Masanori Watanabe

Purification and Identification of Antihypertensive Peptides from Fermented Buckwheat Sprouts

Buckwheat (Fagopyrum esculentum) is rich in antihypertensive compounds. This study investigated the effect of lactic-fermented buckwheat sprouts (neo-FBS) on level, identification, and potency of blood pressure-lowering (BPL) compounds. A single oral dose of 1.0 mg/kg body weight buckwheat sprouts (BS) in spontaneously hypertensive rats did not show significant BPL activity, whereas neo-FBS significantly decreased blood pressure. HPLC of neo-FBS identified two peaks absent in the profile of BS. The peak exhibiting potent BPL activity was fractionated, and six peptides (DVWY, FDART, FQ, VAE, VVG, and WTFR) and tyrosine were identified by LC-MS/MS and Edman degradation. Single oral dose administration of the peptides revealed significant BPL effect of all the peptides, with the most potent being DVWY, FQ, and VVG. DVWY, VAE, and WTFR are novel. This study demonstrates that lactic fermentation of BS produces new, highly potent antihypertensive peptides and increases active compounds GABA and tyrosine already present in BS.

Fermentative l-(+)-lactic acid production from non-sterilized rice washing drainage containing rice bran by a newly isolated lactic acid bacteria without any additions of nutrients

A newly isolated lactic acid producing bacterium Lactobacillus rhamnosus M-23, from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. The maximum lactate yield attained was 59 g/l with a productivity of 1.23 g/l/h and a product optical purity of 95% corresponding to a conversion of 0.85 g of lactic acid per gram of sugar equivalent.

Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: a new approach to estimating ACE-inhibitory activity.

Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional in vitro, in vivo, and a new ex vivo ACE inhibitory assays. Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBPs bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.

Characterization of the native form and the carboxy‐terminally truncated halotolerant form of α‐amylases from Bacillus subtilis strain FP‐133

Two amylases, amylase I and amylase II from Bacillus subtilis strain FP‐133, were purified to homogeneity and characterized. Their stabilities toward temperature, pH, and organic solvents, and their substrate specificities toward polysaccharides and oligosaccharides were similar. Under moderately high salt conditions, both amylases were more stable than commercial B. licheniformis amylase, and amylase I retained higher amylase activity than amylase II. The N‐terminal amino acid sequence, genomic southern blot analysis, and MALDI‐TOFF‐MS analysis indicated that the halotolerant amylase I was produced by limited carboxy‐terminal truncation of the amylase II peptide. The deduced amino acid sequence of amylase II was >95% identical to that of previously reported B. subtilis α‐amylases, but their carboxy‐terminal truncation points differed. Three recombinant amylases — full‐length amylase corresponding to amylase II, an artificially truncated amylase corresponding to amylase I, and an amylase with a larger artificial C‐terminal truncation — were expressed in B. subtilis. The artificially truncated recombinant amylases had the same high amylase activity as amylase I under moderately high salt conditions. Sequence comparisons indicated that an increased ratio of Asp/Glu residues in the enzyme may be one factor responsible for increasing halotolerance.

Bioconversion of biodiesel-derived crude glycerol into lipids and carotenoids by an oleaginous red yeast Sporidiobolus pararoseus KM281507 in an airlift bioreactor

Here we tested the bioconversion of biodiesel-derived crude glycerol by the oleaginous red yeast Sporidiobolus pararoseus KM281507 in two bioreactors types (stirred-tank and airlift). High production yields (biomass, 10.62xa0±xa00.21xa0g/L; lipids, 3.26xa0±xa00.13xa0g/L; β-carotene, 30.64xa0±xa00.05xa0mg/L; total carotenoids, 46.59xa0±xa00.07xa0mg/L) were achieved in a 3.0xa0L airlift bioreactor under uncontrolled pH regimes (initial pH 5.63). Under optimized conditions (6.0 vvm aeration rate; 60xa0±xa05% constant dissolved oxygen [DO] maintained by flushing pure oxygen [O2] into the vessel; 10,000 Lux light irradiation) volumetric production in the airlift bioreactor was further increased (biomass, 19.30xa0±xa01.07xa0g/L; lipids, 6.61xa0±xa00.04xa0g/L, β-carotene, 109.75xa0±xa00.21xa0mg/L; total carotenoids 151.00xa0±xa02.71xa0mg/L). Production was also recorded at a S.xa0pararoseus KM281507 growth rate of 0.16xa0±xa00.00xa0h-1 (lipids, 0.94xa0±xa00.04xa0g/L/d; β-carotene, 15.68xa0±xa00.40xa0mg/L/d; total carotenoids, 21.56xa0±xa00.20xa0mg/L/d). Lipids from S.xa0pararoseus KM281507 had a high unsaturated fatty acid content, with oleic acid (C18:1) accounting for 80% of all fatty acids. This high oleic acid content makes S.xa0pararoseus KM281507 well-suited as a third generation biodiesel feedstock. Our findings show that airlift bioreactors are suitable for bioconversion of crude glycerol into lipids and carotenoids using S.xa0pararoseus KM281507. This approach is advantageous because of its ease of operation, cost efficiency, and low energy consumption.

