10 6 4.8 (0.1) 3 10 6 4.8 (0.5) 3 10 6 4.5 (0.1) 3 10 6
5.8 (0.1) 3 10 6 5.8 (0.5) 3 10 6 5.5 (0.5) 3 10 6 5.0 (0.1) 3 10 6 5.1 (0.5) 3 10 6 5.1 (0.1) 3 10 6 5.0 (0.0) 3 10 6
5.6(0.1) 3 10 6 5.8 (0.8) 3 10 6 5.5 (0.2) 3 10 6 5.0 (0.1) 3 10 6 5.0 (0.1) 3 10 6 5.0 (0.0) 3 10 6 5.0 (0.2) 3 10 6
a
Aerobic plate count (CFU / g)
Values are mean of triplicate determinations and standard deviation, B, C, D, E, F 5 different strains of Bacillus subtilis.
Table 2 Viable counts of five strains of Bacillus subtilis attached to maize granules and stored at 308C a Storage period (months)
B
C
D
E
F
0 1 2 3 4 5 6
4.2 (0.5) 3 10 6 2.0 (0.2) 3 10 6 5.0 (0.0) 3 10 5 1.0 (0.5) 3 10 5 9.2 (0.5) 3 10 4 2.2 (0.5) 3 10 4 6.4 (0.2) 3 10 3
5.5 (0.5) 3 10 6 2.5 (0.2) 3 10 6 4.5 (0.5) 3 10 5 1.5 (0.1) 3 10 5 2.5 (0.5) 3 10 4 9.0 (0.2) 3 10 3 1.0 (0.5) 3 10 3
5.0 (0.5) 3 10 6 1.0 (0.5) 3 10 6 6.5 (0.2) 3 10 5 2.5 (0.5) 3 10 5 9.0 (0.5) 3 10 4 4.8 (0.6) 3 10 3 1.9 (0.1) 3 10 3
5.8 (0.2) 3 10 6 1.0 (0.5) 3 10 6 2.5 (0.2) 3 10 5 6.5 (0.5) 3 10 4 1.0 (0.5) 3 10 4 8.5 (0.1) 3 10 3 1.8 (0.2) 3 10 3
5.8 (0.5) 3 10 6 8.9 (0.4) 3 10 5 6.0 (0.2) 3 10 4 1.2 (0.8) 3 10 4 9.5 (0.5) 3 10 3 2.2 (0.6) 3 10 3 1.0 (0.0) 3 10 3
a
Aerobic plate count (CFU / g)
Values are mean of triplicate determinations and standard deviation, B, C, D, E, F 5 different strains of Bacillus subtilis.
to 10.5061.00 mg N / min (results not shown). The pH of fermentation by the freshly grown Bacillus cells changed from 5.9 to 6.9 with an accompanying increase in protease activity of 4.5060.80 to 14.561.50 mg N / min (results not shown). This implies that the Bacillus cells attached to cowpea granules resulted in the development of stable inocula with desirable fermentation traits for ‘ugba’ production. The pronounced increase in pH from 5.9 to 8.0 in 48 h is also considered to be an advantage as such characters are known to inhibit undesirable microorganism (Stuttgart and Barlie, 1986). From Table 3 it is seen that the amino nitrogen content of ‘ugba’ produced with the attached cells was higher than the value for the product of natural inoculum. During traditional fermentation, the oil bean seeds are hydrated by boiling for at least 12 h each, at steps a and b (Fig. 1) and natural inoculum ferments the substrate at step c, for 96 h, which brings the total processing and fermentation period to about 120 h. In this improved method, seed hydration at a and b
is achieved by autoclaving at 1218C for 30 min each while the immobilised inocula would ferment the substrate in 48 h. Thus, it is possible to obtain good quality ‘ugba’ in less than 60 h. This result is consistent with the observation of Ofuya and Nnajiofor (1989) who worked on the starter, ‘Gastat’ for an improved garri production. The apparent accelerated fermentation associated with the immobilised inocula is probably due to increased cell density per unit reactor and enhanced cell wall permeability and metabolism (Kolot, 1981). In addition, the carrier, cowpea granules, is edible and has no deleterious effect on the product. Cultures were also maintained in an active physiological state for up to 6 months without subculturing which agrees with the report of Kolot (1981) on the strategy of use and selection of microbial carriers for this kind of work. Data on the sensory qualities of ‘ugba’ produced by the different starter cultures showed that products of the immobilised inocula were well accepted. The general acceptability score by the 9-point hedonic scale for the
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
239
