Real-time monitoring of the accretion of Rhizopus oligosporus biomass during the solid-substrate tempe fermentation

We describe a novel method for the real-time estimation of the accretion of blomass during the solid-substrate tempe fermentation of soy beans, lupins and quinoa by Rhizopus oligosporus Salto. The method is based on measurements of the dielectric permittivity at radio-frequencies, using a four-terminal instrument (the Bugmeter). In all cases, excellent Ilnearity is observed during the growth phase between the dielectric permittivity and the hyphal length as determined microscopically.     

Low-temperature scanning electron microscopy of hyphal colonisation of Chenopodium quinoa during fermentation with Rhizopus oligosporus

In a study of tempe made by fermenting quinoa (Chenopodium quinoa Willd.) with Rhizopus oligosporus Saito, the interaction between the substrate and the fungus varied according to the raw material used. Low-temperature scanning electron microscopy confirmed that hyphal infiltration in quinoa was normally limited by the seed coat, but a massive penetration was found in fragments of quinoa where the testa was broken. This finding has implications for the texture and quality of tempe fermentation of grains and pulses.     

Temperature control in solid substrate fermentation through discontinuous rotation

A laboratory-scale system for controlled dynamic solid substrate fermentation was developed and tested. The fermentation takes place in a stainless steel discontinuously rotating drum reactor, under controlled conditions of temperature, gas composition, relative humidity and direction and rate of rotation. The system was tested on a model fermentation of soya beans with Rhizopus oligosporus. In contrast to the traditional tempe fermentation, a granular product is obtained and build-up of heat and mass gradients is restricted. Despite the discontinuous rotation, the fungal growth continues, as evidenced by the production of heat. The rate of cooling depends on the temperature of the gas flushed through the reactor, the gas flow rate and the lenght of the rotation period. As a consequence of the homogeneous temperature control, the fungal heat development continued up to 70 h of fermentation. This is in clear contrast with the traditional tempe fermentation, which is already limited after 36 h by its own heat accumulation. Correspondence to: M. J. R. Nout     

Suppression of penetrative hyphae ofRhizopus oligosporus by membrane filters in a model solid-state fermentation system

Summary Membrane filters overlaid on slabs of a model solid substrate enabled recovery of biomass ofRhizopus oligosporus. Although the presence of the membrane filter affects the growth ofRhizopus oligosporus it provides a useful tool for studying solid-state fermentation.     

Effects of temperature,water activity and gas atmosphere on mycelial growth of tempe fungi Rhizopus microsporus var. microsporus and R. microsporus var. oligosporus

Rhizopus microsporus var. microsporus and var. oligosporus are used in the manufacture of various Asian fermented foods (tempe, black oncom, sufu). In view of solid-substrate fermentation (SSF) control, mycelial growth of strains of both varieties was tested for sensitivity to fluctuations of temperature, water activity and interstitial gas composition. This was achieved by measuring radial growth as well as biomass dry weight of pre-germinated microcolonies on defined media. The optimum conditions were temperature 40 °C, a _w 0.995 and a gas composition of air for the growth of both strains on a model medium. Whereas radial growth rates of var. microsporus and var. oligosporus were similar, biomass growth rates of var. oligosporus were higher than those of var. microsporus under optimum conditions. The temperature-dependent growth of Rhizopus spp. at a _w > 0.98 could be described by the Ratkowsky Equation. Carbon dioxide (5–10% v/v) inhibited the growth of Rhizopus spp. at non-limiting levels of oxygen. The two strains were able to grow at low (0.5% v/v) oxygen levels, but the mycelial density was rather low. No interrelation of water activity and gas composition was observed, but at high water activity the fungi were more sensitive to changes of temperature. The implications for process control are discussed. This revised version was published online in November 2006 with corrections to the Cover Date.     

Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates

Summary The ergosterol content of Rhizopus oligosporus NRRL 5905 varied between 2–24 g/ mg biomass dry matter when grown in laboratory media and was found to be influenced by the substrate composition.When grown on a natural substrate (soya beans) the ergosterol content was considerably higher (estimated at approx. 60–90 g/mg biomass dry matter).In laboratory media, the ergosterol content was also influenced by the extent of aeration and the growth phase of the mycelium; within the range of 25° C–35° C, the incubation temperature did not influence the ergosterol content significantly.In view of these variations, ergosterol should not be used as a chemical index for the quantification of biomass grown in static solid-substrate fermentations with limited mass transfer, e.g. tempe or oncom.     

Influence of solid substrate fermentation on the chemical composition of chickpea

A basic procedure was developed to produce a fermented product by solid substrate fermentation using Rhyzopus oligosporus and chickpea as substrate. Water activity was kept at 0.92 throughout the process. Fermentation increased total, ‘true’ and soluble proteins, soluble solids and soluble carbohydrates, and decreased fiber content and pH. About 12% of solids were lost during 72 h of fermentation. The content of most fatty acids was enhanced by fermentation, whereas peroxide value and tannins declined. The color of the fermented product was not deteriorated after 72 h of fermentation. Scanning electron microscopy studies of microbial growth on the substrate showed penetration of the fungus hyphae and degradation effects on the chickpea cotyledon cells.     

Protein measurement in solid-state fermentation

Summary Membrane filter culture was used as a model system for the investigation of the growth of Rhizopus oligosporus in solid-state fermentation. The biuret method gave more accurate estimations of protein than the Folin-Lowry method. However, due to the change in the protein content of the biomass during the fermentation, protein cannot be considered to be a reliable index of growth in solid-state fermentation.     

Optimization of single cell protein production from cassava starch (Rhizopus oligosporus)

The fermentation pattern of cassava starch utilization was investigated at 37°C using Rhizopus oligosporus UQM 145 F and eight different media. Depending on the medium used, the addition of zinc or zinc plus iron to a combination of calcium plus manganese switches the fermentation from glucose accumulation to biomass (single cell protein) production. Complete starch hydrolyzation was obtained in both cases, with a complete glucose utilization resulting in 24 g biomass containing 30% true protein per 100 g cassava starch (= 7.45 g SCP/100 g substrate) in 24 hours. In the case of glucose accumulation, biomass was kept low and 15.5 g/l glucose representing 57.3% of starch supplied were obtained in 36 hours. R. oligosporus UQM 145 F grows well between 30° and 45°C. At 45°C and pH 5.0, 7.0 g SCP/100 g substrate were obtained, which rose to 8.6 g if cassava starch is replaced by ground cassava tuber.     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

Linoleic acid, α-linolenic acid,and monolinolenins as antibacterial substances in the heat-processed soybean fermented with Rhizopus oligosporus

ABSTRACT

Antibacterial activities against Staphylococcus aureus and Bacillus subtilis were found in an ethanol fraction of tempe, an Indonesian fermented soybean produced using Rhizopus oligosporus. The ethanol fraction contained free fatty acids, monoglycerides, and fatty acid ethyl esters. Among these substances, linoleic acid and α-linolenic acid exhibited antibacterial activities against S. aureus and B. subtilis, whereas 1-monolinolenin and 2-monolinolenin exhibited antibacterial activity against B. subtilis. The other free fatty acids, 1-monoolein, monolinoleins, ethyl linoleate, and ethyl linolenate did not exhibit bactericidal activities. These results revealed that R. oligosporus produced the long-chain polyunsaturated fatty acids and monolinolenins as antibacterial substances against the Gram-positive bacteria during the fungal growth and fermentation of heat-processed soybean.     

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.     

