Sake Mash Research Articles

Determination of Ethanol in Sake Mash by a Chloride Ion-Selective Electrode.

A chloride ion-selective solvent polymeric membrane electrode (Cl-ISE) using quaternary ammonium salts for determination of the ethanol concentration during sake fermentation is described. The ethanol response by a Cl-ISE is based on the change in the transfer activity coefficient of Cl- from water to ethanol. The logarithm value of the transfer activity coefficient increases linearly with increasing ethanol concentration. The Cl-ISE using a mixture of o-nitro-phenyloctyl ether and n-tetradecylalcohol as plasticizers and tetradodecylammonium salts as sensing materials showed good linearities for ethanol (−2.39 mV/%) and for Cl- (−53.6 mV/decade). Furthermore, the interference from co-existent components in sake mash with the response of the Cl-ISE was improved in comparison with that using other plasticizers and sensing materials. The Cl-ISE was found to show good accuracy in the measurement of ethanol in sake mash.

Characterization of a PDR1 mutant allele from a clotrimazole-resistant sake yeast mutant with improved fermentative activity

Clotrimazole-resistant mutants from various sake yeasts show improved fermentative activity in sake mash while retaining their parental advantages for sake making. These mutants also exhibit pleiotropic drug resistance (PDR) phenotypes. To investigate the relationship between the improvement of fermentative activity and PDR phenotypes, a PDR1 mutant allele ( pdr1-h176) encoding a transcription factor was cloned from a clotrimazole-resistant mutant, HL176 ( MATa/ MATα), using PCR amplification. The nucleotide sequences of pdr1-h176 and its wild allele were determined. The mutant allele contained a missense point mutation (L309S) that can confer a PDR phenotype on yeast. This amino acid substitution is located in the conserved motif II in the inhibitory domain of Pdr1p, and is very close to the cluster of three mutation points (P298A, K302Q, and M308I) described by Carvajal et al. (Mol. Gen. Genet., 256, 406–415, 1997) in laboratory strains. A PDR1 wild allele of HL163, the parent strain of HL176, was replaced by pdr1-h176 using gene recombination at the homologous site. The resultant transformants ( PDR1/pdr1-h176) showed the same PDR phenotype as HL176, and they fermented sake mash efficiently even in the final fermentation stage, while HL163 did not. The amino acid substitution (L309S) in pdr1-h176 was considered to be sufficient to improve the fermentative activity of sake yeast, in addition to conferring the PDR phenotype.

乾燥酵母協会701号および協会901号による清酒製造

We made two dry yeast starters of Saccharomyces cerevisiae strains K 701 and K 901.In order to make dry yeast these strains were grown aerobically in a cane molasses medium with a continuous supply of molasses, then the yeast cells were dehydrated in a dram dryer. Those dry yeast starters were used for SAKE brewing in four brewing companies on a production scale of approxinatly 3 ton of total rice weight.Every batch of fermented SAKE mash produced on excellent profile, and amino acidity did not increase even at the end of the fermentation period.This showed that these dry yeast strains aretolerant to a high concentration of ethanol.The SAKE produced had an excellent taste with a light and fruity fresh flavor.These results show that those dry yeast starters are useful for the practical production of SAKE.

Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

Brewing properties of sake yeast whose EST2 gene encoding isoamyl acetate-hydrolyzing esterase was disrupted

The EST2 gene, encoding an isoamyl acetate-hydrolyzing esterase, was disrupted in a diploid strain of Saccharomyces cerevisiae UT-1 (MATa/MATα ura3/ura3 trp1/trp1 EST2/EST2), which is derived from the industrial sake yeast Kyokai no. 701 (strain K-701), by using two disruption plasmids (pDest2U, est2:: URA3; and pDest2T, est2:: TRP1) sequentially. Genomic Southern blot analysis revealed that both loci of the EST2 gene on the chromosome of strain UT-1 were disrupted. The resultant mutants were named UTUT-1 and UTUT-2 (a/MATα ura3/ura3 trp1/trp1 est2::URA3/est2::TRP1). Deficiency in Est2p esterase was also confirmed by activity staining of the gel after native-polyacrylamide gel electrophoresis of cell extracts of the two mutant strains. Small scale sake brewing was carried out using these sake yeasts and the strains they were derived from, and their brewing properties were compared. The fermentation profiles of the four strains (strains K-701, UT-1, UTUT-1, and UTUT-2) were largely similar. The components of the resulting sake were also similar except for the acetate ester concentration, although strains UTUT-1 and UTUT-2 produced approximately 2-times more isoamyl acetate than the wild type K-701. These resuts strongly suggest that the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in the sake mash. Strains UTUT-1 and UTUT-2, deficient in Est2p esterase, are suitable for sake brewing.

