The microflora of soak water during tempeh production from various beans

The microflora of soak water was studied during the soaking of horsebean, pea, chickpea and soybean for tempeh production. Lactic streptococci dominated the flora in both unacidified and acidified soak water. Coliforms and yeasts were found only in unacidified soak water. Growth of micro-organisms in acidified and unacidified soak water resulted in a decrease in pH value of the cooked beans. Microbial acidification during soaking is considered to be important in tempeh production.     

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.     

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.     

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.     

若干种子处理方法对偃松种子生活力的影响

为了提高偃松种子生活力,采用水浸、NaOH溶液浸泡、PEG6000（PEG,聚乙二醇）溶液浸泡、低温冷冻、吸湿/回干5种方法对偃松种子进行处理,研究不同处理中各因素（如溶液浓度、处理时间与温度）对偃松种子生活力的影响,并采用靛蓝染色法测定偃松种子的生活力。结果表明：10%PEG6000溶液、初始温度60℃、浸泡36 h时,偃松种子生活力最强;水浸、低温处理和吸湿/回干处理方法也能提高偃松种子生活力;然而4%~12%NaOH溶液浸泡处理会导致偃松种子生活力下降。因此可将PEG6000溶液处理种子的方法应用于偃松种子萌发及人工种苗培育的实践中,使偃松资源得到更好的可持续开发与利用。     

Impact of Water Column Acidification on Protozoan Bacterivory at the Lake Sediment-Water Interface

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h^-1, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (P = 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (P < 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.     

Phytoplankton in 63 limed lakes in comparison with the distribution in 500 untreated lakes with varying pH

As a result of the different weathering of lime in soils and varying acid deposition, the pH of Swedish oligotrophic lake waters has a broad range from ca. 4 to 8.5. Phytoplankton species occur in all lakes, but the composition and species richness varies in relation to pH. The maximum number of taxa was recorded within the pH-range 7.0–7.6, while the number decreased at higher pH and especially at pH below 5.6. During 1975–1985, thousands of lakes in Sweden were limed with the intention to restore biotic diversity and production. In order to find out whether there are differences in quality between limed and unacidified waters, the distribution of 57 selected phytoplankton taxa in 63 limed lakes was compared with that in 500 unlimed lakes with varying pH. The study shows that most common species in unacidified lakes also occur in the limed ones. However, several species of Cyanophyceae and Conjugatophyceae were missing especially in lakes that were strongly acidified before liming. On the other hand, some taxa were more frequent in the limed lakes.     

Microbial diversity and dynamics of microbial communities during back-slop soaking of soybeans as determined by PCR–DGGE and molecular cloning

Tempe is a traditional fermented food in Indonesia. The manufacture process is quite complex, which comprises two stages, preparatory soaking of soybeans and fungal solid state fermentation. Daily addition of previous soak water (back-slopping) during the soybean soaking step is considered to be crucial in the manufacture of high quality tempe. The microbial diversity and dynamics of the microbial communities evolving during back-slop soaking of soybeans for tempe making was investigated by culture-independent PCR–DGGE and molecular cloning. Both DNA and total RNA were isolated and included in this study, to obtain a view on the succession of total and viable bacteria in the complex microbiota. DGGE profiles indicated that Enterobacter sp., Enterococcus sp., Pseudomonas putida, Leuconostoc fallax, Pediococcus pentosaceus, and Weissella cibaria, were the predominant bacteria. Their occurrence shifted dramatically during the back-slop soaking procedure. This study combined with previous culture-dependent studies could gain a better understanding of the complex microbiota of traditional fermented food and give useful information for its quality control.     

Organogenesis in vitro cultures of embryonic shoots ofAbies balsamea (balsam fir)

Summary Embryonic shoots of 15- to 20-year-oldAbies balsamea (balsam fir) trees were soaked in (a) water for 15 min or 24 hr and (b) water with 1000 mg per l indolebutyric acid (IBA), N-dimethylaminosuccinamic acid (Alar-85), or 1-phenyl-3-methyl-5-pyrazolone (PPZ), singly or in combination with 100 mg per 1 caffeic acid, for 15 min. After the soaking, the embryonic shoots were transferred to a nutrient medium. Nonsoaked (control) embryonic shoots elongated and often formed a basal callus but never showed organogenesis. The soaked embryonic shoots formed new apical buds, with or without bud scales, adventitious dwarf needles or shoots, and root- and embryo-like structures. One of the embryos germinated and formed an irregular shoot. No differences were found between the various soak treatments, except that the 15-min water soak was ineffective. The 24-hr water soak was as effective as the 15-min growth regulator treatments.     

