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Tempeh-like product by groundnut fermentation
Food Microbiology, 1987,4, 121-l 25
Tempeh-like product by groundnut fermentation T. N. Bhavanishankar*, T. Rajashekaran and V. Sreanivasa Murthy Department of Microbiology and Sanitation, Central Food Technological
Research Institute,
Mysore 570 013, India
Received 4 November 1986 In order to increase the nutritive value of partially &fatted groundnut for human consumption, fermentation was carried out using two strains ofRhizopus oligosporus (NRRL 2710 and NRRL 2549). Fermentation increased the soluble solids, amino nitrogen and soluble nitrogen while no change was observed in the crude fibre content. The amino nitrogen, soluble solids, free lysine and methionine contents were highest in the samples fermented with the strain NRRL 2549. Consequently, protein efficiency ratio of the product increased significantly.
Introduction India produces about five million tonnes of groundnut of which around three
million
tonnes are used for oil extraction.
This leaves behind a protein-rich meal. With proper processing, this can be converted to a good protein source for
human consumption. Peanut protein as such is nutritionally inadequate because of the deficiency of essential amino acids such as methionine and lysine. Fermentation is a well known process to increase the nutritive value. Van Veen and Graham (1968) have noticed a slight increase in the digestibility and thiamine content and a considerable increase in the riboflavin content of peanut press cake after fermentation of Neurospora sitophila. Girija Bai et al. (1975) have observed a higher protein efficiency ratio (PER) for groundnut-soybean tempeh fermented by Rhizopus oligosporus than the soybean tempeh fermented by the same organism,
while
Bhavanishankar
et al. (1978) have reported a considerably better release of free amino acids like methionine
and lysine,
a higher
non-
protein nitrogen and free amino nitrogen in the fermented groundnut-soybean mixture
than in the fermented
0740-0020/87/020121
+ 05 $03.0010
soybean.
In the present been made to value of defatted consumption by peh like product oligosporus.
study, an attempt has increase the nutritive groundnut for human fermenting it to a temusing two strains of R.
Materials and Methods Good quality groundnut seeds were procured from the local market and the spoiled seeds, if any, were removed by band picking. Afterwards, the seeds were partially defatted using a hydraulic press and then deskinned.
Organisms The organisms used for the fermentation were Rhizopw oligosporus NRRL 2710 and NRRL 2549 obtained from Northern Regional Research Laboratory, Peoria, Illinois, USA.
Fermentation Partially defatted, deskinned groundnut seeds were soaked in tap water (1: 3) for 1 h. Water was drained and the seeds autoclaved at 15 lb pressure for 20 min. They were cooled to room temperature and inoculated with a spore suspension of a 7-day-old R . oligosporus culture grown on potato-dextrose-agar medium in a Roux bottle. Thirty ml of spore suspension from one Roux bottle was used to inoculate a 500 g sample. The inoculated samples were poured into sterile perforated stainless steel trays and covered with perfor0
1987
Academic
Press
Limited
122
T. N. Bhavanishankar
et al.
ated lids. The samples were allowed to ferment in a humidifier at 31°C for 18 to 22 h at 50% humidity. After fermentation, there was a firm cake like structure due to profuse growth of the fungus on groundnut. The sample was then cut into small cubes (about 1 ems), dipped in 10% saline for about 10 min and moisture was reduced to about 5% by drying in a cross flow drier at 80°C.
Chemical analysis
powder, 100 ml distilled water was added and the mixture shaken on a shaker for 30 min. The sample was then filtered through Whatman No. 1 filter paper and an aliquot of the filtrate was taken for the estimation of nitrogen by micro-kjeldahl method. For protein nitrogen, 10 g powdered sample was treated with 100 ml of 7% trichloroacetic acid; the resulting precipitate was analysed by micro-kjeldahl method. Free lysine and methionine were assayed in the water extracts of the samples by microbiological method (Barton-Wright 1952).
