World Journal of Microbiology and Biotechnology 8, 71-72
Short Communication
Extracellular enzyme activities during cassava fermentation for 'fufu' production O.B. Oyewole* and S.A. Odunfa Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24 h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period. Key words: Enzymes, cassava, fermentation, 'fufu'.
Cassava is an important food crop in Africa where it provides over 50% of the average daily calorific intake in some countries (de Bruijn & Fresco 1989). Cassava root is, however, processed before consumption in order to detoxify it and improve its palatability. Fermentation is an important method for processing cassava and both solid-state and submerged fermentations are employed. In the solid-state fermentation which is used in the production of 'gari', the roots are macerated before fermentation. However, in the submerged fermentation process, whole or large roots, peeled and unpeeted are soaked in water for the fermentation. A n important change that occurs in soaked roots is their retting. The softening of the roots during fermentation aids the diffusion of sugars and the access of fermenting microorganisms to them (Oyewole & Odunfa 1988). The involvement of some enzymes in this softening process has been noted (Okalie & Ugochukwu 1988), but their activities during the production of 'fufu' are yet to be fully investigated. The objective of this work was to determine the activities of amylase, cellulase and pectin methyl esterase during the submerged fermentation of cassava for 'fufu' production.
Materials and Methods Fermentation Matured cassava roots (12 months old) of the local white variety, purchased from a local farm in Abeokuta, Ogun State, Nigeria,
O.B. Oyewole is with the Department of Food Science and Technology, University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria; and S,A. Odunfa is with the Department of Botany and Microbiology, University of Ibadan, Nigeria, *Corresponding author. Fax: 234 39 234650.
were peeled and fermented in the traditional way as described previously (Oyewole & Odunfa 1989). Peeled roots (15 kg) were cut into cylindrical pieces (5 to 7 cm length) before being steeped in water (20 litres) for the fermentation period (96 h). The fermentation process was carried out five times for this study.
Enzyme Extraction Samples of the fermenting roots were collected aseptically at 0 h and at 12 h intervals for the duration of the fermentation for enzyme extractions and assays which were done in duplicate. For amylases and cellulase, the enzyme extracts were made by homogenizing 5 g of the fermenting roots with 25 ml of 0.02 M phosphate buffer (pH 6.5). The hornogenate was filtered through glass wool before being centrifuged at 30,000 x g for I0 min at 4~ The supernatant was used as the enzyme extract. For pectin methyl esterase, the fermenting roots (5 g) were homogenized with 25 ml of 0.25 M Tris-HC1, 0.3 M NaC1 (pH 8.0). The mixture was centrifuged as before. The clear supernatant, which contained the enzyme, was used for the assay.
Enzyme Assays Amylases were assayed as described by Bemfeld (1955). Reducing sugars produced during the reaction of the enzyme solution (1 ml) and the substrate (1% starch) were measured by the dinitrosalicylic acid method. The amylase enzyme activities were expressed as amylase units where one unit is the amount of enzyme in I ml of reactants which released I m g of reducing sugars from I% starch solution at 35~ and pH 6.5. Cellulase was assayed by measuring the reducing sugars elaborated by the dinitrosalicylic method (Bemfeld 1955). The reaction mixture contained I ml of enzyme extract and 9 ml of 0.54% w/v carboxymethylcellulose (CMC) in 0.055 M citrate buffer (pH 5.0) for 1 h at 35~ The cellulase activities were expressed as mg reducing sugars produced per ml of reactants in I h at 35~ The titrimetric method of Rouse (1955) was used in assaying the pectin methyl esterase activities. The reaction mixture contained I0 ml of 1% pectin in 0.1 M NaCI (pH 7.0) and 2 ml of enzyme extract. Pectin methyl esterase activities were expressed in pectin methyl esterase units where one unit was defined as the
(~ 1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Biolechnology, Vol 8, I992
71
O.B. Oyewole and S.A. Odunfa 0.50
030 , E 0,25 E-
T
0.40 r-
0.20 tn
'.~
0.30
0.15 >
5
5 0
0.20 0.10
_o 0.10 >.
0.05 C: ~
'
E 0,00
12
24
36
4.8
60
72
84
96 '0.00 n~
Time [h} Figure 1 Total amylase (0), c~-amylase (A) and pectin methyl esterase (rT) activities during cassava fermentation.
amount of enzyme which requires the addition of a milliequivalent at 0.1 M NaOH per unit time to maintain the reaction of pH 7.0 under the above assay conditions.
