Formation of vitamins by pure cultures of tempe moulds and bacteria during the tempe solid substrate fermentation

The formation of water soluble vitamins (vitamin B₁₂, vitamin B₆, riboflavin, thiamine, nicotinic acid and nicotinamide) during the tempe solid substrate fermentation was investigated. The role of several strains of Rhizopus oligosporus, R. arrhizus , and R. stolonifer and the role of several bacteria in the vitamin formation process were checked. All fungal and bacterial strains were isolated from Indonesian tempe and soaking water samples. The Rhizopus strains formed riboflavin, nicotinic acid, nicotinamide and vitamin B₆. The final concentrations of these substances depended on the different strains involved and on the fermentation time. Isolates of R. oligosporus were generally the best vitamin formers. The moulds did not produce physiologically active vitamin B₁₂. The thiamine content decreased during fermentation. The addition of bacteria, which had been selected in a screening for vitamin B₁₂ production, resulted in an increase of physiologically active vitamin B₁₂. Citrobacter freundii and Klebsiella pneumoniae showed the best formation capabilities. Furthermore, the bacteria produced riboflavin and vitamin B₆ in addition to the moulds. The influence of Rhizopus on the vitamin B₁₂ formation of Cit. freundii was also investigated. The vitamin content of tempe that was fermented with the mould and the bacterium was three times as high as a control fermentation with Cit. freundii only.     

Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins

During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased. During fatty acid synthesis Rhizopus sp. produced only gamma-linolenic acid (GLA) instead of ALA. The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined. There was a distinct increase in the amount of free amino acids during tempe fermentation. Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids. In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans. In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid. The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe. In the presence of the Rhizopus mold, the vitamin B(12) formation by C. freundii was three times higher than that of a fermentation without the mold.     

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.     

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.     

Use of Cheese Whey for Vitamin B12 Production: III. Growth Studies and Dry-Weight Activity1

The patterns of growth and vitamin formation by Propionibacterium shermanii in whey were similar to the patterns established in other substrates. The vitamin formation was observed during the latter part of the fermentation after the organism approached maximal growth. Lactose utilization by the organism corresponded to the logarithmic-growth phase of the organism. Analyses of the dried culture showed a large increase of vitamin B(12) in the fermentation solids compared with unfermented dried whey. A feed analysis showed a notable increase of protein and a large decrease in nitrogen-free extract of the dried fermentation solid compared with dried whey.     

3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species.

Glycerol fermentation by Enterobacter agglomerans revealed that both growth and 1,3-propanediol production ceased after consumption of about 430 mM glycerol, irrespective of the initial glycerol content. This phenomenon was assigned to the production of 3-hydroxypropionaldehyde, which was identified by proton nuclear magnetic resonance and which showed a bacteriostatic effect. The accumulation during glycerol fermentation was also observed with two other enterobacterial species, i.e., Klebsiella pneumoniae and Citrobacter freundii.     

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.     

Effect of Yeast Extract and Vitamin B(12) on Ethanol Production from Cellulose by Clostridium thermocellum I-1-B

Addition to media of yeast extract, a vitamin mixture containing vitamin B(12), biotin, pyridoxamine, and p-aminobenzoic acid, or vitamin B(12) alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by Clostridium thermocellum I-1-B. A similar effect was not observed with C. thermocellum ATCC 27405 and JW20.     

Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii.

The coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis. We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase. Northern blot analyses revealed that both genes were expressed during glycerol fermentation. The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase. The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E. coli strain producing both His6-tagged proteins. Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da. The subunit structure of the native complex is probably alpha2beta2. The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+. The DhaF-DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase. The purified DhaF-DhaG complex of C. freundii efficiently cross-activated the enzyme-cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae. It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.     

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.     

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.     

Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with betaine feeding

The strategy of betaine control for vitamin B(12) large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B(12) biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B(12) production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B(12) fermentation in 120-m(3) fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5-7g/l during 50-140h of fermentation.     

Vitamin B12-dependent propionate production by the ruminal bacterium Prevotella ruminicola 23.

When Prevotella ruminicola 23 was grown in a defined medium containing a vitamin mixture, significant amounts of propionate were formed. Succinate and acetate were the major fermentation acids produced when vitamins were omitted, and further experiments demonstrated that propionate formation was dependent on vitamin B12. When the organism was grown in continuous culture at dilution rates of less than 0.20 h-1, propionate and acetate were the predominant fermentation products and little succinate was formed when vitamin B12 was present. However, at higher dilution rates, propionate formation declined and succinate accumulated. Since cell protein yields were reduced 15 to 25% in the absence of vitamin B12, the pathway for propionate formation may contain an energy-conserving step.     

Vitamin B12-dependent propionate production by the ruminal bacterium Prevotella ruminicola 23.

When Prevotella ruminicola 23 was grown in a defined medium containing a vitamin mixture, significant amounts of propionate were formed. Succinate and acetate were the major fermentation acids produced when vitamins were omitted, and further experiments demonstrated that propionate formation was dependent on vitamin B12. When the organism was grown in continuous culture at dilution rates of less than 0.20 h-1, propionate and acetate were the predominant fermentation products and little succinate was formed when vitamin B12 was present. However, at higher dilution rates, propionate formation declined and succinate accumulated. Since cell protein yields were reduced 15 to 25% in the absence of vitamin B12, the pathway for propionate formation may contain an energy-conserving step.     

低成本绿色循环工艺发酵生产丙酸联产维生素B12

对提取维生素B12后的费氏丙酸杆菌废菌体进行水解处理,考察以菌体水解液作为N源用于丙酸发酵的可行性.利用正交设计得到了提取维生素B12后的废菌体水解优化条件.基于此,构建利用植物纤维床反应器固定化生产丙酸联产维生素B12的低成本绿色循环工艺.结果表明:在4.5L的发酵体系中,单批次总糖质量浓度为200 g/L,发酵进行了5批次共1192h,丙酸生成总量为2 328.75 g,单批次丙酸质量浓度103.50 g/L,丙酸生产效率达0.43 g/(L·h),干菌质量浓度达到19.52 g/L.将菌体注入微好氧发酵罐中发酵获得112.8 mg/L维生素B12.     

An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 mug/mL of vitamin B(12) was achieved.     

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.     

Alkaline lipase production by Citrobacter freundii IIT-BT L139

Around 150 lipase producing bacterial isolates were screened from the local soils enriched with oil. Citrobacrer freundii IIT-BT L139, an isolated microbial strain, produced lipase that had high activity (8.8 U/ml) at pH 9.0 and 40 degrees C. The 16S rDNA phylogenetic studies showed that Citrobacter freundii belongs to the family Enterobacteriaceae and later confirmed by the microbial identification. Suitable C and N sources for lipase production were deduced to be starch and peptone-urea, respectively. In a controlled fermenter (1 L), the lipase activity was found to increase by 36% (12 Uml(-1)). The variation of lipase activity, pH and dissolved oxygen (DO) during growth of the organism in the controlled batch fermenter were monitored. The rheological characteristics of the fermentation broth indicated that it behaved like a Newtonian fluid throughout the fermentation. The fermentation time was comparatively short (60 h). The lipase was also found to be substantially resistant to common detergents. This lipase was, thus, characterized as alkaline, thermostable and solvent stable, which was essentially desirable in pharmaceutical, detergent and other industrial applications or production.     

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.