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.     

Biosynthesis of vitamin B-12 Part I. Role of the ribosomal proteins in vitamin B-12 biosynthesis

1. 70 S ribosomes isolated from strains of Escherichia coli 113-3, K₁₂ and B take part in vitamin B-12 biosynthesis from AdoCbi-GDP, NAD and dimethylbenzimidazole in the presence of enzymes of the cytosol fraction. 2. 70 S ribosomes from E. coli 113-3 bind Ado[⁵⁸Co]Cbi-GDP. This reaction is independent of fusidic acid. 3. Proteins from 5 S RNA complex as well as L2 protein isolated from E. coli 113-3 ribosomes catalyze vitamin B-12 biosynthesis. The main catalytic function in this reaction is performed by protein L18.4. Vitamin B-12 biosynthesis proceeding in the presence of isolated ribosomal proteins is inhibited by fusidic acid, chloramphenicol and vernamycin but not by erythromycin. 5. Vitamin B-12 synthesized in the presence of isolated ribosomal proteins is biologically active.     

DPPH radical-scavenging compounds from dou-chi, a soybean fermented food

Dou-chi, a traditional soybean food fermented with Aspergillus sp., is usually used as a seasoning in Chinese food, and has also been used as a folk medicine in China and Taiwan. As 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengers, four phenol compounds, one isoflavanone, eight isoflavones and one 4-pyrone have been isolated from dou-chi. Among these fourteen compounds, 3'-hydroxydaidzein, dihydrodaidzein and a 4-pyrone compound have not yet been isolated from soybean miso. The structure of the novel 4-pyrone compound, 3-((E)-2-carboxyethenyl)-5-(4-hydroxyphenyl)-4-pyrone-2-carboxylic acid was elucidated by using the same compound as that obtained from the biotransformation of daidzein. 3'-Hydroxydaidzein showed as high DPPH radical-scavenging activity as that of alpha-tocopherol, and 6-hydroxydaidzein had mushroom tyrosinase inhibitory activity with an IC(50) value of 10 muM. The order of estrogenic activity is as follows: genistein > daidzein > 3'-hydroxydaidzein > 8-hydroxygenistein, using a green fluorescent protein expression system. Furthermore, the contents of isoflavones in the fermentation process of dou-chi were measured.     

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).     

Production of corrinoids including vitamin B-12 byMethanosarcina barkeri growing on methanol

Summary A preliminary attempt was made for producing vitamin B-12 byMethanosarcina barkeri strain Fusaro in a fed-batch culture with a methanol minimum medium. After 11 days, total methanol consumption, cell density and corrinoid concentration were 145 g/l, 8.5 g(dry cell weight)/l, and 135 mg/l (73% in supernatant) respectively. Electrophoretic separation revealed that 33% of the total corrinoids was B-12 Factor III (5-hydroxybenzimidazolylcobamide) and the remaining corrinoids were cobinamide (Factor B) and its derivatives.     

Carnitine metabolism in the vitamin B-12-deficient rat.

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.     

Deoxyribonucleoside triphosphate pools in vitamin B-12-deficient Euglenagracilis.

The size of the deoxyribonucleoside triphosphate pools of vitamin B-12-deficient cells of Euglena gracilis, and of vitamin B-12-deficient cells repleted with the vitamin, were measured. We found that the pools were very small, if they exist at all, in deficient cells but expand rapidly with the addition of the vitamin. The sizes of the pools decrease when DNA synthesis is completed, and are very small when the cells begin to divide.     

A novel form of vitamin B-12 and its derivatives.

A new isomeric form of cobalamins is reported. The conversion of cobalamin to cobalamin (the new form) is achieved by substituting the benzimidazole base by a less bulky group like H2O or CN- and modest thermal treatment. The back conversion of adenosylcobalamin to the corresponding regular form occurs in the "base-off" form at room temperature. It seems that the corrin ring becomes quite flexible in the "base-off" form and the freer axial movement of the cobalt atom flips the corrin ring into a different conformation. The change in conformation is borne out by subtle changes in the proton magnetic resonances on the corrin ring and the base, and very marked variation in the emission M?ssbauer spectra. The latter is indicative of appreciable changes in the spatial conformation in the immediate vicinity of the central metal atom. The ultraviolet-visible and infrared spectra of cobalamin are indistinguishable from those of its corresponding regular form. The new conformational isomeric species is present as an impurity in all commercially available cobalamins (including pharmaceutical preparations). It raises the question whether the cobalamins' constitute the real biologically active anti-anemic factor in humans.     

