Rice-Grown Rhizopus oligosporus Inoculum for Tempeh Fermentation

A method of growing Rhizopus oligosporus on cooked rice as the inoculum for the fermentation of soybeans into tempeh was described and evaluated. Isolated R. oligosporus spores on glass beads survived best at low temperature and intermediate humidity. The activity of the rice-grown inoculum to ferment soybeans into tempeh did not decrease appreciably when stored desiccated for one year at 4 C or room temperature. Bacterial contaminants as high as 10⁸ counts per g of cooked soybeans did not seem to affect the 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.     

Gamma-Ray Sterilization and Residual Toxicity Studies of Ground Beef Inoculated with Spores of Clostridium botulinum

Inoculated packs of cooked and raw ground beef were sterilized with gamma radiation from cobalt-60. With inocula of 5,000,000 Clostridium botulinum 213B spores per g of cooked ground beef, 3.8 megarad were required for sterilization; in raw ground beef, 3.72 megarad sterilized the meat when inocula of 1,700,000 C. botulinum 213B spores were used per g. Using C. botulinum 62A spores, cooked ground beef inoculated with 5,200,000 spores per g was sterilized with 3.85 megarad; raw ground beef, inoculated with 2,670,000 spores per g, was sterilized with 3.6 megarad. Cans of meat that were considered sterile by lack of culture growth after incubation for at least 6 months and, in some instances, as long as 5 years, were tested for the presence of botulinus toxin. No toxin was found in any meat taken from inoculated packs prepared from C. botulinum 213B spores; however, all cans of meat that had been inoculated with more than 2,670,000 C. botulinum 62A spores per g of meat, contained type A toxin. It was shown that these latter inocula of heat-shocked spores, by themselves, contained sufficient toxin to kill mice. However, more toxin appeared to be present than could be ascribed to the unirradiated spores alone. This finding is discussed.     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

Image analysis for monitoring the barley tempeh fermentation process

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.     

Production of a highly glucose tolerant β-glucosidase by Paecilomyces variotii MG3: optimization of fermentation conditions using Plackett–Burman and Box–Behnken experimental designs

β-Glucosidases (β-d-glucoside glucohydrolase, 3.2.1.21) are a group of enzymes mainly involved in the hydrolysis of β-glycosidic bonds connecting carbohydrate residues in different classes of β-d glycosides. During cellulose degradation they convert cellobiose and cellooligosaccharides produced by the endo and exoglucanases to glucose. Most of the microbial β-glucosidases are inhibited by glucose. This limits their application in commercial scale cellulose degradation ventures. Solid state fermentation production of a highly glucose tolerant β-glucosidase by a novel isolate of Paecilomyces was optimized using a two step statistical experiment design. In the first step which employed a Plackett–Burman design, the effects of parameters such as moisture, temperature, pH, inoculum concentration, incubation time and different concentrations of (NH₄)₂SO₄, KH₂PO₄, NaCl, peptone and cellobiose were evaluated. The parameters with significant influence on the process were selected and fine tuned in the second step using a Box–Behnken design. The model obtained was validated and a peptone concentration of 2 g/l, inoculum concentration of 1.2 × 10⁶ spores/ml and an incubation period of 96 h were found to be optimum for the maximum production of the enzyme. The optimization resulted in a doubling of the enzyme production by the fungus.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

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.     

Induced Sporicidal Activity of Chlorhexidine against Clostridium difficile Spores under Altered Physical and Chemical Conditions

Background

Chlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with Clostridium difficile, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore’s protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against C. difficile spores.

Principal Findings

C. difficile spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%–0.0004% w/v chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions.

Conclusions

Physical and chemical conditions that alter the protective barriers of C. difficile spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore.     