Analysis of sake mash using multichannel taste sensor

The titrable acidity of and the ethanol concentration in sake mash were measured using a multichannel taste sensor system. A positively charged membrane responded to ethanol and the electric response of the membrane showed good agreement with those determined by gas chromatography in the measurement of sake mash. It was considered that the responses were induced by a change in the activity coefficient of anions. The titrable acidity of sake mash was measured using an electrode with a negatively charged membrane, because the electric response of the membrane showed a strong correlation with the titrable acidity of sake. The result showed good agreement with the value determind by titration, and the agreement was slightly increased by multiple regression together with the electric response of the positively charged membrane. Therefore, it is concluded that this method is useful for determination of the ethanol concentration in and the titrable acidity of sake mash. The results show that the taste sensor can be used for monitoring of those values in sake fermentation.

Factors affecting the increase of inositol content of yeast cells in sake mash : Keiji Furukawa∗, Yuki Nakatsuru, Haruhiko Mizoguchi, and Shodo Hara ( General Research Institute, Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozakinishi-machi, Higashinada-ku, Kobe, Hyogo 658) Seibutsu-kogaku 74: 367–374. 1996

Factors affecting the increase of inositol content of yeast cells in sake mash : Keiji Furukawa∗, Yuki Nakatsuru, Haruhiko Mizoguchi, and Shodo Hara ( General Research Institute, Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozakinishi-machi, Higashinada-ku, Kobe, Hyogo 658) Seibutsu-kogaku 74: 367–374. 1996

Molecular cloning and nucleotide sequence of the isoamyl acetate-hydrolyzing esterase gene ( EST2) from Saccharomyces cerevisiae

A mutant deficient carboxylesterase which hydrolyzes isoamyl acetate ( est2) was isolated from a yeast, Saccharomyces cerevisiae. The EST2 gene was cloned and its nucleotide sequence determined. The EST2 gene has an open reading frame of 714 bp (238 amino acids), and the molecular weight of the gene product calculated from the predicted amino acid sequence was 27,304.66. The EST2 gene product lacks the consensus sequence (Gly-Xaa-Ser-Xaa-Gly) that is observed in the serine type esterase or lipase, although a quite similar sequence (Ala-Cys-Ser-Ala-Gly) was found. Genomic Southern analysis revealed that the EST2 gene was present as a single copy on the chromosomal DNA of laboratory strains of S. cerevisiae as well as the sake brewing yeasts. The est2 mutant accumulated approximately 19 times higher amounts of isoamyl acetate compared with the parent strain in laboratory scale sake brewing. Therefore, the EST2 gene product is likely to play a crucial role in the hydrolysis of isoamyl acetate in sake mash.

精米歩合が清酒醪の窒素量に及ぼす影響

精米歩合が清酒醪の窒素量に及ぼす影響

Effect of yeast fumarase gene ( FUM1) disruption on production of malic, fumaric and succinic acids in sake mash

Organic acids are essential flavor components in alcoholic beverages such as wine and sake (Japanese rice wine). The effects of fumarase deficiency on the productivity of malic, fumaric and succinic acids in Saccharomyces cerevisiae were examined using the FUM1 gene disruptant. The single nuclear gene FUM1 encoding fumarase in S. cerevisiae, DBY746, was disrupted by a site-directed one-step gene disruption technique. The fumarase activity of the isolated FUM1 disruptant was abolished. The FUM1 disruptant having a pet phenotype showed no growth on minimal medium containing glycerol as a carbon source even after prolonged incubation. The productions of organic acids by the FUM1 disruptant in YPD liquid culture containing 10% glucose and in sake mash were analyzed by HPLC. The analytical results of the FUM1 disruptant showed there were no differences in extracellular malate productivity, fumarate productivity had increased, and that succinate productivity was reduced compared with those of the control strain. In the production of sake using the FUM1 disruptant, fermentation ability as monitored by CO 2 evolution was not different from that of the control strain. These results indicate that site-directed FUM1 disruption alters organic acid production without any loss of fermentation ability in yeast.

