Isolation of Aspergillus oryzae mutants producing low levels of 2,4,6-trichloroanisole.

Musty or moldy off-odor in sake severely reduces its quality. Such off-odor is caused by 2,4,6-trichloroanisole (TCA), a compound that is produced by Aspergillus oryzae during sake production by O-methylating the precursor 2,4,6-trichlorophenol (TCP). TCP suppresses the growth of fungi, including A. oryzae, although TCA does not. Therefore, strains that are unable to convert TCP to TCA should be sensitive to TCP in the medium. Nevertheless, A. oryzae with a disrupted O-methyltransferase gene (ΔomtT) grew in a medium containing TCP. In agar medium, we observed no growth difference between the ΔomtT strain and a non-disrupted transformant; however, a significant growth delay was observed with the ΔomtT strain grown in liquid medium containing 0.5 µg/mL of TCP. This strain was more sensitive to low concentrations of TCP, suggesting that omtT contributes to the conversion (detoxification) of TCP in liquid culture. We generated A. oryzae RIB 40 mutants by ultraviolet irradiation and then cultured them in liquid medium containing TCP to obtain strains that did not produce moldy odor. The slow-growing strains were cultured in agar plates and then used to make koji with added TCP. We obtained three strains with lower TCA-producing ability and with sufficient hydrolase activities for sake brewing.

Mutagenesis, breeding, and characterization of sake yeast strains with low production of dimethyl trisulfide precursor.

Dimethyl trisulfide (DMTS) is one of the main components responsible for hineka, the aroma associated with deteriorated Japanese sake during storage. The molecule 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) has been previously identified as a major precursor compound of DMTS. Furthermore, it had been suggested that the yeast methionine salvage pathway is involved in the production of DMTS-P1. In sake brewing tests, DMTS-P1 and the DMTS producing potential (DMTS-pp; DMTS amount of sake after accelerated storage) were significantly reduced in mde1 or mri1 strain, which lack genes of the methionine salvage pathway. Industrial use of the gene-disrupting strains may not be accepted in the Japanese food industry. In order to obtain mde1 or mri1 mutants, we established a method to screen 5'-methylthioadenosine (MTA) non-utilizing strains using minimum culture medium containing methionine or MTA by ethyl methanesulfonate (EMS) mutagenesis with methionine-auxotrophic sake yeast haploid. As expected, mde1 and mri1 mutants were identified among the obtained mutants by an established screening method. The obtained strains had poor fermentation ability in sake brewing tests, so back-crossing was performed on the mutants to obtain mde1 or mri1 homozygous mutants. These strains had improved brewing characteristics, and DMTS-P1 and the DMTS-pp of the produced sake were significantly lower than those of the parent strains. These strains are expected to contribute to improving the maintenance of sake quality during storage.

Construction of sake yeast with low production of dimethyl trisulfide precursor by a self-cloning method.

Dimethyl trisulfide (DMTS) is the primary component responsible for "hineka", the stale aroma of Japanese sake. Deletion of the MRI1 or MDE1 gene of sake yeast, encoding 5'-methylthioribose-1-phosphate isomerase and 5'-methylthioribulose-1-phosphate dehydratase, respectively, has been reported to greatly reduce the amount of DMTS precursor (DMTS-P1) in sake and to suppress the formation of DMTS during storage. In this study, we constructed sake yeast strains lacking MRI1 gene function by a self-cloning method. Two methods were applied: in one, a stop codon was introduced in the MRI1 ORF by point mutation; in the other, the entire MRI1 ORF was deleted from the genome. In both methods, a plasmid vector containing drug-resistance and counter-selectable markers was used to introduce the mutation. We successfully obtained the strains, which did not contain the plasmid sequences, by both methods. Small-scale sake brewing tests using these SC strains (strains obtained by the self-cloning method) found that DMTS-P1 was hardly detected in sake brewed with SC strains, and DMTS production after sake storage was greatly reduced as compared with the parent strain. The components of brewed sake were almost the same between the SC and parent strains. These results suggest that SC strains can produce sake with higher flavor stability without changing the sake brewing properties.

Changes in the aroma compounds of sake during aging.

Changes in the aroma of sake during aging were investigated by aroma extract dilution analysis (AEDA) and quantitative analysis using the stir bar sorptive extraction method. In AEDA, more odor zones were detected in aged sake than in fresh sake. The dilution factors of aldehydes, polysulfides, and some esters were greater in the aged sake, and their increase during aging was confirmed through a quantitative analysis of sake stored for 0-35 years. Among these compounds, 3-methylbutanal, methional, and dimethyltrisulfide (DMTS) were present in aged sake at concentrations exceeding their odor thresholds, and the highest odor active value was observed for DMTS. Sensory tests showed that supplementation with DMTS contributed to both the total odor intensity and the sulfury odor of aged sake aroma.

Contribution of 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake.

Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale Japanese sake, called "hineka". Recently, we isolated one of the precursor compounds of DMTS in sake and identified it as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound. In this work, the contribution of DMTS-P1 to the formation of DMTS was investigated. DMTS-P1 was chemically synthesized from methional in three steps, consisting of the Grignard reaction, followed by oxidation by MnO(2) and an immobilized osmium oxide catalyst. The formation of synthetic DMTS-P1 was confirmed by a comparison of the liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data to that of natural DMTS-P1. Quantitative analysis of DMTS-P1 in sake was developed using LC-MS/MS, and a positive correlation was observed between the concentration of DMTS-P1 in sake and the production of DMTS during storage. These results indicate that DMTS-P1 contributes to the formation of DMTS in sake.

Screening and identification of precursor compounds of dimethyl trisulfide (DMTS) in Japanese sake.

Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale sake, called "hineka"; however, the mechanism underlying the formation of DMTS during the storage of sake has not been elucidated. This paper investigates the precursors of DMTS in sake. An experiment using [methyl-d(3)]-methionine showed that Strecker degradation of methionine plays a minor role in the formation of DMTS. Separation of components in sake by cation exchange resin revealed that DMTS precursors are present in the acidic/neutral fraction rather than in the basic one. Purification of the DMTS precursor compounds was carried out through several chromatographic steps, measuring DMTS-producing potential as an index. High-resolution ESI-MS and 1D/2D NMR experiments enabled the identification of one of the precursor compounds as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one.