Takashi Shinohara

Distribution of phenolic yeasts and production of phenolic off-flavors in wine fermentation

The activity of wine yeasts to decarboxylate ferulic and p-coumaric acids is one of their biological properties related to the production of phenolic off-flavors (POF) in wine-making. We examined POF productivity in 116 strains of wine yeast, 74 strains of wild yeast (Saccharomyces cerevisiae) and 23 strains of non-Saccharomyces yeast, and found that a majority of these yeasts were POF-producing strains. The frequency distribution of POF-producing strains was 81 to 95% in wine yeasts, 85 to 97% in wild yeasts and 78 to 83% in non-Saccharomyces yeasts based on the POF test with addition of ferulic and p-coumaric acids to grape juice medium. The Rhodotorula, Candida, Cryptococcus, Pichia, Hansenula, and Brettanomyces strains had high or moderate POF productivity among the 20 non-Saccharomyces species. The decomposition rate of ferulic acid correlated with POF production and the critical concentration of phenolic acid (free form) in grape must was estimated to be more than 10 mg/l. Segregation of POF phenotype and Southern blot analysis of phenolic wine yeasts suggest that POF production is controlled by the POF gene (PAD1). The results showed the frequent distribution of phenolic yeasts in the wine-making environment. These suggest the importance of controlling POF production by using wine yeast strains of low POF productivity. The grapes must be prepared by a suitable process to prevent the increase in phenolic acid content.

Production and purification of a bacteriocin peptide produced by Lactococcus sp. strain GM005, isolated from Miso-paste.

Lactococcus sp. GM005 was isolated from Miso-paste and was found to produce a bacteriocin with strong antibacterial activity. A culture of Lactococcus sp. GM005, maintained at 30 degrees C and a constant pH of 6.0, exhibited bacteriocin activity eightfold higher than that of a culture grown under pH-uncontrolled conditions. GM005 bacteriocin was purified to homogeneity on SDS-PAGE by hydrophobic column chromatography and gel filtration. The estimated molecular weight of GM005 bacteriocin was approximately 9.6 kDa based on gel-filtration analysis, and was approximately 2.4 kDa based on tricine-SDS-PAGE analysis, indicating a tetrametric structure. N-terminal amino acid analysis revealed that the N-terminal end was blocked. Amino acid composition analysis revealed a high proportion of hydrophobic amino acid residues and lanthionine. This differs from the composition of some antibiotics. GM005 bacteriocin exhibits a bactericidal activity against Lactobacillus sakei JCM1157T.

Restriction fragment length polymorphism analysis of 16S rRNA genes in lactic acid bacteria isolated from red wine

Various lactic acid bacteria isolated from wine and alcoholic drinks attempted to identified by restriction fragment length polymorphism (RFLP). Oenococcus oeni strains exhibited unique RFLP patterns by HaeIII-digestion of 12 reference strains. We performed RFLP analysis using the AccII or HaeIII enzyme for 44 strains isolated from red wine and the results indicated profiles identical to O. oeni type strain. O. oeni does not exhibit interspecific diversity. Thus, the use of RFLP analysis of 16S rDNA is useful in the identification of O. oeni strains isolated from wines.

Selection and hybridization of wine yeasts for improved winemaking properties: Fermentation rate and aroma productivity

Abstract Three strains out of thirty-one wine yeasts (Saccharomyces cerevisiae) were selected for their good winemaking properties (fermentation rate, tolerance of sulfur dioxide, aroma productivity, wine quality) and genetic markers (KHR killer activity, galactose assimilation), and used for hybridization by spore to spore mating. Of the twelve hybrids produced, two, Hy17-108 (RIFY 1001 × RIFY 1067) and Hy41-308 (RIFY 1001 × RIFY 1065), were selected on the basis of a fermentation test. In experimental winemaking the two hybrids demonstrated improved aroma productivity for higher alcohols, aromatic esters and/or fatty acids, while their fermentation rate was nearly the same as that of the parental strains.

Selection and fermentation properties of cryophilic wine yeasts

Abstract Two cryophilic strains, YM-84 and YM-126, were selected by a double-layer agar fermenting technique from 100 strains of the wine yeast, Saccharomyces cerevisiae . The viability (specific growth rate) and fermentability of the two selected strains at low temperatures (7 and 13°C) were superior to those of wine yeast strains W3 and OC-2, indicating the usefulness of the two strains as cryophilic wine yeasts. Experiments using the two selected strains at intermediate temperatures (22 and 30°C) showed that their fermentation ceased prematurely and their ethanol yields were reduced.

Introduction of flocculation property into wine yeasts (Saccharomyces cerevisiae) by hybridization

Abstract Flocculating ability of 173 strains of wine yeasts and 74 strains of wild yeasts ( Saccharomyces cerevisiae ) was examined, and 23 strains were found to be flocculent, but only a wine yeast and 4 wild yeasts had useful flocculating activity. This suggested a minor distribution of flocculent yeasts with winemaking capabilities. Flocculation property of the strains (ABXL-1D, RIFY 1029, RES-5) was introduced into the non-flocculent wine yeasts (RIFY 1001, IAM 4274) by mating. Some suppressions of flocculation gene expression were noticed by the cross. An improved hybrid WWR-2 was obtained by a back cross ([RIFY 1001 × RES-5] × RIFY 1001) and this hybrid demonstrated a practical flocculation and good fermentation property in experimental winemaking. A wine yeast strain with moderately flocculating activity was constructed by the hybridization.

Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts

Abstract An excellent wine yeast, Saccharomyces cerevisiae W3, which had KHR killer, was added as a starter yeast into grape must and behavior of the starter strain and wild yeasts was investigated during fermentation by using KHR killer as a genetic marker. The KHR killer was detected only in the strain W3 and not in other wine and wild yeast strains. Accordingly, the frequency of starter yeast W3 was monitored throughout the fermentation of grape musts by using KHR killer, W3 was discriminated efficiently from wild yeasts during fermentation by KHR killer activity and proved to lead the fermentation as a dominant yeast until their termination.

Experimental white wine making in order to reduce the SO 2 usage

果汁のSO2無添加仕込によって正常な品質のワインをえたが, 果汁のSO2添加仕込 (常法) によるワインは品質が一層安定であった.果汁のナイロン粉末ないしゼラチン処理したワインは, 貯酒中の色調がより安定し, 酒質も良好であった。果汁のべントナイトおよび火入れ処理したワインは貯蔵中の色調が不安定であり, 又酒質は良くなかった。貯酒中の品質安定にSO2は有効であり, その使用は不可欠である。それ故白ワインの醸造において, SO2使用を少くするためには, 仕込のときSO2の使用を必要最小眼にとどめること, 果汁の前清澄, ナイロン粉末ないしゼラチン処理を行うこと, 発酵終了後に適切なSO2量を加えて嫌気的貯酒を行い, 有効SO2 (遊離型SO2) の減少を防止することにあると考える。