Brewing Characteristics Research Articles

Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells.

Sake yeasts belong to the budding yeast species Saccharomyces cerevisiae and have high fermentation activity and ethanol production. Although the traditional crossbreeding of sake yeasts is a time-consuming and inefficient process due to the low sporulation rates and spore viability of these strains, considerable effort has been devoted to the development of hybrid strains with superior brewing characteristics. In the present work, we describe a growth selection system for a- and α-type cells aimed at the crossbreeding of industrial yeasts, and performed hybridizations with sake yeast strains Kyokai No. 6, No. 7 and No. 9 to examine the feasibility of this approach. We successfully generated both a- and α-type strains from all parental strains, and acquired six types of hybrids by outcrossing. One of these hybrid strains was subjected to continuous crossbreeding, yielding the multi-hybrid strain, which inherited the genetic characteristics of Kyokai No. 6, No. 7 and No. 9. Notably, because all of the genetic modifications of the yeast cells were introduced using plasmids, these traits can be easily removed. The approach described here has the potential to markedly accelerate the crossbreeding of industrial yeast strains with desirable properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0216-x) contains supplementary material, which is available to authorized users.

Susceptibility of Hop Cultivars to Downy Mildew: Associations with Chemical Characteristics and Region of Origin

Hop downy mildew (caused by Pseudoperonospora humuli) is a yield-limiting disease in many hop-production regions of the world. In this research, 110 cultivars that are or were widely grown in the United States, Europe, or Australasia were evaluated in western Oregon over three years for their reaction to the shoot infection phase of downy mildew and vigor. There was a large range of downy mildew susceptibility and vigor amongst commercial cultivars, with some cultivars possessing a very high level of resistance. Overall, however, disease resistance and vigor were significantly greater in cultivars originating from Europe than those originating from the United States, Japan, and Australia/New Zealand. Amongst a subset of 79 cultivars, vigor was negatively correlated with levels, in cones, of cohumulone, a chemical constitute of bittering acids typically found in germplasm derived from North America. The generally poor vigor observed in cultivars derived outside of Europe likely is indicative of a lack of tolerance to the crown infection phase of the disease. Thus, the best sources of downy mildew resistance seems to be found in cultivars from the United Kingdom and continental Europe, and such cultivars are typically lower yielding and lack distinctive aroma and flavor characteristics presently desired by craft brewers. Introgression of downy mildew resistance into North American germplasm with high yield and desirable brewing characteristics is needed. Accepted for publication 3 March 2016. Published 7 March 2016.

누룩종류를 달리한 청주 술덧의 이화학적 특성

본 연구는 곰팡이 균종을 달리하여 제조한 다양한 누룩으로 담금한 술덧의 발효 중 품질특성을 비교하여 그 결과를 한국형 청주 제조에 적용하기 위한 이화학적인 기초자료를 확보하는데 그 목적이 있다. 동결건조된 Aspergillus oryzae와 Aspergillus kawachii를 액체 방식으로 각각 배양 후 전통방식으로 제조된 누룩에 각각 또는 혼합 접종시켜 배양후 누룩을 제조하였고, 그 누룩을 이용하여 발효 중 술덧의 품질 변화를 전통방식으로만 성형된 누룩으로 제조된 술덧의 품질과 이화학 적으로 비교 평가하였다. 발효 술덧의 가용성 고형분, 알코올, 비중, 산도 등의 분석을 통해 A. oryzae와 A. kawachii를 각각 단독으로 배양한 누룩을 이용한 발효 술덧에서 발효가 가장 활발하게 일어났음을 알 수 있었다. 또한 술덧의 향과 맛에 가장 큰 영향을 미치는 퓨젤 오일과 에스터 성분 역시 A. oryzae와 A. kawachii를 각각 접종시켜 배양한 누룩으 로 제조한 술덧에서 전통방식의 누룩 또는 A. oryzae와 A. kawachii를 혼합하여 배양한 누룩으로 제조한 술덧에서보다 유의 적으로 높게 나타났다. 본 연구를 통해 기존 전통 누룩만을 이용하여 제조된 술덧에서 발생되는 불완전 발효와 약한 풍미 등을 개선시키기 위해서는 A. oryzae와 A. kawachii를 각각 접종시켜 배양시킨 누룩으로 술을 제조하는 것이 효과적인 것으 로 나타났다.