Estimation of moisture, ash, crude fibre, total nitrogen, total fat, total sugars and soluble solids were carried out according to the procedure given in Official Methods of Analy- Protein efficiency ratio (PER) sis (1970). Amino nitrogen was estimated by PER of unfermented and fermented samples the method of Pope and Stevens (1939). For was determined according to Chapman et al. the estimation of soluble nitrogen, a 10 g (1959). Composition of the diet used for PER sample was taken and ground to a fine studies is given in Table 1. Table 1. Composition
of the diets used for PER studies. Diets
Components (g kg-l)
UFG
Unfermented groundnut (UFG) Fermented groundnut-2710* (FG 2710) Fermented groundnuL2549a (FG 2549) Skim milk powder (SMP) Refined groundnut oil Salt mixtureb Vitamin&d starchc Vitaminized oilc Sago starch
FG2710
FG2549
SMP
240.61 -
240.61
-
248.50
25.78 20.00
25.76 20.00 10.00 10.00 693.63
lO*OO
10.00 693.61
23.61 20.00 10.00 10.00 687.89
280.00 90.00 20.00 10.00 10.00 590.00
a !l’he number denotes NRBL strain number of R. oligosporus used for fermentation. b Hubbel et al. (1937). c Chapman et al. (1959).
Table 2. Composition
of unfermented
Components (%I Moisture Total nitrogen Fat Protein nitrogen
and fermented
(22 h) groundnut
UFG
FG2710
FG2549
5.46 6.65 26.69 6.37
4.34 6.65 26,70 6.30
4.75 6.60 26.70 5.95
UFG, Unfermented groundnut; FG-2710, Groundnut fermented by R. oligosporus FG-2549, Groundnut fermented by R. oligosporus Values are the average of triplicate analysis.
NRRL NRRL
2710; 2549;
samples.
Groundnut
tempeh
123
after (Table 3). The soluble nitrogen, amino nitrogen and soluble solids con-
There was not much difference in total nitrogen, fat and moisture after fermentation. However protein nitrogen was slightly less in the groundnut sample fermented by R. oligosporus NRRL 2710 and considerably less in the sample fermented by the strain NRRL 2549 (Table 2). Total sugar content of fermented samples was found to be slightly less than the unfermented sample after 18 h of fermentation but somewhat higher thereTable 3. Biochemical R. oligosporus
changes at different
tents showed progressive increase as the fermentation advanced. There was no change in the crude fibre content after 22 h of fermentation.
PER of the sample fermented by R. oligosporus NRRL 2710 was found to be slightly
more than that of the unfermen-
ted sample. When the fermenting strain was NRRL 2549, PER was significantly higher than the unfermented sample and the sample fermented by the strain
NRRL 2710 (Table 4). intervals
of groundnut
FG2710 Components (o/o) Total sugar Soluble nitrogen Amino nitrogen Soluble solids Crude fibre Free lysine (mg 100 g-1) Free methionine (mg 100 g-l) Free amino nitrogen (mg 100 g-1)
FG2549
18h
20h
22h
18h
20h
22h
4.64 0.35 0.084 16.00 2.20 4.38
4.47 097 0.336 20.80 2.22 -
5.00 1.04 0.378 23.00 2.26
499 1.47 0.320 27.80 2.40 15.00
4.24 O-98 0.224 21.80 2.10
4.74 1.08 0.336 22.40 2.24 -
4.81 1.48 0.480 31.00 2.30 40.00
-
8.28
1.71
563
0.014
0.049
Table 4. PER of unfermented
SMP UFG FG2710 FG2549 SEM (27 d.f.)
by
UFG
Values are the average of triplicate analyses; UFG, Unfermented groundnut; FG-2710, Groundnut fermented by R. oligosporus FG-2549, Groundnut fermented by R. oligosporus
Diet
fermentation
0.07
NRRL 2710; NRRL 2549.
and fermented
groundnut
samples
Average initial weight (g‘,
Protein consumed (g)
Gain in weight(g)
39.50 39.50 39.50 39.50
24.52 17.78 18.35 18.61 kO.49
72.50 23.10 2490 32.60 52.06
PERa 296 x 1.30 y 1.36~ 1.75 z kO.08
a Means followed by different letters differ significantly (P < 0.05) according to Duncan’s new multiple range test. SMR, Skim milk powder; UFG, Unfermented groundnut; FG2710, Groundnut fermented by R. oligosporus NRFiL 2710; FG-2549, Groundnut fermented by R. oligosporus NRRL 2549.