Dietary self-selection for organic acids by the piglet

Two feeding trials using 48 weaned crossbred piglets each were carried out to determine the effect of acidifying diets with potassium diformate (K-diformate), formic or sorbic acid on dietary preferences in piglets. In Exp. 1 two reference groups were fed either an unacidified diet or a diet containing 2.4% of K-diformate with no choice for selection. Furthermore, piglets in choice group 1 and 2 had the choice between an unacidified diet and a diet supplemented with 1.2 and 2.4% K-diformate, respectively. In Exp. 2, animals of three reference groups received exclusively an unacidified diet or diets supplemented with 1.2% formic acid or 1.2% sorbic acid, respectively. The animals of the choice groups had the choice between an unacidified diet and diets with 1.2% formic acid or 1.2% sorbic acid, respectively. In Exp. 1 average daily feed intake, daily gain and feed conversion ratio were 751?g, 458?g and 1.64?kg/kg, respectively, with no significant differences between treatments. In both choice feeding groups animals chose the diets on offer at random (each around 50%). In Exp. 2 growth and feed intake were not affected by the treatment, but feed conversion ratio was enhanced due to the 1.2% formic acid supplementation. Animals of both organic acid choice groups showed a significant preference for the unacidified diets in each experimental week. The formic acid and sorbic acid diets represented on average only 13.5% and 23.5% of the total feed intake. The present results demonstrate that the inclusion of 1.2% sorbic or formic acid or 2.4% of K-diformate in piglet diets has no negative impact on feed intake, but in a situation of choice feeding, piglets will refuse diets acidified with 1.2% formic or sorbic acid, presumably because of negative taste cues. Acidifying the diets with varying amounts of a K-diformate had no effect on dietary preferences of piglets.     

Dietary self-selection for organic acids by the piglet

Two feeding trials using 48 weaned crossbred piglets each were carried out to determine the effect of acidifying diets with potassium diformate (K-diformate), formic or sorbic acid on dietary preferences in piglets. In Exp. 1 two reference groups were fed either an unacidified diet or a diet containing 2.4% of K-diformate with no choice for selection. Furthermore, piglets in choice group 1 and 2 had the choice between an unacidified diet and a diet supplemented with 1.2 and 2.4% K-diformate, respectively. In Exp. 2, animals of three reference groups received exclusively an unacidified diet or diets supplemented with 1.2% formic acid or 1.2% sorbic acid, respectively. The animals of the choice groups had the choice between an unacidified diet and diets with 1.2% formic acid or 1.2% sorbic acid, respectively. In Exp. 1 average daily feed intake, daily gain and feed conversion ratio were 751 g, 458 g and 1.64 kg/kg, respectively, with no significant differences between treatments. In both choice feeding groups animals chose the diets on offer at random (each around 50%). In Exp. 2 growth and feed intake were not affected by the treatment, but feed conversion ratio was enhanced due to the 1.2% formic acid supplementation. Animals of both organic acid choice groups showed a significant preference for the unacidified diets in each experimental week. The formic acid and sorbic acid diets represented on average only 13.5% and 23.5% of the total feed intake. The present results demonstrate that the inclusion of 1.2% sorbic or formic acid or 2.4% of K-diformate in piglet diets has no negative impact on feed intake, but in a situation of choice feeding, piglets will refuse diets acidified with 1.2% formic or sorbic acid, presumably because of negative taste cues. Acidifying the diets with varying amounts of a K-diformate had no effect on dietary preferences of piglets.     

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.     

Studies on the absorption spectra of plant pigments in estuaries

Summary Absorption spectra of acetone-soluble plant pigments were obtained from several sources, including the overlying waters, epibenthic layers and sediments of two New England estuaries, the remains of Zostera marina and its epiphytes, and pure cultures of marine diatoms. Comparisons of the absorption spectra and various absorbance ratios revealed no significant differences in the extracts of pigments sampled from the overlying waters and epibenthic layers; the extracts from both layers differed significantly, however, from the extracts of sedimentary pigments. Unacidified extracts of pigments from healthy diatom cultures and detritus rich in epiphytic diatoms resembled those of the overlying waters and epibenthic layers; acidified extracts from the above two sources and unacidified and acidified extract from non-living detritus resembled those of the sediments. Good correlations between field and laboratory studies are indicated, and the uses of absorbance ratios as ecological indicators are discussed.     

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.     

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.     

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.     

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.     

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.