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.     

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.     

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.     

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.     

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 solubilized porcine ileal intrinsic factor receptor and on a 340 000-dalton component binding vitamin B-12

A 340 000-dalton component “C-III” was found when Triton X-100-containing extracts of ileal mucosa were incubated with human or porcine intrinsic factor vitamin B-12 preparations. It was not formed when abnormal human intrinsic factor, unable to attach to the intrinsic factor receptor, was used. Prolonged storage promoted the transfer of vitamin B-12 to it from the vitamin B-12-intrinsic factor receptor species C-I and C-II. The component was also present in ileal extracts prepared with or without detergent and it bound vitamin B-12 directly. Immunologically and by electrofocusing it could be classified as a cobalophilin but its molecular dimensions were larger than described for cobalophilin. It thus represents a novel vitamin B-12 binding protein, possibly a macromolecular acceptor of vitamin B-12 which accepts vitamin B-12 bound via intrinsic factor to the ileal intrinsic factor receptor.In the presence of EDTA or at low pH, vitamin B-12-intrinsic factor did not bind to any of the receptor species and under the same conditions it could all be dissociated from the receptor complexes but not from C-III. The dissociated receptor was able to recombine with vitamin B-12-intrinsic factor and it appeared to bind free and vitamin B-12-bound intrinsic factor in vivo.     

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.     

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.     

Vitamin B-12 Status during Pregnancy and Child’s IQ at Age 8: A Mendelian Randomization Study in the Avon Longitudinal Study of Parents and Children

Vitamin B-12 is essential for the development and maintenance of a healthy nervous system. Brain development occurs primarily in utero and early infancy, but the role of maternal vitamin B-12 status during pregnancy on offspring cognitive function is unclear. In this study we assessed the effect of vitamin B-12 status in well-nourished pregnant women on the cognitive ability of their offspring in a UK birth cohort (ALSPAC). We then examined the association of SNPs in maternal genes FUT2 (rs492602) and TCN2 (rs1801198, rs9606756) that are related to plasma vitamin B-12, with offspring IQ. Observationally, there was a positive association between maternal vitamin B-12 intake and child’s IQ that was markedly attenuated after adjustment for potential confounders (mean difference in offspring IQ score per doubling of maternal B-12 intake, before adjustment: 2.0 (95% CI 1.3, 2.8); after adjustment: 0.7 (95% CI −0.04, 1.4)). Maternal FUT2 was weakly associated with offspring IQ: mean difference in IQ per allele was 0.9 (95% CI 0.1, 1.6). The expected effect of maternal vitamin B-12 on offspring IQ, given the relationships between SNPs and vitamin B-12, and SNPs and IQ was consistent with the observational result. Our findings suggest that maternal vitamin B-12 may not have an important effect on offspring cognitive ability. However, further examination of this issue is warranted.     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

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.     

Genetic Variation in Vitamin B-12 Content of Bovine Milk and Its Association with SNP along the Bovine Genome

Vitamin B-12 (also called cobalamin) is essential for human health and current intake levels of vitamin B-12 are considered to be too low. Natural enrichment of the vitamin B-12 content in milk, an important dietary source of vitamin B-12, may help to increase vitamin B-12 intake. Natural enrichment of the milk vitamin B-12 content could be achieved through genetic selection, provided there is genetic variation between cows with respect to the vitamin B-12 content in their milk. A substantial amount of genetic variation in vitamin B-12 content was detected among raw milk samples of 544 first-lactation Dutch Holstein Friesian cows. The presence of genetic variation between animals in vitamin B-12 content in milk indicates that the genotype of the cow affects the amount of vitamin B-12 that ends up in her milk and, consequently, that the average milk vitamin B-12 content of the cow population can be increased by genetic selection. A genome-wide association study revealed significant association between 68 SNP and vitamin B-12 content in raw milk of 487 first-lactation Dutch Holstein Friesian cows. This knowledge facilitates genetic selection for milk vitamin B-12 content. It also contributes to the understanding of the biological mechanism responsible for the observed genetic variation in vitamin B-12 content in milk. None of the 68 significantly associated SNP were in or near known candidate genes involved in transport of vitamin B-12 through the gastrointestinal tract, uptake by ileum epithelial cells, export from ileal cells, transport through the blood, uptake from the blood, intracellular processing, or reabsorption by the kidneys. Probably, associations relate to genes involved in alternative pathways of well-studied processes or to genes involved in less well-studied processes such as ruminal production of vitamin B-12 or secretion of vitamin B-12 by the mammary gland.     