Table 3 Effect of different starter cultures on the amino-nitrogen content of African oil bean seed during fermentation a Fermentation period (d)
0 1 2 3 4 5 6 a
Amino-nitrogen content (mg N / g DM) Control
Natural Inoculum
Bacillus subtilis freshly grown
Immunolitised inoculum of Bacillus subtilis in cowpea
0.6 (0.0) 1.4 (0.1) 2.6 (0.2) 3.8 (0.2) 2.4 (0.0) 2.2 (0.0) 2.0 (0.1)
0.5 (0.0) 2.0 (0.0) 9.2 (0.2) 11.0 (0.5) 15.5 (0.8) 9.0 (0.5) 9.1 (0.5)
0.5 4.8 9.5 15.6 15.8 13.2 13.5
0.5 10.5 18.5 17.8 17.2 16.5 16.0
(0.0) (0.1) (0.1) (0.2) (0.5) (0.5) (1.0)
(0.0) (0.5) (0.2) (0.5) (0.6) (0.5) (0.5)
Values are means of triplicate determinations and standard deviation.
products were 6.5061.0 for the products of natural inoculum, 6.861.5 for product of freshly grown Bacillus subtilis and 7.161.0 for immobilised inocula. The good quality of ‘ugba’ obtained with the immunolitised inoculum is very encouraging because of its applicability within the indigenous technology of the developing countries. It is also expected that the use of this type of starter culture will enhance process standardization and uniform product quality.
References Achinewhu, S.C., 1983. Protein quality of African oil bean seed (Pentaclethra macrophylla Bentham). J. Food Sci. 48, 1374– 1375. Achinewhu, S.C., Ryley, I., 1986. Effect of fermentation on the Thiamin, Riboflavin, and Niacin contents of melon seed (citrulus vulgaris) and African oil bean seed (Pentaclethra macrophylla Bentham). Food Chem. 20, 243–252. Collins, C.H., Lyne, P.M., 1984. In: Microbiological Methods, 4th Edition. Butterworth, London, pp. 113–210. Harrigan, W.F., McCance, M.E., 1976. In: Laboratory Methods in Food and Dairy Microbiology. Academic Press, London, pp. 284–297. Isu, N.R., Njoku, H.O., 1997. An evaluation of the microflora associated with fermented African oil bean (Pentaclethra macrophylla Bentham) seeds during ‘ugba’ production. Plant Foods Human Nutr. 51, 14–157.
Isu, N.R., 1995. Studies on the accelerated production and preservation of ‘ugba’ (Pentaclethra macrophylla Bentham). PhD Thesis, University of Port Harcourt, Nigeria. Kolot, B.F., 1981. Microbial Carriers – Strategy for selection. Process Biochemistry, August–September. Kunitz, M., 1947. Crystalline soyabean trypsin, inhibitor. J. Gen. Physiol. 30, 291–310. Mba, A.V., Njike, M.C., Oyenuga, V.A., 1974. Proximate chemical composition and amino acid content of Nigerian oil seeds. J. Sci. Food Agric. 25, 1547–1553. Ngo, T.T., Pham, A.P., Yam, C.F., Lenhoff, H.H., 1982. Interference in the determination of ammonia with the hypochlorite– alkaline–phenol method of Bethelot. J. Anal. Chem. 54, 26– 35. Njoku, H.O., Ogbulie, J.N., Nnubia, C.O., 1990. Microbiological study of the traditional processing of African oil bean seeds (Pentaclethra macrophylla) for ‘ugba’. Prod. J. Food Microbiol. 1, 1–8. Obeta, J.A., 1983. A note on the microorganisms associated with the fermentation of seeds of African oil bean tree (Pentaclethra macrophylla). J. Appl. Bacteriol. 54, 433–455. Ofuya, C.O., Nnajiofor, C., 1989. Development and evaluation of a starter culture for the industrial production of garri. J. Appl. Bacteriol. 66, 37–42. Pham, C.B., Del-Rosario, G.F., 1983. Preparation of protein hydrolysate from defatted coconut and soya bean meals. Effect of process variables on the amino-nitrogen released and flavour development. J. Food Technol. 18, 21–25. Sharpe, M.E., 1979. In: 2nd Edition. Identification Methods For Microbiologists, Vol. 14. Academic Press, London. Stuttgart, J.C., Barlie, 1986. Mycolgia memoir. In: Hessettine, C.W., Wary, H.L. (Eds.), Indigenous Fermented Food of Nonwestern Origin, pp. 212–217. J. Cramer, Berlin.
