Reduction of incubation time for tempeh fermentation by use of pregerminated inoculum

Spores ofRhizopus oligosporus, pregerminated 8–12 h on cooked white rice, were used to inoculate cooked soybeans, reducing the incubation time of the tempeh fermentation by up to 4 h.A potato extract-yeast extract-glucose broth containing germinated spores as inoculum for tempeh reduced incubation time by 4–5 h. Reduction of up to 17% of the tempeh fermentation time using spores pregerminated in a solid substrate may be of considerable economic value to soyfoods manufacturers.     

Penetration of Rhizopus oligosporus into Soybeans in Tempeh

Histological observations were made on the penetration of hyphae of Rhizopus oligosporus into soybean cotyledons in tempeh, an Indonesian soybean food. Hyphal penetrations averaged one per 1,400 μm² (±390 μm²) on the curved (outer) cotyledon surface and one per 1,010 μm² (±340 μm²) on the flat (inner) one. Hyphae infiltrated to a depth of 742 μm, or about 25% of the average width of a soybean cotyledon. This previously unreported degree of penetration offers partial explanation for the rapid physical and chemical changes in soybeans during tempeh fermentation.     

Oats tempeh

Oats was used as a substrate in tempeh fermentation. The time needed to obtain sufficient mold growth was at least 30 hours at 31°C (Rhizopus oligosporus NRRL 2710). pH was decreasing during the first 32 hours of incubation reaching pH = 5.30. Fermentation of oats led to an increase in water soluble nitrogen, but it did not change protein nitrogen content. R. oligosporus proteinases of optimum pH = 5.50 are postulated to play an important role in oats tempeh fermentation. When a mixture of oats and soybean (1:1) was used, mold growth was faster and the cake tougher. Mixing cereals with legumes to produce good tempeh is recommended.     

An original habitat of tempeh molds

Tempeh is a traditional Indonesian food made from soybeans fermented with Rhizopus species. Some researchers believe the original habitat of the tempeh molds may be closely related to fresh leaves of Hibiscus species because these leaves artificially infected with the tempeh molds are used to start tempeh fermentation in cottage-scale factories. To verify this hypothesis, we investigated the occurrence of Rhizopus species in Hibiscus leaves and identified the isolated Rhizopus strains precisely. Rhizopus oryzae, one of the tempeh molds, occurred in sample leaves of some Hibiscus species with considerable frequency. This result implies that tempeh molds that lived in Hibiscus leaves might have fermented soybeans accidentally when used to wrap the cooked soybeans. The original habitat of the tempeh mold could be fresh leaves of Hibiscus species.     

Phytic acid changes in soybeans fermented by traditional inoculum and six strains of Rhizopus oligosporus

Tempeh was prepared from Delmar variety soybeans inoculated with the traditional Indonesian inoculum (usar) and six pure culture strains of Rhizopus oligosporus. The strains BTU3K1 and CT11K2 produced the best quality tempeh. The phytic acid content of soybeans was reduced from 1mD07% in whole dry soybeans to 0mD67–0mD75% in tempeh. Most mould strains did not have a significantly different effect on reducing the phytic acid content in tempeh, although the traditional inoculum was significantly different to strains CTU7K2, BTU3K1, BT1K4 and CT11K2.     

Vitamin B12 production by Citrobacter freundii or Klebsiella pneumoniae during tempeh fermentation and proof of enterotoxin absence by PCR.

The influence of some fermentation parameters on vitamin B12 formation by strains of Citrobacter freundii and Klebsiella pneumoniae isolated from Indonesian tempeh samples during tempeh fermentation was investigated. A decrease in fermentation temperature from 32 to 24 degrees C led to a decrease in vitamin B12 formation. Inoculation of soybeans with different numbers of cells of C. freundii at the beginning of solid-substrate fermentation showed that only the velocity of vitamin formation and not the final amount of vitamin formed depended on the number of cells. The addition of cobalt and 5,6-dimethylbenzimidazole increased the vitamin B12 content of tempeh. Nevertheless, levels of incorporation of the two precursors into the vitamin B12 molecule were very low. Neither C. freundii nor K. pneumoniae possessed the genes encoding the enterotoxins Shiga-like toxin SLT IIA, heat-labile enterotoxin LT Ih, and heat-stable enterotoxin ST Ih, as indicated by PCR. This result supports the suggested use of these two strains to form vitamin B12 during tempeh fermentation in Indonesia.     