Simultaneous recovery and purification of rice protein and phosphorus compounds from full-fat and defatted rice bran with organic solvent-free process

We studied a process that enables simultaneous recovery of protein and phosphorus compounds from rice bran. Phosphorus substances in full-fat and defatted rice bran such as phytic acid and inorganic ions were solubilized under acidic conditions in the first step. After that, inorganic and/or organic phosphate salts were recovered in insoluble form under weak alkaline conditions. Furthermore, protein fractions obtained after phosphorus compounds had been removed were solubilized under alkaline conditions. After solubilization, protein fractions with high content were recovered by isoelectric precipitation (IP) followed by electrolyzed water treatment (EWT). The highest protein content (52.3xa0w/w%) was attained when machine defatted rice bran was treated through the process. Energy-dispersive X-ray spectroscopy (EDX) and inductively coupled plasma atomic emission spectrometry (ICP-AES) analyses demonstrated efficient desalting from the protein fractions by EWT and higher phosphorus contents (15.1-16.4xa0w/w% P) in the phosphorus fractions compared with commercial phosphate rock. In addition, no heavy metal ions in either protein or phosphorus fractions were detected. These results suggest that the newly developed process is suitable for practical recovery of highly concentrated protein and phosphorus compounds from rice bran without enzymes or chemicals such as organic solvents, buffering agents, and surfactants.

Direct bioconversion of rice residue from canteen waste into lipids by new amylolytic oleaginous yeast Sporidiobolus pararoseus KX709872

ABSTRACT The new amylolytic oleaginous red yeast, Sporidiobolus pararoseus KX709872, produced both α-amylase (540u2009±u20090.09u2009mU/mL) and amyloglucosidase (23u2009±u20090.00u2009mU/mL) and showed good ability to directly convert rice residue from canteen waste to biomass and lipids. Effects of medium composition and cultivation conditions on growth and lipid accumulation for strain KX709872 were investigated under shaking flask and upscaling levels. At Cu200a:u200aN ratio of 25u200a:u200a1, pH 5.45, 22.36°C, and 199.40u2009rpm for 7 days, volumetric production of biomass and lipids, lipid content, and lipid productivity reached 17.69u2009±u20090.44, 8.35u2009±u20090.19u2009g/L, 49.48u2009±u20090.41% (w/w), and 1.67u2009±u20090.11u2009g/L/day, respectively. Production of lipids was also implemented in 5.0-L stirred tank bioreactor with 2.5u2009L of optimized medium at 300u2009rpm and 3.0 vvm for 5 days. Volumetric production of biomass and lipids, lipid content, and lipid productivity were 16.33u2009±u20090.49, 8.75u2009±u20090.13u2009g/L, 56.61u2009±u20090.04% (w/w), and 2.19u2009±u20090.03u2009g/L/day, respectively. Meanwhile, the fatty acids of lipids from strain KX709872 had high oleic acid content (60−62%) which was similar to those of vegetable oils, indicating that these lipids are promising as an alternative biodiesel feedstock. Moreover, the biodiesel derived from lipids of strain KX709872 had properties satisfying the criteria of ASTM D6751 and EN 14214 standards.

Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis

ObjectivesA bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential.ResultsHalotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions.ConclusionsChanging the pI of a bacterial serine protease may be an effective strategy to improve the enzyme’s halotolerance.

An integrated process for xylooligosaccharide and bioethanol production from corncob

An integrated process for xylooligosaccharides (XOs) and bioethanol production from corncob was investigated. XOs were produced by a consecutive process of KOH treatment and hydrolysis by an in-house thermostable endo-xylanase from Streptomyces thermovulgaris. XO yields of 0.15u202fg/gKOH-treated corncob (22.13u202fg/L) and 0.52u202fg/graw corncob of cellulose-rich corncob (CRC) were obtained. After 96u202fh of enzymatic hydrolysis, CRC hydrolysate contained 62.16, 51.21, 10.03 and 0.92u202fg/L of total sugar, glucose, xylose and arabinose, respectively. Bioethanol production by separate hydrolysis and fermentation (SHF) using CRC hydrolysate, and by simultaneous saccharification and fermentation (SSF) using CRC was studied at 40u202f°C for thermotolerant Candida glabrata. SHF showed an ethanol yield of 0.28u202fg/gCRC (21.92u202fg/L) and ethanol productivity of 0.304u202fg/L/h with 93% theoretical yield. Surprisingly, by SSF, those parameters were 0.27u202fg/gCRC (31.32u202fg/L), 0.33u202fg/L/h and 89%, respectively. This integrated process might be a new cost-effective approach for corncob valorization.

Extracellular protease derived from lactic acid bacteria stimulates the fermentative lactic acid production from the by-products of rice as a biomass refinery function

A lactic acid producing bacterium, Lactobacillus rhamnosus M-23, newly isolated from a rice washing drainage storage tank was found to produce l-(+)-lactic acid from a non-sterilized mixture of rice washing drainage and rice bran without any additions of nutrients under the simultaneous saccharification and fermentation (SSF) process. This strain has the ability to utilize the non-sterilized rice washing drainage and rice bran as a source of carbohydrate, saccharifying enzymes and nutrients for lactic acid production. Observation of extracellular protease activity in SSF culture broth showed that a higher protease activity was present in strain M-23 than in other isolated lactic acid producing bacteria (LABs). To investigate the structural changes of solid particles of rice washing drainage throughout LAB cultivation, scanning electron microscopic (SEM) observation and Fourier transform infrared-spectroscopy (FT-IR) analysis were performed. The results of the SEM observation showed that the surface material could be removed from solid particles of rice washing drainage treated by culture broth (supernatant) of strain M-23, thus exposing the crystal structure of the starch particle surface. The results of the FT-IR analysis revealed that the specific transmittance decrease of the CC and CO stretching and OH group of the solid particles of the rice washing drainage were highly correlated with the produced lactic acid concentration and extracellular protease activity, respectively. These results demonstrate the high lactic acid producing ability of strain M-23 from a non-sterilized mixture of rice washing drainage and rice bran under the SSF condition due to the removal of proteinaceous material and exposure of the starch particle surface by extracellular protease.