Fujiharu Yanagida

Anti-hypertensive substances in fermented soybean, natto.

Natto is a traditional Japenese fermented food made by fermenting boiled soy beans withBacillus natto. Its contents of inhibitors against the angiotensin converting enzyme (ACE, EC3.4.15.1) were investigated. Relatively strong inhibitory activity (IC50:0.4 mg/ml, 11.8 inhibition units/g natto) was detected in natto extracts and the inhibitory activity observed in the viscous fraction was more potent than in the bean extract. Two groups of inhibitors in the viscous material, high and low molecular weight inhibitors, were resolved by dialysis test. The inhibitor of high molecular weight was a protein with low IC50 value (0.12 mg/ml). The two types of low molecular weight inhibitors were detected in ethanol extracts (IC50: 0.53 mg/ml and 0.95 mg/ml) and they were found to be stable over a wide range of pH and temperature up to 100°C. They were different in the mode of ACE inhibition. One is competitive, and the other noncompetitive against the hydrolysis of Bz-Gly-His-Leu by ACE.

Cloning and characterization of groESL operon in Acetobacter aceti.

Abstract The groESL operon of Acetobacter aceti was cloned and sequenced. We observed that GroES and GroEL of A. aceti had high amino acid sequence homologies to GroES and GroEL of Escherichia coli and Bacillus subtilis . The upstream region of the groESL operon contained the heatshock promoter, which was previously reported in α-purple proteobacteria, and the highly conserved inverted repeat sequence. Phylogenetic analysis revealed that the A. aceti GroES and GroEL are very closely related to those of other α-purple proteobacteria. Transcription of this operon in A. aceti was induced by heat shock as well as by exposure to ethanol and acetic acid, which are present during fermentation of acetic acid. A. aceti that overexpressed the groESL was more resistant than the control strain to Stressors such as heat, ethanol, or acetic acid, indicating that GroES and GroEL are closely associated with the characteristic nature of Acetobacter and play an important role in acetic acid fermentation.

A Gene Encoding Phosphatidylethanolamine N-Methyltransferase from Acetobacter aceti and Some Properties of its Disruptant

Phosphatidylcholine (PC) is a major component of membranes not only in eukaryotes, but also in several bacteria, including Acetobacter. To identify the PC biosynthetic pathway and its role in Acetobacter sp., we have studied Acetobacter aceti IFO3283, which is characterized by high ethanol oxidizing ability and high resistance to acetic acid. The pmt gene of A. aceti, encoding phosphatidylethanolamine N-methyltransferase (Pmt), which catalyzes methylation of phosphatidylethanolamine (PE) to PC, has been cloned and sequenced. One recombinant plasmid that complemented the PC biosynthesis was isolated from a gene library of the genomic DNA of A. aceti. The pmt gene encodes a polypeptide with molecular mass of either 25125, 26216, or 29052 for an about 27-kDa protein. The sequence of this gene showed significant similarity (44.3% identity in the similar sequence region) with the Rhodobacter sphaeroides pmtA gene which is involved in PE N-methylation. When the pmt gene was expressed in E. coli, which lacks PC, the Pmt activity and PC formation were clearly demonstrated. A. aceti strain harboring an interrupted pmt allele, pmt::Km, was constructed. The pmt disruption was confirmed by loss of Pmt and PC, and by Southern blot analyses. The null pmt mutant contained no PC, but tenfold more PE and twofold more phosphatidylglycerol (PG). The pmt disruptant did not show any dramatic effects on growth in basal medium supplemented with ethanol, but the disruption caused slow growth in basal medium supplemented with acetate. These results suggest that the lack of PC in the A. aceti membrane may be compensated by the increases of PE and PG by an unknown mechanism, and PC in A. aceti membrane is related to its acetic acid tolerance.

Purification and characterization of intracellular esterases related to ethylacetate formation in Acetobacter pasteurianus

Acetobacter sp. produce ethylacetate during acetic acid fermentation. We found that high ethylacetate-producing strains contained more intracellular esterase than low ethylacetate-producing strains. Acetobacter pasteurianus N-23, a high ethylacetate-producing strain, formed two major intracellular esterases (esterase-1 and esterase-2) that were regulated by ethanol. These two esterases were purified to homogeneity by means of hydrophobic interaction, ion exchange, gel filtration, and hydroxyapatite chromatographies. The two esterases hydrolyzed various esters, but only esterase-1 showed ethylacetate synthesis activity at pH 3. The molecular weights of esterase-1 and esterase-2 were 72 kDa and 44 kDa, respectively, as determined by gel filtration, and 38 kDa and 42 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Esterase-1 was inhibited by cysteine enzyme inhibitors such as diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. These esterases may play some role in the production of ethylacetate by Acetobacter sp. during vinegar fermentation.