124
T. N. Bhavanishankar et al.
Discussion
amino acids (lysine and methionine) by the strain NRRL 2549 during fermentaAs it is expected, protein content of tion cannot also be ruled out. .partially defatted, unfermented groundTotal sugar content after 18 h of nut is quite high (about 40%). Protein fermentation was found to decrease nitrogen content of the fermented sam- slightly as compared to the unfermented ples suggests that there is proteolysis sample. This may be due to utilization of during fermentation and that R. oligos- the sugars by the growing organisms as a porus NRRL 2549 has got higher source of carbon. After 20 and 22 h of proteolytic activity than the strain fermentation, the sugar content was NRRL 2710 when the fermenting sub- found to increase which may be due to strate is groundnut (Table 2). This obser- degradation of complex carbohydrates by vation supports our earlier results (Bha- the organisms (Table 3). Soluble solids vanishankar et al. 1978) where it was are almost doubled in the fermented reported that a higher PER of the fer- sample where the fermenting strain was mented groundnut + soybean mixture, R. oligosporus NRRL 2549. as compared to the fermented soybean or The significantly higher PER obtained the unfermented control, was due to for groundnut fermented by the strain better attack on groundnut protein by R. NRRL 2549 may be due to substantial oligosporus NRRL 2549 resulting in increase in the free amino acids (lysine increased free lysine and methionine. and methionine) and soluble nitrogen Higher proteolytic activity of the strain which are readily available for easier NRRL 2549 is further evidenced by assimilation. Wang and Hesseltine higher soluble nitrogen and amino nit- (1966) have reported that R. oligosporus rogen contents of the sample fermented NRRL 2710 gives most satisfactory temby the strain NRRL 2549 as against the peh product when soybean or wheat was sample fermented by the strain NRRL used as the substrate. However, accord2710 (Table 3). Amino nitrogen content ing to our findings, R. oligosporus NRRL and free lysine and methionine were also 2549 is a better strain than NRRL 2710 highest in the sample fermented by R. when groundnut is used as the substrate. bligosporus NRRL 2549. Murata et al. Thus, fermentation with the selected (1967) have observed similar increase in strain of R. oligosporus can help to free amino acids after fermentation of improve the nutritive value of groundsoybean. Possibility of synthesis of the nut meal.
References Barton-Wright, E. C. (1952) The microbiological assay of vitamin B complex and amino acids. London, Sir Isaac Pitman & Sons Ltd, pp. 121-126. Bhavanishankar, T. N., Shantha, N. V., Rajashekaran, T., Sreedharan, V. P. and Sreenivasa Mm-thy, V. (1978) Studies on tempeh made from groundnut and soybean mixture. Proceedings of the First Indian Convention of Food Scientists and Technologists, Mysore, India, p. 95. Chapman, D. G., Castillo, R. and Campbell, J. A. (1959) Evaluation of protein in foods. 1. A method for the determination of protein efficiency ratio. Can. J. Biochem. Physiol. 37, 679-686. Girija Bai, R., Prabha, T. N., Ramachandra Rao, T. N., Sreedharan, V. P. and Sreedhara, N. (1975) Studies on tempeh: Part I. Processing and nutritional evaluation of tempeh from a mixture of soybean and groundnut. J. Food Sci. Technol. 12,135-138. Hubbel, R. B., Mendel, L. B. and Wakeman, A. J. (1937) A new salt mixture for use in experimental diets. J. Nutr. 14, 273-285.
Groundnut tempeh 125 Murata, K., Ikehata, H. and Miyamoto, T. (1967) Studies on the nutritional value of Tempeh. J. Food Sci. 32,58&586. Official Methods of Analysis, Association of Official Analytical Chemists, 11th Ed., (1970). Washington DC, Benjamin Franklin Station, 20044. Pope, C. G. and Stevens, M. F. (1939) The determination of amino nitrogen using a copper method. Biochem. J. 33,1070-1077. Van Veen, A. G., Graham, D. C. W. and Steinkraus, K. H. (1968) Fermented peanut press cake. Cereal Sci. Today. 13,96-99. Wang, H. L. and Hesseltine, C. W. (1966) Wheat tempeh. Cereal Chem. 43,563-570.