Results and Discussion Amylases and pectin methyl esterase were detected during the fermentation of cassava for 'fufu' production (Figure 1). The possible involvement of these carbohydrate degrading enzymes has been suggested previously (Okolie & Ugochukwu 1988). Amylase activities have been demonstrated in some Bacillus spp. isolated from fermenting cassava (Amund & Ogunsua 1987). The amylases appear, therefore, to be involved in the initial breakdown of cassava starch into simple sugars. The decline in the activities of the amylases from 12 to 24 h of fermentation may be due to the disappearance of the amylolytic microorganisms from the fermenting spectrum or due to other inhibitions. Cellulose activities were relatively lower than that of amylases during the fermentation (data not shown) and were about 0.05 to 0.09 cellulose units per ml for the period of fermentation. Pectin methyl esterase and cellulase production during cassava fermentation appear to play important roles in the softening of the cassava roots as is the case with other plant materials (Hultine & Levine 1965). In view of the contributions of these enzymes, their application in large scale fermentation of cassava could be useful in optimizing the process.
72
WorldJournalof Microbiologyand Biotechnologv,Vol 8, 1992
Acknowledgement This work was supported through a grant from the International Foundation for Science, Stockholm, Sweden.
References Around, O.O. & Ogunsua, O.A. 1987 Extracellular amylase production by cassava fermenting bacteria. Journal of Industrial Microbiology 2, 123-127. Bernfeld, P. 1955 Amylases, alpha and beta. In Methods in Enzymology, eds Colowick, S.P. & Kaplan, N.O., pp. 149-158. London: Academic Press. de Bruijn, G.H. & Fresco, L.O. 1989 The importance of cassava in world food production. Netherlands Journal of Agricultural Sciences 37, 21-34. Hultine, H.O. & Levine, A.S~ 1965 Pectin methyl esterase in the ripening of banana. Journal of Food Science 30, 917-921. Okolie, P.N. & Ugochukwu, E.N. 1988 Changes in activities of cell wall degrading enzymes during fermentation of cassava (Manihot esculenta Crantz) with Citrobacterfreudii. Journal of the Science of Food and Agriculture 44, 51-61. Oyewole, O.B. & Odunfa, S.A. 1988 Microbiological studies on cassava fermentation for "lafun" production. Food Microbiology 5, 125-133. Oyewole, O.B. & Odunfa, S.A. 1989 Effects of fermentation on the carbohydrate, mineral and protein contents of cassava during "fufu" production. Journal of Food Composition and Analysis 2, 170-170. Rouse, A.H. 1955 Pectinesterase and pectin commercial citrus juices as determined by the method used at the Citrus Experimentation Station. Bulletin, University of Florida, Agricultural Experimentation Station, Florida.
(Received in revised form I5 May I991; accepted 23 May I991)
Short Communication
Extracellular enzyme activities during cassava fermentation for 'fufu' production O.B. Oyewole* and S.A. Odunfa Amylase and pectin methyl esterase activities increased rapidly during the early period of the fermentation of cassava for 'fufu' production, attaining their peak activities after 12 and 24 h, respectively. Cellulase activity was lower and approximately constant for most of the fermentation period. Key words: Enzymes, cassava, fermentation, 'fufu'.
Cassava is an important food crop in Africa where it provides over 50% of the average daily calorific intake in some countries (de Bruijn & Fresco 1989). Cassava root is, however, processed before consumption in order to detoxify it and improve its palatability. Fermentation is an important method for processing cassava and both solid-state and submerged fermentations are employed. In the solid-state fermentation which is used in the production of 'gari', the roots are macerated before fermentation. However, in the submerged fermentation process, whole or large roots, peeled and unpeeted are soaked in water for the fermentation. A n important change that occurs in soaked roots is their retting. The softening of the roots during fermentation aids the diffusion of sugars and the access of fermenting microorganisms to them (Oyewole & Odunfa 1988). The involvement of some enzymes in this softening process has been noted (Okalie & Ugochukwu 1988), but their activities during the production of 'fufu' are yet to be fully investigated. The objective of this work was to determine the activities of amylase, cellulase and pectin methyl esterase during the submerged fermentation of cassava for 'fufu' production.
Materials and Methods Fermentation Matured cassava roots (12 months old) of the local white variety, purchased from a local farm in Abeokuta, Ogun State, Nigeria,
O.B. Oyewole is with the Department of Food Science and Technology, University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria; and S,A. Odunfa is with the Department of Botany and Microbiology, University of Ibadan, Nigeria, *Corresponding author. Fax: 234 39 234650.
were peeled and fermented in the traditional way as described previously (Oyewole & Odunfa 1989). Peeled roots (15 kg) were cut into cylindrical pieces (5 to 7 cm length) before being steeped in water (20 litres) for the fermentation period (96 h). The fermentation process was carried out five times for this study.
Enzyme Extraction Samples of the fermenting roots were collected aseptically at 0 h and at 12 h intervals for the duration of the fermentation for enzyme extractions and assays which were done in duplicate. For amylases and cellulase, the enzyme extracts were made by homogenizing 5 g of the fermenting roots with 25 ml of 0.02 M phosphate buffer (pH 6.5). The hornogenate was filtered through glass wool before being centrifuged at 30,000 x g for I0 min at 4~ The supernatant was used as the enzyme extract. For pectin methyl esterase, the fermenting roots (5 g) were homogenized with 25 ml of 0.25 M Tris-HC1, 0.3 M NaC1 (pH 8.0). The mixture was centrifuged as before. The clear supernatant, which contained the enzyme, was used for the assay.