Role of intracellular esterases in the production of esters by Acetobacter pasteurianus

Esters are the major flavor compounds produced by Acetobacter sp. during vinegar production. The two genes encoding the esterases in the bacteria were disrupted, and the effects of the disruptions studied. When cultured in the presence of ethanol, the est1 gene-disrupted mutant (DE1K) did not produce any ethyl acetate or isoamyl acetate. However, the disruption of est2 did not affect the ester production. Ethyl acetate production by N-23 (pME122P) and DE1K (pME122P), which contain est1, was 1.7-fold higher than that by the wild type, N-23. On analyzing the relationship between ethyl acetate production and the extracellular ethanol and acetic acid concentrations, we found that the highest amount of ethyl acetate was produced when the molar ratio of ethanol and acetic acid was 1:1. These results indicate that the ester production by Acetobacter sp. is mostly catalyzed by the intracellular esterase, esterase-1, with ethanol and acetic acid used as the substrates.

Anti-Hypertensive Substances in Viscous Material of Fermented Soybean (NATTO)

The angiotensin converting enzyme (ACE) inhibitor in the viscous material of natto was investigated. The relatively strong inhibitory activities were detected both in the water soluble and the water insoluble but ethanol soluble fraction in the viscous material. The former contained the inhibitory substance with the high molecular weight and considered to be a proteineous substance. The latter had the inhibitor of the small molecular weight (ca 600), which was stable over the wide range of pH and thermostable. It showed competitive inhibition against the hydrolysis of Bz-Gly-His-Leu by ACE.

A permease exhibiting a dual role for lysine and biotin uptake in Saccharomyces cerevisiae

Among Saccharomyces cerevisiae strains each defective in one of 11 amino acid permeases, a lysine permease disruptant (DK) exhibited about 2-fold reductions in maximum cell density and fermentation ability compared to the parent in a synthetic medium. These unusual properties of DK were found to result from the requirement of biotin for growth, in contrast to the parent whose growth was not dependent on external biotin. The rate of 14C-labeled biotin uptake and the intracellular free biotin content of DK were 2-2.5 fold lower than in the parent. We suggest that lysine permease in S. cerevisiae has the ability to transport both lysine and biotin.

The Chemical Constituents and Sensory Test of Commercial Cookeed Curry

市販調理済カレー25点の化学的成分分析に主成分分析を適用した.また,14点のカレーについて香味の官能試験を行ない,カレーの特徴を解析した.(1) 化学的成分分析においては,各成分の平均含量は,水分72.81%,タンパク質4.38%,脂肪8.84%,灰分1.90%,繊維1.64%,全糖9.63%,直糖0.75%,ショ糖1.88%,デンプン6.30%,過酸化物価2.77,チオバルビツール酸価0.61であった。各成分共,含量のバラツキが多かった.(2) 化学的成分分析の主成分分析では,第1主成分は炭水化物と水分の成分として,第2主成分は油脂の品質表示と甘味を表わす成分であった.(3) 香味の官能試験において,香りについては香辛料や肉の香りが強くない方が良く,色については明るい方が良く,褐色の強いものが悪かった.味については旨味があり,甘味と辛味のバランスがとれているものが良かった.評点法による香味の官能試験より,5つの特徴あるグループに大別された.

A study on Curry. Part I. The chemical constituents and sensory test of commercial cookeed Curry.

市販調理済カレー25点の化学的成分分析に主成分分析を適用した.また,14点のカレーについて香味の官能試験を行ない,カレーの特徴を解析した.(1) 化学的成分分析においては,各成分の平均含量は,水分72.81%,タンパク質4.38%,脂肪8.84%,灰分1.90%,繊維1.64%,全糖9.63%,直糖0.75%,ショ糖1.88%,デンプン6.30%,過酸化物価2.77,チオバルビツール酸価0.61であった。各成分共,含量のバラツキが多かった.(2) 化学的成分分析の主成分分析では,第1主成分は炭水化物と水分の成分として,第2主成分は油脂の品質表示と甘味を表わす成分であった.(3) 香味の官能試験において,香りについては香辛料や肉の香りが強くない方が良く,色については明るい方が良く,褐色の強いものが悪かった.味については旨味があり,甘味と辛味のバランスがとれているものが良かった.評点法による香味の官能試験より,5つの特徴あるグループに大別された.

Studies on the acetic acid bacteria and their utilization (part 9)

食酢醸造中の遊離アミノ酸について検討した。米酢の場合を除いて各種の食酢では2～3増加するアミノ酸も見られるが, 殆んどのアミノ酸は減少した。特に減少の著しいアミノ酸はGlu, Asp, Proと Alaであった。全アミノ酸量は仕込時の量に較べ38～60%と減少した。米酢の場合には本小仕込実験では米粒を含んだ酒を使用したので全アミノ酸量は1.6～2.6倍に増加した。これは含まれていた蛋白質の分解によるものと考えられる。