Katsumi Hashizume

Organ-Specific Transcription of Putative Flavonol Synthase Genes of Grapevine and Effects of Plant Hormones and Shading on Flavonol Biosynthesis in Grape Berry Skins

In order to investigate the control mechanism of flavonol biosynthesis of grapevine, we obtained five genomic sequences (FLS1 to FLS5) of putative flavonol synthase genes from Vitis vinifera cv. Cabernet Sauvignon. The mRNA of five FLSs accumulated in flower buds and flowers, while the mRNA of FLS2, FLS4, and FLS5 accumulated in small berry skins and then decreased toward veraison. At the ripening stage, the mRNA of only FLS4 and FLS5 accumulated again. This change in mRNA accumulation did not contradict the flavonol accumulation in the berry skins. Shading of the berries completely inhibited the increase in flavonol content and mRNA accumulation of FLS4, but did not affect the mRNA accumulation of FLS5. The effects of light and plant hormones on flavonol accumulation were different from those on anthocyanin accumulation. Thus flavonol biosynthesis appears to be under a different control system from that of anthocyanin biosynthesis.

Influence of Maceration Temperature in Red Wine Vinification on Extraction of Phenolics from Berry Skins and Seeds of Grape (Vitis vinifera)

The extraction of phenolics from berry skins and seeds of the grape, Vitis vinifera cv. Cabernet Sauvignon, during red wine maceration and the influence of different temperature conditions (cold soak and/or heating at the end of maceration) were examined. Phenolics contained mainly in berry skins, viz., anthocyanin, flavonol, and epigallocatechin units within proanthocyanidins, were extracted during the early stage of maceration, whereas those in seeds, viz., gallic acid, flavan-3-ol monomers, and epicatechin-gallate units within proanthocyanidins, were gradually extracted. In addition to their localization, the molecular size and composition of the proanthocyanidins possibly influenced the kinetics of their extraction. Cold soak reduced the extraction of phenolics from the seeds. Heating at the end of maceration decreased the concentration of proanthocyanidins. Thus, modification of the temperature condition during maceration affected the progress of the concentration of phenolics, resulting in an alteration of their make-up in the finished wine.

Structural characteristics, properties, and in vitro digestibility of rice

ABSTRACT Using rice samples derived from normal rice cultivars and endosperm starch mutant, we investigated key factors contributing to the enzyme digestibility of steamed rice grains. The chemical composition of polished rice grains, structural features of endosperm starch, and enzyme digestibility of steamed rice grains were examined. The protein content of polished rice grains was 4.6–9.1%, amylose content was 4–27%, the DPn of purified amylose was 900–1,600, the amylopectin short/long chain ratio was 1.2–5.9, and the enzyme digestibilities of steamed polished rice grains were 0.9–12.6 °Brix. Amylose content and RVA parameters (viscosity, breakdown, and setback) correlated significantly with enzyme digestibility of steamed rice grains. Multiple regression formulas were constructed to predict digestibility of steamed rice grain as a function of the molecular characteristics of the starch. When both amylose content and the short/long chain amylopectin ratio were used as predictor variables, they accounte...

Methoxypyrazine Content of Japanese Red Wines

Three 2-methoxy-3-alkylpyrazines (MPs) in Japanese red wine and grape samples were determined by GC-EIMS, using 2-methyl-3-n-propylpyrazine as an internal standard. MPs in the Cabernet Sauvignon red wines were derived not only from the pulp but also from other parts of the grape berries. All of the Cabernet Sauvignon red wines made annually from 1975 to 1994 contained 2-methoxy-3-isobutylpyrazine (isobutylMP), although those made from well-ripened grapes had a low isobutylMP level. It is suggested that the climatic conditions of September might effect the isobutylMP level of Cabernet Sauvignon grapes and red wines. The mean isobutylMP level of Cabernet Sauvignon, Cabernet Franc, and Merlot Japanese commercial red wines was significantly higher than the mean isobutylMP level of Muscat Bailey A and Zweigeltrebe.

N-Linked Oligosaccharides of Aspergillus awamori Feruloyl Esterase Are Important for Thermostability and Catalysis

A unique N-linked glycosylation motif (Asn79-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward α-naphthylbutyrate (C4), α-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased K m and decreased k cat for N79A, and to a considerably decreased k cat for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.