Mass production of spores of lactic acid‐producing Rhizopus oryzae NBRC 5384 on agar plate

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato‐dextrose‐agar medium was studied aiming at starting its L (+)‐lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar‐on‐bottom or agar‐on‐top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar‐on‐top mode gave larger amount of spores than in an agar‐on‐bottom mode at 30°C for 7‐day cultivation. Scale‐up of the agar plate culture from 26.4 to 292 cm² was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m³ industrial submerged fermentation started directly from 2 × 10⁵ spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full‐scale industrial submerged fermentation started directly from spore inoculation omitting pre‐culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10⁸ cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10⁶–10⁷ cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus . In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5·5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett–Burman experimental design. Subsequently, optimal conditions were obtained using the Box–Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett–Burman design and Box–Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.     

Effect of pea and soybean extracts on the growth of five clostridium strains

Water extracts of pea and soybean stimulated the growth of five tested gas-producing Clostridium strains. When pea and soybean tempeh extracts were used the inhibition effect took place. It is postulated that an antibacterial compound is formed during tempeh fermentation. The stimulating effect might be connected with flatus formation by legumes.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Deriving fractional rate of degradation of logistic-exponential (LE) model to evaluate early in vitro fermentation

Water-soluble components of feedstuffs are mainly utilized during the early phase of microbial fermentation, which could be deemed an important determinant of gas production behavior in vitro. Many studies proposed that the fractional rate of degradation (FRD) estimated by fitting gas production curves to mathematical models might be used to characterize the early incubation for in vitro systems. In this study, the mathematical concept of FRD was developed on the basis of the Logistic-Exponential (LE) model, with initial gas volume being zero (LE₀). The FRD of the LE₀ model exhibits a continuous increase from initial (FRD₀) toward final asymptotic value (FRD_F) with longer incubation time. The relationships between the FRD and gas production at incubation times 2, 4, 6, 8, 12 and 24 h were compared for four models, in addition to LE₀, Generalization of the Mitscherlich (GM), c^th order Michaelis–Menten (MM) and Exponential with a discrete LAG (EXP_LAG). A total of 94 in vitro gas curves from four subsets with a wide range of feedstuffs from different laboratories and incubation periods were used for model testing. Results indicated that compared with the GM, MM and EXP_LAG models, the FRD of LE₀ model consistently had stronger correlations with gas production across the four subsets, especially at incubation times 2, 4, 6, 8 and 12 h. Thus, the LE₀ model was deemed to provide a better representation of the early fermentation rates. Furthermore, the FRD₀ also exhibited strong correlations (P < 0.05) with gas production at early incubation times 2, 4, 6 and 8 h across all four subsets. In summary, the FRD of LE₀ model provides an alternative to quantify the rate of early stage incubation, and its initial value could be an important starting parameter of rate.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10(8) cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10(6)-10(7) cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus. In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5.5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Two-stage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p<0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R(2) of 0.9963 (polygalacturonase) and 0.9806 (spores) (p<0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 x 10(7) total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 x 10(7)spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 x 10(7)spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.     

Fermentative production of spiramycins

Fermentative production of spiramycins by Streptomyces ambofaciens has been performed using fermentation media of different chemical compositions. Medium I was selected from nine media as the best for production of high titres of spiramycins. Biochemical changes which occurred during fermentative production of spiramycins revealed that adjustment of the initial pH value of the medium was very important. The initial pH value of the fermentation medium which allowed the organism to produce a good yield of antibiotic was 6.5. The fermentation period affected the formation of spiramycins, and the maximum incubation period required for the fermentation process was 120 h. The role of inoculum on spiramycin yield showed that it was better to inoculate the fermentation medium with vegetative cells of Streptomyces ambofaciens rather with spores. The carbon source influenced spiramycin biosynthesis: dextrin was the best carbon source and stimulated the organism to form high titres of antibiotics. The best concentrations of dextrin and glucose for increased antibiotic yields were 25 and 15 gl^?1, respectively. Organic sources in the fermentation medium were more efficient than inorganic nitrogen sources for spiramycin formation. Fodder yeast was the best organic nitrogen source in fermentative production of spiramycins. The maximal concentrations of fodder yeast, soybean meal, peptone, Ca(NO₃)₂ and NH₄NO₃ for increased antibiotic yield were 6.5, 6.0, 4.0, 10.0 and 4.0 gl^?1, respectively.