Recovery from vitamin B-12-induced unbalanced growth. The shortened cell cycle and the deoxyribonucleoside triphosphate pools.

The deoxyribonucleoside triphosphate pools are undetectable in vitamin B-12-deficient cells of Euglena gracillis, but appear rapidly after the replenishment with the vitamin. They reach a maximum size that is about 6 times that of normal exponentially growing cells, but decrease to almost zero as the cells divide. The pools expand again during the post-replenishment shortened cell cycle. However, the expansion takes place during rather than before the resumption of DNA synthesis. The maximum sizes reached are still larger than in normal cells. By using the protein-synthesis inhibitor cycloheximide and determining the pool size, we found that vitamin-deficient cells apparently accumulate a large amount of ribonucleoside triphosphate reductase apoenzyme, which lacks the vitamin B12 coenzyme. We showed that the production of the deoxyribonucleoside triphosphates is not closely coupled to DNA synthesis under our experimental conditions, and that the concentration of the deoxyribonucleoside triphosphate pools per unit of DNA synthesized is almost constant for all stages of growth examined.     

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.     

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37°C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6±0.2 g mannitol, 61.9±0.1 g lactic acid, and 40.4±0.3 g acetic acid from 300 g fructose per liter in 67 h.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

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.     

Application of vitamin B(12)-targeting site on Lactobacillus helveticus B-1 to vitamin B(12) assay by chemiluminescence method

Lactobacillus helveticus B-1 is assumed to have a vitamin B(12)-targeting (or B(12)-binding) site on the cells, since the binding reaction of vitamin B(12) with L. helveticus B-1 cells proceeded instantly and quantitatively. This reaction is specific to complete B(12) compounds, cobalamins, and can be used for a vitamin B(12) assay method by chemiluminescence. The calibration graph was linear from 0.1 to 10.0 ng/mL. The B(12) contents in oyster and sardine were 75.9 and 39.4 microg/100g, respectively. These values were very close to those obtained using a chemilumi-ADVIA Centaur immunoassay system with intrinsic factor and to those obtained by microbiological assays.     

Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri

Summary Production of vitamin B-12 compounds from methanol was carried out by Methanosarcina barkeri Fusaro, an anaerobic methanogen. The methanogen released about 40% to 70% of corrinoids irrespective of the culture medium used. The use of cysteine instead of Na₂S as the sole sulphur source for cell growth led to an increase in the cobalt chloride concentration in the culture medium up to 16 times the normal (0.6 mg·l^-1) without medium precipitation. This in turn resulted in an intracellular vitamin B-12 content of 5.6 mg·g dry cell^-1, the rest being discharged into the culture supernatant; this was 87 mg·l^-1, 73% of the total corrinoids after 20 repeated intermittently fed cultures and the final cell concentration was 5.8 g dry cell·l^-1. Taking advantage of this, continuous production of extracellular vitamin B-12 compounds was attempted with a fixed-bed bioreactor (carrier: diatomaceous clay). At a steady state operation at space velocity of 9 to 11 day^-1, the concentration of the discharged corrinoid was 6.8 to 7.9 mg·l^-1, having a vitamin B-12 activity of about 4 mg·l^-1. Total cell mass retained in the reactor was 39.6 g dry cell l-reactor^-1. Identification of the corrinoids revealed that 19% of the total corrinoids was comprised of the vitamin B-12 Factor III (5-hydroxybenzimidazolyl cobamide) and the remainder were mainly the base-free vitamin B-12 Factor B (cobinamide and its derivatives).     

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.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus grown on different grains

Aflatoxin production by Aspergillus parasiticus on corn, soybean, and cottonseed in the absence or presence of added sodium phytate was examined. No variation in aflatoxin concentrations was found in raw, chemically sterilized, or autoclaved soybeans whereas a five-fold reduction in total aflatoxins was found in cottonseed after addition of 330 g sodium phytate to 10 g of autoclaved material. However, phytate did not affect aflatoxin production on non-sterile cottonseeds, although in corn a slight inhibition was found. Extraction of raw soybeans with hexane allowed production of 20-fold more aflatoxins, but levels were still lower than those found on rice or corn. Part of this relative inhibition in soybeans may arise from a heat-unstable, polar solvent-soluble, dialyzable factor present in soybeans. Our results support the conclusion that phytate is not the factor in soy responsible for its relative resistance to aflatoxin formation.