Tempeh-like product by groundnut fermentation T. N. Bhavanishankar*, T. Rajashekaran and V. Sreanivasa Murthy Department of Microbiology and Sanitation, Central Food Technological
Research Institute,
Mysore 570 013, India
Received 4 November 1986 In order to increase the nutritive value of partially &fatted groundnut for human consumption, fermentation was carried out using two strains ofRhizopus oligosporus (NRRL 2710 and NRRL 2549). Fermentation increased the soluble solids, amino nitrogen and soluble nitrogen while no change was observed in the crude fibre content. The amino nitrogen, soluble solids, free lysine and methionine contents were highest in the samples fermented with the strain NRRL 2549. Consequently, protein efficiency ratio of the product increased significantly.
Introduction India produces about five million tonnes of groundnut of which around three
million
tonnes are used for oil extraction.
This leaves behind a protein-rich meal. With proper processing, this can be converted to a good protein source for
human consumption. Peanut protein as such is nutritionally inadequate because of the deficiency of essential amino acids such as methionine and lysine. Fermentation is a well known process to increase the nutritive value. Van Veen and Graham (1968) have noticed a slight increase in the digestibility and thiamine content and a considerable increase in the riboflavin content of peanut press cake after fermentation of Neurospora sitophila. Girija Bai et al. (1975) have observed a higher protein efficiency ratio (PER) for groundnut-soybean tempeh fermented by Rhizopus oligosporus than the soybean tempeh fermented by the same organism,
while
Bhavanishankar
et al. (1978) have reported a considerably better release of free amino acids like methionine
and lysine,
a higher
non-
protein nitrogen and free amino nitrogen in the fermented groundnut-soybean mixture
than in the fermented
0740-0020/87/020121
+ 05 $03.0010
soybean.
In the present been made to value of defatted consumption by peh like product oligosporus.
study, an attempt has increase the nutritive groundnut for human fermenting it to a temusing two strains of R.
Materials and Methods Good quality groundnut seeds were procured from the local market and the spoiled seeds, if any, were removed by band picking. Afterwards, the seeds were partially defatted using a hydraulic press and then deskinned.
Organisms The organisms used for the fermentation were Rhizopw oligosporus NRRL 2710 and NRRL 2549 obtained from Northern Regional Research Laboratory, Peoria, Illinois, USA.
Fermentation Partially defatted, deskinned groundnut seeds were soaked in tap water (1: 3) for 1 h. Water was drained and the seeds autoclaved at 15 lb pressure for 20 min. They were cooled to room temperature and inoculated with a spore suspension of a 7-day-old R . oligosporus culture grown on potato-dextrose-agar medium in a Roux bottle. Thirty ml of spore suspension from one Roux bottle was used to inoculate a 500 g sample. The inoculated samples were poured into sterile perforated stainless steel trays and covered with perfor0
1987
Academic
Press
Limited
122
T. N. Bhavanishankar
et al.
ated lids. The samples were allowed to ferment in a humidifier at 31°C for 18 to 22 h at 50% humidity. After fermentation, there was a firm cake like structure due to profuse growth of the fungus on groundnut. The sample was then cut into small cubes (about 1 ems), dipped in 10% saline for about 10 min and moisture was reduced to about 5% by drying in a cross flow drier at 80°C.
Chemical analysis
powder, 100 ml distilled water was added and the mixture shaken on a shaker for 30 min. The sample was then filtered through Whatman No. 1 filter paper and an aliquot of the filtrate was taken for the estimation of nitrogen by micro-kjeldahl method. For protein nitrogen, 10 g powdered sample was treated with 100 ml of 7% trichloroacetic acid; the resulting precipitate was analysed by micro-kjeldahl method. Free lysine and methionine were assayed in the water extracts of the samples by microbiological method (Barton-Wright 1952).