Short communication
Improvement of the traditional processing and fermentation of African oil bean (Pentaclethra macrophylla Bentham) into a food snack – ‘ugba’ a, b ,1 N.R Isu *, C.O. Ofuya a
Microbiology and Biotechnology Unit, School of Biological Sciences, College of Sciences, Abia State University, P.M.B. 2000 Uturu, Abia State, Nigeria b Department of Microbiology, University of Port Harcourt, Port Harcourt, Nigeria Received 20 May 1999; received in revised form 6 April 2000; accepted 20 April 2000
Abstract Inocula for the improvement of the traditional production of ‘ugba’ were developed by attaching pure cultures of Bacillus subtilis responsible for the natural fermentation of the African oil bean seeds on cowpea granules. Changes in pH, amino-nitrogen and protease activity were used as fermentation indicators. In comparison with the natural fermentation, changes in these process variables were more pronounced. Results also showed that the production time could be significantly reduced. In addition, the overall product quality of ‘ugba’ produced by the developed inocula was good and highly acceptable. The cultures were stable and viable for over 6 months on the granules of cowpea. 2000 Elsevier Science B.V. All rights reserved. Keywords: Fermentation; Bacillus; Biological carriers; ‘Ugba’
1. Introduction ‘Ugba’, a popular, fermented and protein-rich food product with appealing organoleptic qualities is produced by an agelong, primitive and house-hold process of solid-state fermentation of the seeds of African oil bean tree (Pentaclethra macrophylla Bentham). The steps involved in production of ‘Ugba’ are shown in Fig. 1. Usually boiling for 12 h *Corresponding author. 1 This article is entirely dedicated to the memory of Dr. C.O. Ofuya who passed on while supervising part of this project.
is used and not autoclaving as indicated in the figure. The process takes five to ten days involving rudimentary utensils and produces just enough ‘ugba’ for immediate family consumption and occasionally for retail marketing. It serves as a snack or sidedish if fermented for 5–6 days or as soup condiment after 7–10 days of fermentation. It has been reported that ‘ugba’ is not only rich in essential amino-acids but also in carbohydrates, lipids and vitamins (Mba et al., 1974; Achinewhu, 1983; Achinewhu and Ryley, 1986). The traditional fermentation process is believed to be carried out by natural microorganisms arising from utensils, hand-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00318-4
236
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
2. Materials and methods
2.1. Isolation and identification of Bacillus subtilis
Fig. 1. Processing of African oil bean seed (Pentaclethra macrophylla Bentham).
ling and packaging materials (Obeta, 1983; Njoku et al., 1990). As a result, the process is unpredictable and often leads to ‘ugba’ of varied qualities. Previous studies however, showed that although many microorganisms are associated with the fermentation, only the Bacillus species are necessary for ‘ugba’ production (Njoku et al., 1990; Isu, 1995). ‘Ugba’ has the potential of serving as a low-cost, palatable and nutritious food item for over 40 million people who eat it (Mba et al., 1974) It is believed that improved processing and fermentation methods must be sought, that will not compromise the product safety and quality. It has also become evident that major improvements in the fermentation process will depend on developing bacterial cultures with commercially desirable fermentation traits and establishing conditions for their predominance of the process. This is necessary for a meaningful scale up. The objective of this study, therefore, was to develop stable inocula with desirable fermentation traits for an improved and facilitated production of ‘ugba’.
