The process of vanillin production during awamori brewing proceeds in this manner：1） Feruloyl oligosaccharide（FAO） is released from hemicellulose by xylanase（Xln） ; 2） Free ferulic acid（FA） is released from FAO by ferulic acid esterase（FAE） ; 3） FA is converted to 4-vinylguaiacol（4VG） by phenolic acid decarboxylase（PAD） from Aspergillus luchuensis; 4） 4VG is converted to vanillin by natural oxidization during the aging process. However, it has been unknown when these reactions occur during brewing. In this study, we investigated when Xln and FAE produce FAO and FA during brewing. As a result, it is suggested that these enzymes are expressed during koji making, but most of these enzyme reactions are carried out in the moromi. Moreover, PAD was not induced by the FA in moromi, indicating that the FA in moromi was converted to 4VG by the PAD that was accumulated during koji making. Comparing polished rice with a polishing ratio of 90%（PR90） koji and that with a polishing ratio of 98%（PR98） koji, no difference appeared in Xln activity, but PR98 koji had higher FAE and PAD activity and released more of their products. Therefore, it is suggested that with more rice bran content, the higher are the activities of FAE and PAD, along with a greater amount of free FA and 4VG.

In total, 43 strains of wine yeast were screened for their application in sake brewing, focusing on their ethanol yield in sake mash and 4-vinylguaiacol（4-VG） production. Of the 43 strains, 10 did not produce 4-VG and 34 demonstrated an ethanol yield as high as the K901 strain. The detection of the 10 strains that did not produce 4-VG suggested that the 4-VG production test is an important and efficient method for screening wine yeast strains for sake brewing. The brewing characteristics of six wine yeast strains（RIB1026, RIB1045, RIB1049, RIB1062, RIB1063, and RIB1069）, which exhibited no 4-VG production and high ethanol production, were investigated for small-scale sake brewing. These strains demonstrated ethanol yields of 16.1%—18.85% in sake mash, indicating their potential application in the fermentative production of sake. Moreover, these strains showed a characteristic production ability for organic acids and aroma components, and did not show the killer phenotype versus K-9. These data indicate that the six selected wine yeast strains in this study do not influence other sake-brewing yeast strains used in breweries and that they have the ability to produce characteristic sake.

In this study, the relationship between starch gelatinization properties and starch retrogradation properties were investigated taking the prediction of steamed rice solubility in sake brewing into account. We defined the starch retrogradation property as the relationship between concentrations of rice flour and retrogradation rates obtained by the DORFT method. Forty-four rice samples --including 4 cultivars harvested in 2015-2019-- were employed and the correlations between gelatinization peak temperature, amylopectin ratio, gelatinization peak viscosity, and starch retrogradation property were determined. As a result, gelatinization peak temperature obtained by DSC had a high correlational coefficient of 0.536 with the starch retrogradation property, indicating that it could be possible to estimate the starch retrogradation by the gelatinization peak temperature. On the other hand, the correlational coefficient between gelatinization peak viscosity obtained by RVA and the starch retrogradation property was 0.615, which is higher than that of the gelatinization peak temperature. It might be more useful for the estimation of starch retrogradation to take not only amylopectin but also amylose into consideration because starch retrogradation advanced the re-association in the presence of both amylose and amylopectin.