Genetically modified soybeans: false-positive detection in fermented natural soybean (tempe)

Tempe was prepared using mixtures of natural soybean and genetically modified Roundup Ready (RUR) soybean fermented with natural Rhizopus sp. The amount of RUR soybean was quantified using an ELISA plate test. The RUR signal decreased during fermentation. In the control experiments on fermentation of non-RUR soybean, the tempe gave a false-positive RUR signal. The cross-reacting substance was generated only in non-RUR soybean during fermentation by Rhizopus sp., Rhizopus oligosporus, R. oryzae, Mucor rouxii and Aspergillus awamori.     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

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     

Agar plate growth studies of Rhizopus oligosporus and Aspergillus oryzae to determine their suitability for solid-state fermentation

Summary Colony radial growth rates of Rhizopus oligosporus and Aspergillus oryzae were compared under various conditions on agar plates containing cassava starch. Both organisms grew well on cassava starch as their sole source of carbon and energy, although growth was stimulated by the addition of yeast extract and peptone. Neither organism utilized ungelatinized starch effectively. The optimum initial pH for R. oligosporus was 7, although good growth was obtained at pH 5 when ammonium sulfate was partially replaced by urea. A. oryzae grew well over a range of initial pH values from 5 to 8. Growth of R. oligosporus was inhibited by NaCl concentrations above 0.5% (w/v) while A. oryzae was unaffected up to 4% NaCl. The best colony radial growth rate obtained for R. oligosporus was 1.01 mm/h, which was far superior to that obtained for A. oryzae (0.29 mm/h). R. oligosporus was chosen as the more suitable organism for future studies of the protein enrichment of cassava by solid-state fermentation.     

Growth of thiobacillus ferrooxidans under electrochemical reduction of inorganic energy source used

An electrolytic cell was designed and constructed for the formentation of T. ferroxidans using Fe²⁺ as the energy-supplying substrate. The Fe³⁺ produced by T. ferrooxidans by fermentation is continuously reduced to Fe ₂₊ in the electrolytic cell. A suitable version of the electrolytic cell permitted the elimination of most inorganic solid matter (precipitate) from the fermentation process. The fermentation of T. ferrooxidans was carried out with and without the electrolytic cell. Fermentation with the cell yielded significant rises both in the maximum obtainable growth rate and the biomass concentration. The experimental of data are discussed and the theoretical substrate consumption coefficient was calculated for Fe²⁺ as a function of the pH and the coefficient was compared with the experimental results.     

Modeling of <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial growth kinetics and optimal fed-batch fermentation for beauvericin production

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l⁻¹ glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l⁻¹) than in the batch culture (194 mg l⁻¹). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.     

The effect of urea on growth of moulds and biomass yield in solid-state cultivation

Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergillus oryzae and Rhizopus oligosporus, led to a higher concentration of NH₄ ⁺ and pH but a decrease in biomass production.The authors are with the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia.     

Degradation of quinolizidine alkaloids of lupin by Rhizopus oligosporus

Rhizopus oligosporus has proven beneficial in the detoxification of lupin seeds. The fermentation process is mainly affected by the initial pH in the medium. In the range of growth of mold, there are maximum enzymatic activities in pH of 3.5 and 5.5. Metabolism change occurs at these pH levels; therefore, we studied the growth, pH changes, dry matter intake, and alkaloid degradation within 48 h of fermentation. Cultures of lupin agar (LA) with pH of 3.5 and 5.5 were made in Petri dishes with lupin flour. Results showed pH directly affects the degradation of alkaloids and fungal growth. Detoxification levels achieved were 16.58 and 63.23 % in treatments LA 3.5 and LA 5.5, respectively. Fungal growth was 0.919 mg/cm² in LA 3.5 and 1.081 mg/cm² in LA 5.5. Maximum degradation rate in LA 5.5 was given between 16 and 20 h, which coincided with maximum fungal growth. Despite having similar dry matter intake in both treatments, a pH of 3.5 did not show the same degree of detoxification. The analysis with exponential, yield of growth, yield of dry matter intake and luedeking and piret equations, confirm the relation between intake and growth with detoxification. Dry matter intake equation predicts with R ² of 0.94 the detoxification in LA 5.5. A pH of 5.5 is directly related with detoxification and fungal development.