This was carried out according to Isu and Njoku (1997) as follows: Five packages of locally produced ‘ugba’ purchased from local producers were transported in sterile polyethylene bags to the laboratory and were prepared for analysis on arrival. Samples (5 g) from each package were aseptically placed in sterile stomacher bags (Seward, London, UK) and 45 ml of sterile 0.1% Peptone water (Oxoid) containing 1% Tween 80 were added. Homogenization was carried out by stomaching for 90 s in a colworth stomacher (Seward). After ten fold dilutions, 0.1 ml of the 10 26 dilution was inoculated on each of the dry, sterile surfaces of three tryptone soy agar (Oxoid) plates by the spread plate technique. Plates were incubated at 308C for 18 h and were observed and counted. Representatives of the predominant and less frequently occurring colony types were purified by repeated subculturing, Gram-stained and checked for purity. Standard microbiological techniques (Collins and Lyne, 1984) were used for preliminary identification of isolates. Gram positive, straight or curved rod like separate cells with endospores isolated on nutrient agar (Oxoid) were chosen. Further tests for the identification of the Bacillus subtilis were carried out using the classification scheme based on Sharpe (1979) and Harrigan and McCance (1976). Stock cultures of the purified Bacillus isolates were maintained on nutrient agar (Oxoid) slopes and stored at 48C for further use. Cultures were resuscitated by plating a loopful from the slopes on tryptone soy agar (TSA Oxoid) and incubating at 308C for 18 h. Five strains of Bacillus subtilis were used for further studies.
2.2. Biological carriers The biological carriers used in the study were sterile granules of cowpea (Phaeseolus vulgaris) and that of maize (Zea mays) white variety. They were produced and sterilized according to Isu (1995) as follows: The seeds (100 g of each) were autoclaved at 1218C for 15 min; dried at 808C for 24 h and dry-milled using a warring blender (Moulinex, UK). Particles (500–700 mm) were obtained by sieving
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
with laboratory test sieve (Endocotts Ltd., UK). The different fractions were collected separately, dispensed into 1 g portions in bijoux bottles and sterilized at 808C for 8 h in a hot air oven (Gallenkamp, UK).
2.3. Immobilized inocula Five strains of Bacillus subtilis were employed. Suspensions of the cultures grown on TSA were made in 0.05 M potassium phosphate buffer (pH. 7.0) and standardized using a spectrophotometer (Gallenkamp SPT-260, UK). Using sterile Pasteur pipettes, 0.05 ml of each culture containing about 5 3 10 7 bacterial cells were aseptically added to 1.0 g of the sterile cowpea or maize granules in bijoux bottles. They were thoroughly mixed and aseptically dried at 298C618C using sterile air from a monopoint dryer (Bra un Ag., Type 4547, Germany). The dry attached cells were stored at 48C and used as immobilised inocula for ‘ugba’ production. The viability of the immobilised cells was assessed monthly for 6 months by resuspending 0.1 g in 9.9 ml sterile deionized water, mixing and determining colony forming units per gram (CFU / g) by spread plating onto TSA (Oxoid) and incubating at 308C for 48 h. Cowpea granules which showed the highest CFU were used for further studies.
2.4. Processing and fermentation of African oil bean seeds African oil bean seeds obtained from a local market in Umuahia, Abia State, Nigeria, were processed by a modified method of the traditional process summarized in Fig. 1. Different starter cultures, natural inocula freshly grown vegetative cells (on TSA) as well as immobilised cells were each used to inoculate sterile African oil bean slices in high density polyethene bags. Fifty grams of sterile oil bean slices were inoculated with ca. 10 6 cells or 0.1 g of the immobilised cells and allowed to ferment at 308C for 6 days. Fermentation was monitored using changes in pH, amino nitrogen and protease activity of the substrate as indicators. Sterile oil bean slices inoculated with 0.1 g of sterile cowpea granules served as control.
237
2.4.1. pH determination The pH of the fermenting substrate was determined by the method of Ngo et al. (1982) using a pH meter (Pye Unicam, UK). 2.4.2. Amino nitrogen determination The method of Pham and Del-Rosario (1983) was employed in determining amino nitrogen. 2.4.3. Protease activity determination The protease activities of the different starter cultures were determined by the modified casein digestion method described by Kunitz (1947). 2.4.4. Evaluation of product quality The sensory qualities of ‘ugba’ as snack were evaluated by a taste panel comprising persons familiar with the product, using a 9-point hedonic scale. The products were assessed for flavour, texture, colour and general acceptability, using characters previously described (Njoku et al., 1990). In the scoring, 9 5 excellent / best while 1 5 not good / worst.