English

There is a gradual shift to substitute barley with sorghum in brewing industry to reduce the cost of doing business and make beer products more competitive. This study evaluates the sorghum genotypes for desirable malting and brewing characteristics. Biochemical characteristics assayed for 131 sorghum [Sorghum bicolor (L) Moench] accessions included total starch, amylopectin, amylose, proteins, tannins contents, germination energy and germination capacity. Results indicate that starch contents ranged from 22.8 - 81.2%, amylose from 11.5 - 30.2% while the amylopectin content ranged from 6.6 - 59.8%. Generally, amylose contents of sorghum genotypes were lower than their amylopectin contents, with a ratio of 1:2. The mean protein content for the sorghum accessions was 9.4% with a range of 3 - 18%, while that of barley was from 7.7 - 9.8%. Germination energy and germination capacity for sorghum ranged from 82.9 - 99.8% and 74.0 to 99.5%, respectively. Barley varieties showed germination energy and capacity greater than 98%. Sorghum tannin contents ranged from 2.55 mg/100 ml to as high as 100 mg/100 ml while barley varieties had tannin contents of 8.9 to 10.3 mg/100 ml. Two genotypes, SDSA 1x ICSR 43 and SP 993520-1 were the most favorable for brewing.   Key words: Sorghum, starch, protein, tannin, germination energy, malting and brewing.

청수 포도의 양조특성에 미치는 세포벽분해효소와 침용시간의 영향

We investigated the effect of the cell-wall-degrading enzyme and its skin contact time on the brewing characteristics of Cheongsoo grape. The easy of juice extraction was excellent at the cell-wall-degrading enzyme and skin contact treatments, and the aroma was best after five days of skin contact treatment. Furthermore, the juice yields of the Chengsoo grape were more dramatically increased by the cell-wall-degrading enzyme and skin contact treatments than by the control. The data on the pH, total acidity, and soluble solids did not significantly differ among the treatments, and the pH range from 3.1 to 3.4; the total acidity from 0.5% to 0.6% (as tartaric acid); and the soluble solids, from 6.7 to 7.1 . The alcohol content of cell-wall-degrading enzyme treatment was highest with 13.3%. The total polyphenol was gradually increased with the longer skin contact time, and was highest after 10 days of skin contact treatment, at 306.4 mg/L. The main organic acids detected in the Cheongsoo wine were malic and tartaric acid, and citric, succinic and lactic acid were also detected. Our results show that the cell-wall-degrading enzyme and skin contact treatments were better in terms of the easy of juice extraction and significantly increased the juice yield and the volatile compound of the Cheongsoo wine.

