Masachika Takashio

Breeding of Brewer's yeast by hybridization between a top-fermenting yeast Saccharomyces cerevisiae and a cryophilic yeast Saccharomyces bayanus

To improve the fermentability of a top-fermenting yeast at low-temperature, we performed hybridization trials between four top-fermenting Saccharomyces cerevisiae strains and a cryophilic yeast Saccharomyces bayanus YM84 with good fermentability at low-temperature. The hybrids selected using 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside were checked with pulsed-field gel electrophoresis and their brewing performance at the low-temperature of 10.5 degrees C was observed using small-scale (2 l) fermentation trials.

Characterization of Factors Involved in the Production of 2(E)-Nonenal during Mashing

To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 70°C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2(E)-nonenal; however, this effect was significantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2(E)-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10(E),12(Z)-octadecadienoic and 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid into 2(E)-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 70°C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment.

Analysis of an inactivated Lg-FLO1 gene present in bottom-fermenting yeast

During the course of investigating a flocculation-related gene of a bottom-fermenting yeast, we identified a new Lg-FLO1 homologue which contains the N-terminal domain of the Lg-FLO1 gene. The results of the partial DNA sequence analysis of the amplified product obtained by inverse-PCR suggested that the homologue contains a sequence present in the YIL169c (chr. IX of Saccharomyces cerevisiae). Southern blot analyses using the VTH1, HXT12, SDL1 and UBP7 genes as probes for chr. IX strongly indicated that an approximately 20-kb region from the YIL169c ORF to the left telomere in chr. IX translocated to the Lg-FLO1 ORF region in chr. VIII of bottom-fermenting yeast. This translocation might convert a flocculent cell to a non-flocculent one.

Identification and Functional Analyses of Two cDNAs That Encode Fatty Acid 9-/13-Hydroperoxide Lyase (CYP74C) in Rice

Fatty acid hydroperoxide lyase (HPL), a member of cytochrome P450 (CYP74), produces aldehydes and oxo-acids involved in plant defensive reactions. In monocots, HPL that cleaves 13-hydroperoxides of fatty acids has been reported, but HPL that cleaves 9-hydroperoxides is still unknown. To find this type of HPL, in silico screening of candidate cDNA clones and subsequent functional analyses of recombinant proteins were performed. We found that AK105964 and AK107161 (Genbank accession numbers), cDNAs previously annotated as allene oxide synthase (AOS) in rice, are distinctively grouped from AOS and 13-HPL. Recombinant proteins of these cDNAs produced in Escherichia. coli cleaved both 9- and 13-hydroperoxide of linoleic and linolenic into aldehydes, while having only a trace level of AOS activity and no divinyl ether synthase activity. Hence we designated AK105964 and AK107161 OsHPL1 and OsHPL2 respectively. They are the first CYP74C family cDNAs to be found in monocots.

Adsorption to or desorption of beer components from a lipid membrane related to sensory evaluation

The relationship between the adsorption to or desorption of beer from a lipid membrane and sensory evaluation was studied using a lipid-coated quartz crystal microbalance connected to a flow injection system. The adsorption and duration of adsorption of commercial beers showed a significant correlation with their body and smoothness in a sensory evaluation, respectively. Isohumulones, tartaric acid, NaCl, glutamic acid, and tannic acid were adsorbed onto the lipid membrane. Di- and trihydroxyoctadecenoic acids increased the duration of adsorption of the beer components onto the lipid membrane but not the extent of adsorption. They decreased the astringent duration of beer and the smoothness in the sensory evaluation but did not affect the intensity of bitterness or astringency or the body. It seems that this system, which modifies the electrostatic and hydrophobic interactions of the beer components with the tongue and throat surfaces, can mainly evaluate bitterness and/or astringency which significantly affect the body and smoothness of beer.

Characterization of 9-fatty acid hydroperoxide lyase-like activity in germinating barley seeds that transforms 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid into 2(E)-nonenal.

Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r=0.53, P<0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.

Identification of hop varieties using specific primers derived from RAPD markers

A useful method to identify hop varieties by genomic analysis is presented. Utilizing the simplicity of the random amplified polymorphic DNA (RAPD) procedure, genetic markers for the identification of hop varieties were screened. To make up for the deficiencies of RAPD, specific primers were designed based on the sequences of the RAPD markers. Twelve typical hop varieties were successfully distinguished using these specific primers. The method was also successfully used for the detection of the mixed samples.