3. Results and discussion The dominant microorganism was Bacillus subtilis, but Bacillus coagulans, Bacillus pumilus and Bacillus megaterium were also observed. As seen from Tables 1 and 2 immobilisation showed better survival of Bacillus subtilis on cowpea than on maize. The effect of the different starter cultures on the process variables of the fermentation of African oil bean seeds to ‘ugba’ showed that the attached immobilised cells fermented the substrate in 48 h as against 96 h by the natural inoculum and 72 h by the freshly grown cells in broth culture. Fermentation by the immobilised cells were characterized by a more pronounced increase in pH, from 5.9 to 8.0 in 48 h, accompanied by a corresponding increase in protease activity from 4.560.85 mg nitrogen per minute (mg N / min) to 27.6561.50 mg N / min (results not shown). This character has been reported to be important for an efficient production process for ‘ugba’ (Achinewhu and Ryley, 1986). During the same period, the change in pH of the substrate fermenting by the natural inoculum was from 5.9 to 6.5 while protease activity increased from 4.5060.80
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
238
Table 1 Viable counts of five strains of Bacillus subtilis attached to cowpea granules and stored at 308C a Storage period (months)
B
C
D
E
F
0 1 2 3 4 5 6
4.2 (0.2) 3 10 6 4.0 (0.4) 3 10 6 4.0 (0.0) 3 10 6 4.1 (0.1) 3 10 6 4.0 (0.5) 3 10 6 3.8 (0.8) 3 10 6 3.9 (0.1) 3 10 6
5.5 (0.5) 3 10 6 5.5 (0.8) 3 10 6 5.2 (0.0) 3 10 6 5.2 (0.2) 3 10 6 5.0 (0.5) 3 10 6 5.0 (0.8) 3 10 6 5.0 (0.2) 3 10 6
5.0 (0.1) 3 10 6 5.0 (0.5) 3 10 6 5.0 (0.8) 3 10 6 5.0 (0.5) 3 10 6 4.8 (0.1) 3 10 6 4.8 (0.5) 3 10 6 4.5 (0.1) 3 10 6
5.8 (0.1) 3 10 6 5.8 (0.5) 3 10 6 5.5 (0.5) 3 10 6 5.0 (0.1) 3 10 6 5.1 (0.5) 3 10 6 5.1 (0.1) 3 10 6 5.0 (0.0) 3 10 6
5.6(0.1) 3 10 6 5.8 (0.8) 3 10 6 5.5 (0.2) 3 10 6 5.0 (0.1) 3 10 6 5.0 (0.1) 3 10 6 5.0 (0.0) 3 10 6 5.0 (0.2) 3 10 6
a
Aerobic plate count (CFU / g)
Values are mean of triplicate determinations and standard deviation, B, C, D, E, F 5 different strains of Bacillus subtilis.
Table 2 Viable counts of five strains of Bacillus subtilis attached to maize granules and stored at 308C a Storage period (months)
B
C
D
E
F
0 1 2 3 4 5 6
4.2 (0.5) 3 10 6 2.0 (0.2) 3 10 6 5.0 (0.0) 3 10 5 1.0 (0.5) 3 10 5 9.2 (0.5) 3 10 4 2.2 (0.5) 3 10 4 6.4 (0.2) 3 10 3
5.5 (0.5) 3 10 6 2.5 (0.2) 3 10 6 4.5 (0.5) 3 10 5 1.5 (0.1) 3 10 5 2.5 (0.5) 3 10 4 9.0 (0.2) 3 10 3 1.0 (0.5) 3 10 3
5.0 (0.5) 3 10 6 1.0 (0.5) 3 10 6 6.5 (0.2) 3 10 5 2.5 (0.5) 3 10 5 9.0 (0.5) 3 10 4 4.8 (0.6) 3 10 3 1.9 (0.1) 3 10 3
5.8 (0.2) 3 10 6 1.0 (0.5) 3 10 6 2.5 (0.2) 3 10 5 6.5 (0.5) 3 10 4 1.0 (0.5) 3 10 4 8.5 (0.1) 3 10 3 1.8 (0.2) 3 10 3
5.8 (0.5) 3 10 6 8.9 (0.4) 3 10 5 6.0 (0.2) 3 10 4 1.2 (0.8) 3 10 4 9.5 (0.5) 3 10 3 2.2 (0.6) 3 10 3 1.0 (0.0) 3 10 3
a
Aerobic plate count (CFU / g)
Values are mean of triplicate determinations and standard deviation, B, C, D, E, F 5 different strains of Bacillus subtilis.