Enzyme Assays Amylases were assayed as described by Bemfeld (1955). Reducing sugars produced during the reaction of the enzyme solution (1 ml) and the substrate (1% starch) were measured by the dinitrosalicylic acid method. The amylase enzyme activities were expressed as amylase units where one unit is the amount of enzyme in I ml of reactants which released I m g of reducing sugars from I% starch solution at 35~ and pH 6.5. Cellulase was assayed by measuring the reducing sugars elaborated by the dinitrosalicylic method (Bemfeld 1955). The reaction mixture contained I ml of enzyme extract and 9 ml of 0.54% w/v carboxymethylcellulose (CMC) in 0.055 M citrate buffer (pH 5.0) for 1 h at 35~ The cellulase activities were expressed as mg reducing sugars produced per ml of reactants in I h at 35~ The titrimetric method of Rouse (1955) was used in assaying the pectin methyl esterase activities. The reaction mixture contained I0 ml of 1% pectin in 0.1 M NaCI (pH 7.0) and 2 ml of enzyme extract. Pectin methyl esterase activities were expressed in pectin methyl esterase units where one unit was defined as the
(~ 1992 Rapid Communications of Oxford Ltd World Journal of Microbiology and Biolechnology, Vol 8, I992
71
O.B. Oyewole and S.A. Odunfa 0.50
030 , E 0,25 E-
T
0.40 r-
0.20 tn
'.~
0.30
0.15 >
5
5 0
0.20 0.10
_o 0.10 >.
0.05 C: ~
'
E 0,00
12
24
36
4.8
60
72
84
96 '0.00 n~
Time [h} Figure 1 Total amylase (0), c~-amylase (A) and pectin methyl esterase (rT) activities during cassava fermentation.
amount of enzyme which requires the addition of a milliequivalent at 0.1 M NaOH per unit time to maintain the reaction of pH 7.0 under the above assay conditions.
Results and Discussion Amylases and pectin methyl esterase were detected during the fermentation of cassava for 'fufu' production (Figure 1). The possible involvement of these carbohydrate degrading enzymes has been suggested previously (Okolie & Ugochukwu 1988). Amylase activities have been demonstrated in some Bacillus spp. isolated from fermenting cassava (Amund & Ogunsua 1987). The amylases appear, therefore, to be involved in the initial breakdown of cassava starch into simple sugars. The decline in the activities of the amylases from 12 to 24 h of fermentation may be due to the disappearance of the amylolytic microorganisms from the fermenting spectrum or due to other inhibitions. Cellulose activities were relatively lower than that of amylases during the fermentation (data not shown) and were about 0.05 to 0.09 cellulose units per ml for the period of fermentation. Pectin methyl esterase and cellulase production during cassava fermentation appear to play important roles in the softening of the cassava roots as is the case with other plant materials (Hultine & Levine 1965). In view of the contributions of these enzymes, their application in large scale fermentation of cassava could be useful in optimizing the process.
72
WorldJournalof Microbiologyand Biotechnologv,Vol 8, 1992
Acknowledgement This work was supported through a grant from the International Foundation for Science, Stockholm, Sweden.
References Around, O.O. & Ogunsua, O.A. 1987 Extracellular amylase production by cassava fermenting bacteria. Journal of Industrial Microbiology 2, 123-127. Bernfeld, P. 1955 Amylases, alpha and beta. In Methods in Enzymology, eds Colowick, S.P. & Kaplan, N.O., pp. 149-158. London: Academic Press. de Bruijn, G.H. & Fresco, L.O. 1989 The importance of cassava in world food production. Netherlands Journal of Agricultural Sciences 37, 21-34. Hultine, H.O. & Levine, A.S~ 1965 Pectin methyl esterase in the ripening of banana. Journal of Food Science 30, 917-921. Okolie, P.N. & Ugochukwu, E.N. 1988 Changes in activities of cell wall degrading enzymes during fermentation of cassava (Manihot esculenta Crantz) with Citrobacterfreudii. Journal of the Science of Food and Agriculture 44, 51-61. Oyewole, O.B. & Odunfa, S.A. 1988 Microbiological studies on cassava fermentation for "lafun" production. Food Microbiology 5, 125-133. Oyewole, O.B. & Odunfa, S.A. 1989 Effects of fermentation on the carbohydrate, mineral and protein contents of cassava during "fufu" production. Journal of Food Composition and Analysis 2, 170-170. Rouse, A.H. 1955 Pectinesterase and pectin commercial citrus juices as determined by the method used at the Citrus Experimentation Station. Bulletin, University of Florida, Agricultural Experimentation Station, Florida.
(Received in revised form I5 May I991; accepted 23 May I991)