Production of vitamin B-12 in tempeh, a fermented soybean food

Several varieties of soybeans contained generally less than 1 ng of vitamin B-12 per g. It was found that use of a lactic fermentation typical of tropical conditions during the initial soaking of the soybeans did not influence the vitamin B-12 content of the resulting tempeh. Pure tempeh molds obtained from different sources did not produce vitamin B-12. It was found that the major source of vitamin B-12 in commercial tempeh purchased in Toronto, Canada, was a bacterium that accompanies the mold during fermentation. Reinoculation of the pure bacterium onto dehulled, hydrated, and sterilized soybeans resulted in the production of 148 ng of vitamin B-12 per g. The presence of the mold, along with the bacterium, did not inhibit or enhance production of vitamin B-12. Nutritionally significant amounts of vitamin B-12 were also found in the Indonesian fermented food, ontjom.     

Production of vitamin B-12 in tempeh, a fermented soybean food.

Several varieties of soybeans contained generally less than 1 ng of vitamin B-12 per g. It was found that use of a lactic fermentation typical of tropical conditions during the initial soaking of the soybeans did not influence the vitamin B-12 content of the resulting tempeh. Pure tempeh molds obtained from different sources did not produce vitamin B-12. It was found that the major source of vitamin B-12 in commercial tempeh purchased in Toronto, Canada, was a bacterium that accompanies the mold during fermentation. Reinoculation of the pure bacterium onto dehulled, hydrated, and sterilized soybeans resulted in the production of 148 ng of vitamin B-12 per g. The presence of the mold, along with the bacterium, did not inhibit or enhance production of vitamin B-12. Nutritionally significant amounts of vitamin B-12 were also found in the Indonesian fermented food, ontjom.     

The Effects of Temperature on Pratylenchus scribneri and P. alleni Populations on Soybeans and Tomatoes

In soil temperature tests, rates of Pratylenchus scribneri and P. alleni reproduction were measured at various lemperatures on ''Clark 63'' and ''Cutler 71'' soybeans and ''Rutgers'' tomatoes. Recovered P. scribneri equaled or exceeded initial inoculum levels at temperatures of 27.5 C or higher on soybeans, and at 20 C or higher on tomatoes. Population increases were greatest at 3.5 C on both hosts. Populations increased on soybeans, but not on tomatoes, when soil temperature was raised from 25 to 35 C for either 3 or 9 days. Recovered P. alleni were less than the initial inoculum at 27.5 C but higher at 32 and 37.5 C and at 27.5 C on tomatoes, the lowest temperature tested for this nematode. In the field, soil temperatures 10 cm deep in eastern Kansas soybean growing areas reach 27.5 C only occasionally and for relatively short periods, which probably explains the relatively low and variable populations of P. scribneri and P. alleni on soybeans there.     

Studies on the Nutritional Value of Tempeh

1. The nutritional value of tempeh in comparison with that of unfermented soybeans was studied in rat feeding experiment. It was observed that the PER value of tempeh (fresh or stored) was not significantly different from that of the unfermented soybeans (fresh or stored).

2. The peroxide value of the oil of stored tempeh powder was only 10% of that of stored soybean powder. Red blood cells of rats receiving tempeh were less than 20% in hemolysis by dialuric acid whereas hemolysis was 100% for the rats receiving the unfermented soybeans. Serum tocopherol was 0.14±0.05 mg/dl in the former group, but 0.07±0.05 mg/dl in the latter. Liver TBA values were 0.20±0.05 O. D./g and 0.65±0.13 O. D./g in the tempeh and the unfermented soybeans group, respectively.