to 10.5061.00 mg N / min (results not shown). The pH of fermentation by the freshly grown Bacillus cells changed from 5.9 to 6.9 with an accompanying increase in protease activity of 4.5060.80 to 14.561.50 mg N / min (results not shown). This implies that the Bacillus cells attached to cowpea granules resulted in the development of stable inocula with desirable fermentation traits for ‘ugba’ production. The pronounced increase in pH from 5.9 to 8.0 in 48 h is also considered to be an advantage as such characters are known to inhibit undesirable microorganism (Stuttgart and Barlie, 1986). From Table 3 it is seen that the amino nitrogen content of ‘ugba’ produced with the attached cells was higher than the value for the product of natural inoculum. During traditional fermentation, the oil bean seeds are hydrated by boiling for at least 12 h each, at steps a and b (Fig. 1) and natural inoculum ferments the substrate at step c, for 96 h, which brings the total processing and fermentation period to about 120 h. In this improved method, seed hydration at a and b
is achieved by autoclaving at 1218C for 30 min each while the immobilised inocula would ferment the substrate in 48 h. Thus, it is possible to obtain good quality ‘ugba’ in less than 60 h. This result is consistent with the observation of Ofuya and Nnajiofor (1989) who worked on the starter, ‘Gastat’ for an improved garri production. The apparent accelerated fermentation associated with the immobilised inocula is probably due to increased cell density per unit reactor and enhanced cell wall permeability and metabolism (Kolot, 1981). In addition, the carrier, cowpea granules, is edible and has no deleterious effect on the product. Cultures were also maintained in an active physiological state for up to 6 months without subculturing which agrees with the report of Kolot (1981) on the strategy of use and selection of microbial carriers for this kind of work. Data on the sensory qualities of ‘ugba’ produced by the different starter cultures showed that products of the immobilised inocula were well accepted. The general acceptability score by the 9-point hedonic scale for the
N.R Isu, C.O. Ofuya / International Journal of Food Microbiology 59 (2000) 235 – 239
239
Table 3 Effect of different starter cultures on the amino-nitrogen content of African oil bean seed during fermentation a Fermentation period (d)
0 1 2 3 4 5 6 a
Amino-nitrogen content (mg N / g DM) Control
Natural Inoculum
Bacillus subtilis freshly grown
Immunolitised inoculum of Bacillus subtilis in cowpea
0.6 (0.0) 1.4 (0.1) 2.6 (0.2) 3.8 (0.2) 2.4 (0.0) 2.2 (0.0) 2.0 (0.1)
0.5 (0.0) 2.0 (0.0) 9.2 (0.2) 11.0 (0.5) 15.5 (0.8) 9.0 (0.5) 9.1 (0.5)
0.5 4.8 9.5 15.6 15.8 13.2 13.5
0.5 10.5 18.5 17.8 17.2 16.5 16.0
(0.0) (0.1) (0.1) (0.2) (0.5) (0.5) (1.0)
(0.0) (0.5) (0.2) (0.5) (0.6) (0.5) (0.5)
Values are means of triplicate determinations and standard deviation.
products were 6.5061.0 for the products of natural inoculum, 6.861.5 for product of freshly grown Bacillus subtilis and 7.161.0 for immobilised inocula. The good quality of ‘ugba’ obtained with the immunolitised inoculum is very encouraging because of its applicability within the indigenous technology of the developing countries. It is also expected that the use of this type of starter culture will enhance process standardization and uniform product quality.
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