Estimation of moisture, ash, crude fibre, total nitrogen, total fat, total sugars and soluble solids were carried out according to the procedure given in Official Methods of Analy- Protein efficiency ratio (PER) sis (1970). Amino nitrogen was estimated by PER of unfermented and fermented samples the method of Pope and Stevens (1939). For was determined according to Chapman et al. the estimation of soluble nitrogen, a 10 g (1959). Composition of the diet used for PER sample was taken and ground to a fine studies is given in Table 1. Table 1. Composition
of the diets used for PER studies. Diets
Components (g kg-l)
UFG
Unfermented groundnut (UFG) Fermented groundnut-2710* (FG 2710) Fermented groundnuL2549a (FG 2549) Skim milk powder (SMP) Refined groundnut oil Salt mixtureb Vitamin&d starchc Vitaminized oilc Sago starch
FG2710
FG2549
SMP
240.61 -
240.61
-
248.50
25.78 20.00
25.76 20.00 10.00 10.00 693.63
lO*OO
10.00 693.61
23.61 20.00 10.00 10.00 687.89
280.00 90.00 20.00 10.00 10.00 590.00
a !l’he number denotes NRBL strain number of R. oligosporus used for fermentation. b Hubbel et al. (1937). c Chapman et al. (1959).
Table 2. Composition
of unfermented
Components (%I Moisture Total nitrogen Fat Protein nitrogen
and fermented
(22 h) groundnut
UFG
FG2710
FG2549
5.46 6.65 26.69 6.37
4.34 6.65 26,70 6.30
4.75 6.60 26.70 5.95
UFG, Unfermented groundnut; FG-2710, Groundnut fermented by R. oligosporus FG-2549, Groundnut fermented by R. oligosporus Values are the average of triplicate analysis.
NRRL NRRL
2710; 2549;
samples.
Groundnut
tempeh
123
after (Table 3). The soluble nitrogen, amino nitrogen and soluble solids con-
There was not much difference in total nitrogen, fat and moisture after fermentation. However protein nitrogen was slightly less in the groundnut sample fermented by R. oligosporus NRRL 2710 and considerably less in the sample fermented by the strain NRRL 2549 (Table 2). Total sugar content of fermented samples was found to be slightly less than the unfermented sample after 18 h of fermentation but somewhat higher thereTable 3. Biochemical R. oligosporus
changes at different
tents showed progressive increase as the fermentation advanced. There was no change in the crude fibre content after 22 h of fermentation.
PER of the sample fermented by R. oligosporus NRRL 2710 was found to be slightly
more than that of the unfermen-
ted sample. When the fermenting strain was NRRL 2549, PER was significantly higher than the unfermented sample and the sample fermented by the strain
NRRL 2710 (Table 4). intervals
of groundnut
FG2710 Components (o/o) Total sugar Soluble nitrogen Amino nitrogen Soluble solids Crude fibre Free lysine (mg 100 g-1) Free methionine (mg 100 g-l) Free amino nitrogen (mg 100 g-1)
FG2549
18h
20h
22h
18h
20h
22h
4.64 0.35 0.084 16.00 2.20 4.38
4.47 097 0.336 20.80 2.22 -
5.00 1.04 0.378 23.00 2.26
499 1.47 0.320 27.80 2.40 15.00
4.24 O-98 0.224 21.80 2.10
4.74 1.08 0.336 22.40 2.24 -
4.81 1.48 0.480 31.00 2.30 40.00
-
8.28
1.71
563
0.014
0.049
Table 4. PER of unfermented
SMP UFG FG2710 FG2549 SEM (27 d.f.)
by
UFG
Values are the average of triplicate analyses; UFG, Unfermented groundnut; FG-2710, Groundnut fermented by R. oligosporus FG-2549, Groundnut fermented by R. oligosporus
Diet
fermentation
0.07
NRRL 2710; NRRL 2549.
and fermented
groundnut
samples
Average initial weight (g‘,
Protein consumed (g)
Gain in weight(g)
39.50 39.50 39.50 39.50
24.52 17.78 18.35 18.61 kO.49
72.50 23.10 2490 32.60 52.06
PERa 296 x 1.30 y 1.36~ 1.75 z kO.08
a Means followed by different letters differ significantly (P < 0.05) according to Duncan’s new multiple range test. SMR, Skim milk powder; UFG, Unfermented groundnut; FG2710, Groundnut fermented by R. oligosporus NRFiL 2710; FG-2549, Groundnut fermented by R. oligosporus NRRL 2549.