3. Subsitution of whole egg for tempeh to supply 30% of protein in the diet improved the quality of the protein as measured by the protein efficiency ratio. An equal improvement was accomplished by supplementation of tempeh with lysine, methionine, and threonine in such amounts as the level of these amino acids equal to that in the tempeh-egg diet.     

Heat and mass transfer effects in static solid-substrate fermentations: design of fermentation chambers

An experimental device was constructed to allow nearly simultaneous measurements to be made on temperature and gas composition at different depths in a solid-substrate fermentation bed. The time-dependent values of temperature, mol % O(2) and mol % CO(2) were measured at five positions in beds 6.35 cm (2.5 in.) deep. With a tempeh fermentation (Rhiopus oligosporus growing on soybeans) the temperature gradient could be as steep as 3 degrees C/cm during active mold growth and concentration of CO(2) could reach 21 vol. % in the bottom layer.     

Effect of water activity on production and activity of Rhizopus oligosporus polysaccharidases

Summary During tempeh fermentation, Rhizopus oligosporus produced polysaccharidases to degrade soya bean cell walls; the maximum activity for all polysaccharidases tested occurred 20–30 h after inoculation. R. oligosporus was also grown in a soya bean extract model medium to which glycerol was added to control water activity (a _w). The overall activities of the major enzymes produced by the fungus, polygalacturonase, endocellulase and xylanase, appeared to be strongly influenced by a _w. The production of enzymes as well as their specific activities were affected by a _w. The optimum a _w for polygalacturonase and xylanase activity coincided with that for mycelial growth, namely 0.99–1.00. In contrast, the optimum a _w for (endo)cellulase was 0.98, at which mycelial growth was significantly reduced. Correspondence to: M. Sarrette     

Stimulation by potassium ions of the growth of Rhizopus oligosporus during liquid-and solid-substrate fermentations

Soya beans and several other beans and cereals have been used as substrates for tempe fermentation with the fungus Rhizopus oligosporus Saito. Except for the presence of alkaloids, the chemical composition of lupins (Lupinus mutabilis Sweet) is similar to that of soya beans. Therefore the potential of lupins for tempe production in regions with a long tradition of lupin consumption is promising. The preparation of the fermentation substrate when using bitter lupins (which contain significan quantities of alkaloids) as starting material includes a debittering stage to remove the alkaloids. However, we found that the debittering process yielded lupins that did not support the mycelial growth required in the tempe fermentation. We discovered that potassium is preferentially leached out during the debittering process. The effect of potassium on fungal biomass formation was monitored using a computerized system that determines biomass accretion by measurement of the electrical capacitance at radio frequencies. The importance of potassium for the growth of R. oligosporus was confirmed in liquid cultures. A linear relationship was found between biomass yield and K⁺ concentration in the range of 1 to 10 mg/l. The present report represents one of the few demonstrations of a mineral deficiency during the growth of a fungus on a natural, solid substrate.     

Comparison of Compositions of Odor Components of Natto and Cooked Soybeans

Natto, a traditional Japanese food product, was prepared from cooked soybeans by fermentation and its odor concentrate was obtained with a simultaneous distillation and extraction system. It was compared to those obtained from soybeans cooked for 0 ~ 3, 3 ~ 5.5 and 5.5 ~ 8 hr, respectively, by gas chromatography and gas chromatography-mass spectrometry. In the odor concentrates of the cooked soybeans, hexanal, (E)-2-hexenal and hexanol contributing to the green and grassy odor of soybeans disappeared or decreased while the cooking was in progress. 2-Pentylfuran and 1-octen-3-ol contributing to the beany odor remained even if the soybeans were cooked for 8 hr and were fermented into Natto. In the odor concentrate of Natto, pyrazines and sulfur containing compounds were important contributors to the characteristic odor of Natto. As the beany odor was not detected for Natto, it was concluded that the pyrazines and sulfur containing compounds cause the characteristic odor of Natto and mask the beany odor.     