Lysinibacillus chungkukjangi sp. nov., isolated from Chungkukjang, Korean fermented soybean food

One bacterial strain 2RL3-2^T was isolated from Chungkukjang, a traditional Korean fermented food made from soybeans, and determined to be a Gram-positive, aerobic, spore-forming rod. Growth of the novel strain was optimal at 30°C and pH 7.0. The 16S rRNA gene of strain 2RL3-2^T showed the highest level of sequence similarity to Lysinibacillus sinduriensis BLB-1^T (99.0%), Lysinibacillus massiliensis 4400831^T (97.1%), Lysinibacillus xylanilyticus XDB9^T (97.0%), and Lysinibacillus odysseyi 34hs-1^T (96.8%). Phylogenetic analysis showed that strain 2RL3-2^T formed a robust cluster with L. sinduriensis BLB-1^T, L. massiliensis 4400831^T, and L. odyssey 34hs-1^T. The major fatty acids were anteiso-C15:0 (47.3%), iso-C_16:0 (16.3%), and anteiso-C_17:0 (11.3%), and the only menaquinone was MK-7. Diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine were the major polar lipids, along with an unknown phospholipid and two unknown lipids. The peptidoglycan type was A4α, with an interpeptide bridge of l-Lys-d-Asp. DNA-DNA hybridization values between strain 2RL3-2^T and closely related Lysinibacillus species were below 43±4%. Therefore, based on phenotypic, chemotaxonomic, and phylogenetic characteristics, it was determined that strain 2RL3-2^T represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus chungkukjangi sp. nov. is proposed. The type strain is 2RL3-2^T (=KACC 16626^T =NBRC 108948^T).     

The interaction between vitamin B-12 and micelles in aqueous solution.

The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.     

Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean food in northern Thailand

AIMS: To clarify the diversity of Bacillus subtilis strains in Thua nao that produce high concentrations of products useful in food manufacturing and in health-promoting compounds. METHOD AND RESULTS: Production of amylase, protease, subtilisin NAT (nattokinase), and gamma-polyglutamic acid (PGA) by the Bacillus subtilis strains in Thua nao was measured. Productivity of protease NAT by these strains tended to be higher than by Japanese commercial natto-producing strains. Molecular diversity of isolated strains was analysed via randomly amplified polymorphic DNA-PCR fingerprinting. The strains were divided into 19 types, including a type with the same pattern as a Japanese natto-producing strain. CONCLUSION: B. subtilis strains that could be a resource for effective production of protease, amylase, subtilisin NAT, or PGA were evident in Thua nao produced in various regions in northern Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study clearly demonstrated the value of Thua nao as a potential resource of food-processing enzymes and health-promoting compounds.     

传统大豆发酵食品中大豆低聚糖的生理功能研究

大豆低聚糖是大豆中可溶性碳水化合物的总称,在大豆和传统大豆发酵食品中含量丰富。近年研究发现大豆低聚糖在改善肠胃道功能、抗肿瘤、调节血脂等方面具有着重要的生理活性,因此引起了国内外研究人员的广泛关注。大豆低聚糖的研究,不仅能为功能性大豆制品的开发提供理论基础,也能为人们合理的膳食提供参考。本文综述了近年来国内外对大豆低聚糖生理保健机能的研究进展。     

Selection of starter cultures for the production of kinema,a fermented soybean food of the Himalaya

Kinema, a traditional fermented soybean food, serves as a low-cost source of protein in the diet of the people of the Himalaya. The traditional method of kinema preparation results in a product with inconsistent quality. Forty five strains of spore-forming bacteria were isolated from nine samples of kinema collected from markets in the Darjeeling hills and Sikkim in India. Of these, 10 strains, identified as Bacillus subtilis (Ehrenberg) Cohn, were selected as possible starter cultures on the basis of enzyme activities and the production of slimy material. Protease activity (U/ml), -amylase activity (U/ml) and relative viscosity of the selected strains were 6.5 to 81.5, 0.1 to 9.3, and 1.1 to 20.1, respectively. Kinema produced by these strains showed nitrogen contents (expressed as a percentage of the total nitrogen content) of: water-soluble N, 48.4 to 76.5; TCA-soluble N, 16.0 to 27.6; formal N, 5.0 to 12.5; ammonia-N, 4.4 to 7.8%. Reducing sugar was 1.1 to 2.0% of the wet weight and relative viscosity was from 2.0 to 35.2. Correlation matrices of the biochemical parameters and sensory attributes of the kinema produced by these B. subtilis strains were statistically analysed. Strains KK-2:B10 and GK-2:B10 of B. subtilis were the best starter cultures for improved kinema production.     

A novel form of vitamin B-12 and its derivatives

A new isomeric form of cobalamins is reported. The conversion of cobalamin to cobalamin′ (the new form) is achieved by substituting the benzimidazole base by a less bulky group like H₂O of CN^? and modest thermal treatment. The back conversion of adenosylcobalamin′ to the corresponding regular form occurs in the ‘base-off’ form at room temperature. It seems that the corrin ring becomes quite flexible in the ‘base-off’ form and the freer axial movement of the cobalt atom flips the corrin ring into a different conformation. The change in conformation is borne out by subtle changes in the proton magnetic resonances on the corrin ring and the base, and very marked variation in the emission Mössbauer spectra. The latter is indicative of appreciable changes in the spatial conformation in the immediate vicinity of the central metal atom.The ultraviolet-visible and infrared spectra of a cobalamin′ are indistinguishable from those of its corresponding regular form.The new conformational isomeric species is present as an impurity in all commercially available cobalamins (including pharmaceutical preparations). It raises the question whether the cobalamins′ constitute the real biologically active anti-anemic factor in humans     

The interaction between vitamin B-12 and micelles in aqueous solution

The apparent pK for benzimidazole displacement of a number of cobalamins is markedly affected by the presence of sodium lauryl sulfate micelles. However, micelles of cetyltrimethylammonium bromide or Triton X have little or no effect on the pK. By measuring the apparent pK as a function of sodium lauryl sulfate concentration, the association constants between the micelles and both base on and base off methylcobalamin were calculated. This calculation indicates that the base off form is strongly associated with the micelle while the base on form is not.     