Structural and Retrogradation Properties of Rice Endosperm Starch Affect Enzyme Digestibility of Steamed Milled-Rice Grains Used in Sake Production

ABSTRACT Structural and physicochemical characteristics of endosperm starch from milled rice grains of seven Japanese cultivars used in sake production were examined. Amylose content was 15.2–20.2%, number-average degree of polymerization (DPn) of amylose was 900–1,400, and the ratio of short-to-long chain amylopectin was 2.7–3.5, respectively. The degree of retrogradation of purified starch stored for seven days at 4°C after gelatinization was 20–31%. The degree of retrogradation correlated negatively with the ratio of short-to-long chain amylopectin. The effect of holding time after steaming on enzyme digestibility and starch retrogradation of steamed rice grains was investigated. The longer the holding time after steaming, the greater the extent of retrogradation, and the less the degree of enzymatic digestibility. The decreased rate of enzyme digestibility correlated with amylopectin chain length distribution. Samples with short-chain amylopectin exhibited a slow decrease in enzyme digestibility. It w...

Taste-Guided Fractionation and Instrumental Analysis of Hydrophobic Compounds in Sake

The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.

Analyses of peptides in sake mash: forming a profile of bitter-tasting peptides.

Some oligopeptides and amino acids have a strong influence on the sensory qualities of sake, but the formation process of such compounds in sake mash has not been well elucidated. In this study, we investigated the formation process of bitter-tasting peptides derived from rice proteins in sake mash, because knowledge about their formation may contribute to the quality control of sake. We analyzed rice protein hydrolysates in sake mash, as well as in the enzymatic digest of steamed rice grains digested by either sake-koji or by crude enzyme extracted from sake-koji. SDS-PAGE showed that a smaller amount of polypeptides (>M.W. 10,000) accumulated in the supernatant of sake mash than in either enzymatic digest. The concentration of peptides in the supernatant of sake mash increased gradually from the early stages of fermentation. Five bitter-tasting peptides (No. 9, <QLFNPS; No. 13, <QLFNPSTNP; No. 17, <QLFNPSTNPWH; No. 18, <QLFNPSTNPWHSP; No. 20, <QLFGPNVNPWHNP), which were previously found in sake mash, were not found in significant amounts in sake-koji. On the other hand, these peptides accumulated at the early stages of both sake mash fermentation and the enzymatic digests, although the levels in sake mash were higher than those in the digests. The present study demonstrated that the 5 bitter-tasting peptides formed in high concentrations when steamed rice grains were digested under conditions of sake mash fermentation with yeast.

Stimulatory effect of ferulic acid on the production of extracellular xylanolytic enzymes by Aspergillus kawachii.

Production of extracellular β-1,4-xylanase, α-L-arabinofuranosidase, feruloyl esterase, and acetyl xylan esterase from Aspergillus kawachii was higher in a culture supplemented with ferulic acid than in a counterpart. Culture supernatant grown on oat spelt xylan supplemented with ferulic acid exhibited an increase in ferulic acid-releasing activity from insoluble arabinoxylan relative as compared to that from the ferulic acid-free culture.

Factors affecting phenolic acid liberation from rice grains in the sake brewing process.

Phenolic acid (ferulic and p-coumaric acid) liberation from rice grains was examined using rice samples containing phenolic acid at different levels, using two sake mash simulated digestion tests to elucidate influencing factors. Phenolic acid levels in a digest made from steamed rice using dialyzed rice koji enzymes were smaller than levels in a rice koji self-digest. Differences in phenolic acid levels among rice samples in the rice koji self-digest were larger than levels in a digest of steamed rice. In the rice koji self-digest, phenolic acid levels in the ingredient rice grains or in the formed digest related to feruloylesterase (FE) activity in the rice koji. Addition of exogenous FE to rice koji self-digestion increased phenolic acid levels, while addition of xylanase (Xyl) showed weak effects. A concerted effect of FE and Xyl was not clearly observed. Addition of ferulic acid to koji made from α-rice grains raised FE activity, but it did not increase the activity of other enzymes. A similar phenomenon was observed in an agar plate culture of koji mold. These results indicated that ferulic acid levels in ingredient rice grains correlate with FE activities of koji, as a resulut, they affect the phenolic acid levels in sake mash.