Optimization of Process Variables for Lipase Biosynthesis from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> NRRL 5905 Using Evolutionary Operation Factorial Design Technique

Extracellular lipase was produced from Rhizopus oligosporus NRRL 5905 through solid state fermentation (SSF). To provide an optimum fermentation conditions for maximum lipase yield, five process variables (temperature, liquid–solid ratio, pH, incubation period and spore concentration) were optimized using evolutionary operation (EVOP) factorial design technique taking into account the interaction between the process variables. Optimization through EVOP resulted in around 3 fold increase in lipase activity (77 U gds⁻¹) at a liquid–solid ratio of 1.5:1, fermentation temperature of 35°C, initial fermentation pH 6, incubation period 5 days and a spore concentration of 10⁸ spores ml⁻¹.     

Evaporative temperature and moisture control in a rocking reactor for solid substrate fermentation

Summary In the solid substrate fermentation of cooked yellow corn grits with Rhizopus oligosporus in a rocking drum fermenter, temperature was controlled by blowing air through the substrate, forcing water evaporation. The rate of evaporation was controlled by the relative humidity of the air, according to the rate of heat generation during fermentation. Moisture content was maintained constant by spraying cold water on the substrate regulated by the water balance equation of the system. Both controls were operated by computer programs. The rocking motion in the reactor allowed even distribution of air and water in the substrate without disturbing the growing mycelia.     

Response of broiler chicks to dietary full-fat soybeans extruded at different temperatures prior to or after grinding

Whole (WSB) or ground (GSB) full-fat soybeans were extruded at 80, 100, 120 or 140°C and incorporated into broiler diets at 30% inclusion level during a 3-week trial.Extrusion of soybeans at 140 °C improved feed intake compared to those extruded at lower temperatures (P < 0.05). However, there were no significant effects of the extrusion temperature on weight gain, feed:gain ratio or mortality rate (P > 0.05). Also, there were no significant effects of whether the extrusion was undertaken prior to or after grinding of the soybeans on feed intake, weight gain, feed:gain ratio, or mortality rate (P > 0.05). There was no significant interaction between the extrusion temperature and the form extruded on any of the performance parameters (P > 0.05).Although increase in extrusion temperature reduced the trypsin inhibitor activity (TIA) of the soybeans, there were no significant effects of the extrusion temperature or type of soybean extruded on nitrogen, fat, calcium or phosphorus retention or diet ME (P > 0.05).It is concluded that low temperature extrusion of full fat soybeans does not reduce TIA to a satisfactory level (< 10 mg g⁻¹ sample) that will enable high dietary inclusion of such extruded soybeans for broilers.     

Exoglucanase production by Aspergillus niger grown on wheat bran

β-Exoglucanase production on the lignocellulosic material, wheat bran, by Aspergillus niger under solid state fermentation (SSF) on a laboratory scale was investigated. Different fermentation parameters, such as moisture content, initial pH, temperature, depth of the substrate, and inoculum size on exoglucanase production were optimized. Moisture content of 40 %, pH of 7.0, substrate depth of 1.0 cm, inoculum size of 2?×?10⁶ spores/g of wheat bran, and temperature at 30 °C were optimal for maximum production of exoglucanase. Maximum yields of exoglucanase with 28.60 FPU/g of wheat bran were obtained within 3 days of incubation under optimal conditions.     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

Protease production by Rhizopus oligosporus in solid-state fermentation

Rice bran was superior to other proteinaceous substrates for protease production by Rhizopus oligosporus ACM 145F in solid-state fermentation. Maximum protease yield was after 72 h. The optimal initial moisture content was 47% (a _w=0.97). Dried, ground and resuspended fermented rice was the most pratical and effective inoculum preparation, although, in the laboratory, spore suspensions prepared directly from agar slants were more convenient. Inoculum density (from 10² to 10⁷ spores/g substrate) and age (3, 5, 7 and 9 days) had little effect on protease yield.The authors are with the Department of Chemical Engineering, The University of Queensland, Brisbane, Queensland, 4072, Australia