Release of vitamin B-12 in vitro from intrinsic factor-vitamin B-12 complex Purification and characterization of a releasing factor from rat ileal brush border

Vitamin B-12 is released from the purified gastric intrinsic factor-[⁵⁷Co]vitamin B-12 (intrinsic factor- [⁵⁷Co]vitamin B-12) complex, when incubated with rat intestinal mucosa. Maximum specific activity for splitting the complex is localized in ileal brush border. Release of [⁵⁷Co]vitamin B-12 is not due to its mere exchange during incubation with endogenous non-radioactive vitamin B-12. The splitting process has specific requirement for Ca²⁺ and ATP and it is thermolabile, time- as well as temperature-dependent. It is also inactivated by the presence of p-chloromercuribenzoate. Further, the vitamin B-12-releasing factor has been isolated from solubilized brush border and is purified 70-fold by (NH₄)₂SO₄ precipitation, gel filtration and Con. A-Sepharose 4B affinity chromatography. In SDS-polyacrylamide gel electrophoresis, it is resolved into a single band of about 25 kDa, indicating its purity. The releasing factor exhibits maximum activity at pH 7.4; isoelectric focusing reveals only one major form with pI 7.52. With intrinsic factor-[⁵⁷Co]vitamin B-12-complex as the substrate, apparent K_m and V_max values obtained are 128.2·10⁻¹² M/1 and 117.6 pg·h⁻¹ 100 μg protein, respectively. Amino acid and carbohydrate analyses reveal the glycoprotein nature of the factor. Intrinsic factor-[⁵⁷Co]vitamin B-12 complex is not susceptible to unspecific proteolytic digestion/ Similarly, the releasing factor does not hydrolyse other proteins. Thus, the observed substrate-specificity of the releasing factor differentiates it from other known proteolytic enzymes of ileal brush borders.     

Angiotensin I-converting enzyme inhibitory peptides isolated from tofuyo fermented soybean food

Angiotensin I-converting enzyme (ACE) inhibitory activity was observed in a tofuyo (fermented soybean food) extract with an IC(50) value of 1.77 mg/ml. Two ACE inhibitors were isolated to homogeneity from the extract by adsorption and gel filtration column chromatography, and by reverse-phase high-performance liquid chromatography (HPLC). The purified substances reacted with 2,4,6-trinitrobenzensulfonic acid sodium salt. The amino acid sequences of these inhibitors determined by Edman degradation were Ile-Phe-Leu (IC(50), 44.8 microM) and Trp-Leu (IC(50), 29.9 microM). The Ile-Phe-Leu sequence is found in the alpha- and beta-subunits of beta-conglycinin, while the Trp-Leu sequence is in the B-, B1A- and BX-subunits of glycinin from soybean. Both of the peptides are non-competitive inhibitors. The inhibitory activity of Trp-Leu was completely preserved after a treatment with pepsin, chymotrypsin or trypsin. Even after successive digestion by these gastrointestinal proteases, the activity remained at 29% of the original value.     

Uptake of a microbially-produced vitamin (B12) by soybean roots

Vitamin B12 (Cyanocobalamin) is one of the vitamins believed to be produced exclusively by microorganisms. Although soil is a rich source of vitamin B12, systematic study as to possible uptake of this vitamin by the plant roots is lacking. This study was undertaken to investigate, under water culture conditions, the uptake of [⁵⁷Co]-cyanocobalamin by soybean (Glycine max (L.) Merr.). In the range of 10 to 3200 mol L^–1, uptake of vitamin B12 was a linear function of the vitamin concentration in the nutrient solution. Depending on the vitamin concentration, 12 to 34% of the total absorbed vitamin was transported to the plant shoots, with proportionally more vitamin B12 transported at higher vitamin concentrations. Aeration of the rooting medium with nitrogen gas significantly increased the total uptake and the percentage of vitamin transported to the shoots. Addition of respiration inhibitor dinitrophenol to the nutrient solution did not affect the total uptake or the partitioning of the vitamin. Root temperature (5–30°C) did not affect the total uptake but significantly altered the partitioning of the vitamin between the roots and the shoots. Foliar-applied vitamin B12 was not translocated to any considerable degree to other plant parts, indicating that phloem transport does not contribute to the distribution of this vitamin within the plant. It is suggested that adding manure (which is rich in this vitamin) to the soil could increase soil and thus plant content of vitamin B12. This could be of importance in raising the intake of this vitamin by people living by choice or necessity on vegetarian diets who are usually threatened by vitamin B12 deficiency.