124
T. N. Bhavanishankar et al.
Discussion
amino acids (lysine and methionine) by the strain NRRL 2549 during fermentaAs it is expected, protein content of tion cannot also be ruled out. .partially defatted, unfermented groundTotal sugar content after 18 h of nut is quite high (about 40%). Protein fermentation was found to decrease nitrogen content of the fermented sam- slightly as compared to the unfermented ples suggests that there is proteolysis sample. This may be due to utilization of during fermentation and that R. oligos- the sugars by the growing organisms as a porus NRRL 2549 has got higher source of carbon. After 20 and 22 h of proteolytic activity than the strain fermentation, the sugar content was NRRL 2710 when the fermenting sub- found to increase which may be due to strate is groundnut (Table 2). This obser- degradation of complex carbohydrates by vation supports our earlier results (Bha- the organisms (Table 3). Soluble solids vanishankar et al. 1978) where it was are almost doubled in the fermented reported that a higher PER of the fer- sample where the fermenting strain was mented groundnut + soybean mixture, R. oligosporus NRRL 2549. as compared to the fermented soybean or The significantly higher PER obtained the unfermented control, was due to for groundnut fermented by the strain better attack on groundnut protein by R. NRRL 2549 may be due to substantial oligosporus NRRL 2549 resulting in increase in the free amino acids (lysine increased free lysine and methionine. and methionine) and soluble nitrogen Higher proteolytic activity of the strain which are readily available for easier NRRL 2549 is further evidenced by assimilation. Wang and Hesseltine higher soluble nitrogen and amino nit- (1966) have reported that R. oligosporus rogen contents of the sample fermented NRRL 2710 gives most satisfactory temby the strain NRRL 2549 as against the peh product when soybean or wheat was sample fermented by the strain NRRL used as the substrate. However, accord2710 (Table 3). Amino nitrogen content ing to our findings, R. oligosporus NRRL and free lysine and methionine were also 2549 is a better strain than NRRL 2710 highest in the sample fermented by R. when groundnut is used as the substrate. bligosporus NRRL 2549. Murata et al. Thus, fermentation with the selected (1967) have observed similar increase in strain of R. oligosporus can help to free amino acids after fermentation of improve the nutritive value of groundsoybean. Possibility of synthesis of the nut meal.
References Barton-Wright, E. C. (1952) The microbiological assay of vitamin B complex and amino acids. London, Sir Isaac Pitman & Sons Ltd, pp. 121-126. Bhavanishankar, T. N., Shantha, N. V., Rajashekaran, T., Sreedharan, V. P. and Sreenivasa Mm-thy, V. (1978) Studies on tempeh made from groundnut and soybean mixture. Proceedings of the First Indian Convention of Food Scientists and Technologists, Mysore, India, p. 95. Chapman, D. G., Castillo, R. and Campbell, J. A. (1959) Evaluation of protein in foods. 1. A method for the determination of protein efficiency ratio. Can. J. Biochem. Physiol. 37, 679-686. Girija Bai, R., Prabha, T. N., Ramachandra Rao, T. N., Sreedharan, V. P. and Sreedhara, N. (1975) Studies on tempeh: Part I. Processing and nutritional evaluation of tempeh from a mixture of soybean and groundnut. J. Food Sci. Technol. 12,135-138. Hubbel, R. B., Mendel, L. B. and Wakeman, A. J. (1937) A new salt mixture for use in experimental diets. J. Nutr. 14, 273-285.
Groundnut tempeh 125 Murata, K., Ikehata, H. and Miyamoto, T. (1967) Studies on the nutritional value of Tempeh. J. Food Sci. 32,58&586. Official Methods of Analysis, Association of Official Analytical Chemists, 11th Ed., (1970). Washington DC, Benjamin Franklin Station, 20044. Pope, C. G. and Stevens, M. F. (1939) The determination of amino nitrogen using a copper method. Biochem. J. 33,1070-1077. Van Veen, A. G., Graham, D. C. W. and Steinkraus, K. H. (1968) Fermented peanut press cake. Cereal Sci. Today. 13,96-99. Wang, H. L. and Hesseltine, C. W. (1966) Wheat tempeh. Cereal Chem. 43,563-570.