Reduction of pyruvic acid in sake mash by enzymatic reaction of pyruvate decarboxylase in permeabilized cells : Note—H aruhiko M izoguchi∗ and S hodo H ara ( General Institute of Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozaki-nishimachi, Higashinada-ku, Kobe, Hyogo 658) Seibutsu-kogaku 73: 37–42. 1995

Reduction of pyruvic acid in sake mash by enzymatic reaction of pyruvate decarboxylase in permeabilized cells : Note—H aruhiko M izoguchi∗ and S hodo H ara ( General Institute of Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozaki-nishimachi, Higashinada-ku, Kobe, Hyogo 658) Seibutsu-kogaku 73: 37–42. 1995

Properties of sake yeast mutants resistant to isoamyl monochloroacetate

Isoamyl monochloroacetate ( i-AmOCIAc) inhibited the growth of sake yeast. AOF10, which is resistant to isoamyl monofluoroacetate and has low esterase activity toward isoamyl acetate ( i-AmOAc), also showed resistance to i-AmOClAc. Monochloroacetic acid formed by an esterase through the hydrolysis of i-AmOClAc was more effective at glycolysis inhibition than i-AmOClAc. Forty five mutants resistant to i-AmOClAc were isolated from a parental haploid strain (HL-69) derived from sake yeast. Three had less than 20% of the original activity of the esterase. All mutants fermented sake mash as well as the parent strain, HL-69, and accumulated 1.3 to 1.7-fold the levels of i-AmOAc and 1.8 to 2.4-fold the levels of isobutyl acetate accumulated by HL-69. Surprisingly one mutant (CL-90), which accumulated 1.7-fold the amount of i-AmOAc and 1.3-fold the amount of ethyl acetate, exhibited increased alcohol acetyltransferase (EC 2.3.1.84) activity (1.6-fold). Esterase patterns were also examined by gel electrophoresis and activity staining. Changes in the intensity of the strongest band ( EST2) were closely related to the activity of the esterase toward i-AmOAc.

清酒醪の発酵温度とアミノ酸の動向

清酒醪の発酵温度とアミノ酸の動向

膜蒸留を利用した清酒醪アルコール分のオンライン測定

膜蒸留を利用した清酒醪アルコール分のオンライン測定

グルタチオン含有清酒の製造方法

An easy process for producing glutathione-containing sake was devised without using particular yeast strains. The sake containing glutathione was obtained by heating normally made sake mash for a short period folloed by rapid cooling. The best condition was heating mash at 60°C for a time as short as possible.

Breeding of a useful strain of sake yeast for Ginjo-sake brewing

Abstract A useful sake-fermenting yeast for brewing ginjo -sake was bred by protoplast fusion. First, 61 native strains were isolated from ginjo -sake mash. From these isolates, two strains, YNS50 and YNS59, were selected. These two strains possessed many of the characteristics required for ginjo -sake brewing in a complementary manner. Strain YNS50 produced high amounts of the flavor components isoamyl acetate and ethyl caproate and low amounts of acids. Strain YNS59, on the other hand, exhibited good growth and fermentation at low temperatures (∼10°C) and had high resistance to ethyl alcohol. Next, a lysine auxotrophic mutant (YDH17) of YNS50 and ϱ-mutant (YDH8) of YNS59 with a marker useful for the selection of the fusants were constructed. Finally, prototrophs arising from the complementation of each marker by protoplast fusion were obtained. A strain (strain YNF74) that stably maintained all of the useful characteristics derived from each wild type strain was screened from 120 fusants. In a sub-industrial scale sake brewing test, we demonstrated that strain YNF74 exhibited its superior characteristics in brewing ginjo -sake. The sake produced by strain YNF74 achieved high evaluation in a tasting test.

Identification of enzymic digestion and fermentation rates in sake mash using the extended kalman filter method

Identification of enzymic digestion and fermentation rates in sake mash using the extended kalman filter method

On-line monitoring of alcohol concentration in sake mash by use of liquid-membrane-liquid-type membrane distillation and a semiconductor-type alcohol sensor

On-line monitoring of alcohol concentration in sake mash by use of liquid-membrane-liquid-type membrane distillation and a semiconductor-type alcohol sensor

Application of fuzzy control theory to the sake brewing process

Abstract Know-how of skilled operators concerning the management of the traditional sake brewing process was analyzed using fuzzy control theory, and arranged as production rules having the following four fuzzy variables: if (DB = DBi) & (DD = DDi) & (AL = ALi) then (CT = CTi). Membership functions of DB and DD were arranged in real-time using a kinetic model proposed by us previously. Based on these control rules, a fuzzy control system for regulating the moromi (sake mash) temperature was constructed. A computer simulation of sake brewing indicated that the proposed control system could be applied to control the temperature along the desired curve of traditional sake brewing.

Performance of Sake mash brewing with fuzzy control

Performance of Sake mash brewing with fuzzy control