증류주의 품질 최적화를 위한 효모선발 연구

본 연구의 목적은 쌀을 원료로 하여 증류주 제조시 쌀 특성에 적합한 최적의 증류주 효모를 선발하는데 있다. 사용된 5종류의 효모는 전반적으로 정상적인 발효패턴을 보였으나 송천효모(Y1)와 증류주 효모(Y5)가 다른 효모에 비해 우수한 발효력을 보였다. 또한 발효후 술덧을 분석한 결과 고급알코올은 증류주 효모(Y4, Y5)에서, 에스터는 송천효모(Y1)와 증류주효모(Y4, Y5)에서 유의적으로 높게 검출된 반면, 유기산 함량은 증류주 효모(Y3, Y4, Y5)에서 유의적으로 높게 나타났다. 발효술덧을 동증류기를 이용한 상압증류한 증류주 분석 결과 송천효모(Y1)와 증류주 효모(Y5)가 최종 알코올 농도가 높아 제조수율에 유리한 것으로 판단되며, 고급알코올과 에스터 등 향기 성분은 발효술덧 분석 결과와 같이 고급알코올은 증류주 효모(Y4, Y5)에서, 에스터는 송천효모(Y1)와 증류주 효모(Y4, Y5)에서 유의적으로 높게 나타나 발효술덧의 향기 패턴이 증류주에 대부분 전이되는 것으로 판단된다. 효모별 발효특성과 발효술덧 및 증류주의 물리화학적 특성, 관능평가를 종합한 결과 증류주 효모인 siha aktivehefe 6 brennereihefe(Y5)가 쌀을 이용한 증류주 품질 최적화에 가장 적합한 효모로 선발되었다. The aim of this study was to select yeast strain for quality optimization of distilled spirit which is prepared using rice. Five yeasts strains were compared for their brewing characteristics and showed normal fermentation pattern, but songcheon yeast(Y1) and distillery yeast(Y5) revealed higher fermentation ability than other samples tested. The analyzed results of fermented mashing showed that the distillery yeasts(Y4, Y5) had significantly much higher alcohol content, and songcheon yeast(Y1), distillery yeasts(Y4, Y5) had significantly higher ester content than other samples tested respectively, while the distillery yeasts(Y3, Y4, Y5) had significantly higher organic acid content than other samples tested. The analyzed results of distilled spirits which were distilled using copper distillery apparatus showed that the songcheon yeast(Y1) and distillery yeast(Y5) had a higher yield compared to other samples tested. In addition, the results of the aroma compounds such as ester and higher alcohol of distilled spirits among the five yeasts tested were similar to the analyzed results of fermented mashing. Siha aktivhefe 6 brennereihefe(Y5) indicated the highest overall preference including sensory evaluation and was selected as best yeast strain for quality optimization of distilled spirit which is prepared using rice.

1400년대~1600년대 고문헌에 기록된 술의 고찰 - 순곡주류 중 단양주를 중심으로 -

The purpose of this study was to investigate the characteristics and brewing methods of Danyangju, which is one of the traditional single brewed rice liquors using Korean ancient literatures publishes from the 1400's to 1600's. Total 32 Danyangju products and 55 brewing methods were found by reviewing the ancient literatures. Danyangju products could be classified into four groups depending on type of grain used: 1) 13 Danyangju products with non-glutinous rice, 2) 14 products with waxy rice, 3) 4 products with mixture of waxy and nonwaxy rice, and 4) 2 products with barley. In this paper, not only ingredients, formula and utensils needed for preparation of Danyangju, but also brewing characteristics and terminology were reviewed. The findings on Danyangju in this study would be useful to improve the brewing methods and quality of Korean traditional liquors.

Aspergillus oryzae strains isolated from traditional Korean Nuruk: Fermentation properties and influence on rice wine quality

Forty-seven strains of Aspergillus oryzae isolates from Korean nuruks were compared for their brewing characteristics. A. oryzae YI-A6 and YI-A7 showed the highest acid α-amylase, glucoamylase, and carboxypeptidase activities, respectively. Sixteen isolates with high amylolytic or proteolytic enzyme activities were selected for investigation of their rice wine fermentation characteristics. After 12 days of brewing at 15°C, ethanol concentrations were 10.2–14.3% for A. oryzae strains. Fermentation rates were the highest for YI-A7. Most rice wine samples fermented with nuruk strains had lower concentrations of off-flavor compounds than the control did. All mean sensory attribute values significantly differed among samples. Pearson correlation coefficients showed that glucoamylase activity was positively correlated to both ethanol productivity and overall harmony (p<0.01). Thus, glucoamylase activity was identified as the best factor for screening Aspergillus strains for use in rice wine brewing.

Breeding Research on Sake Yeasts in Japan: History, Recent Technological Advances, and Future Perspectives

Sake is an alcoholic beverage of Japan, with a tradition lasting more than 1,300 years; it is produced from rice and water by fermenting with the koji mold Aspergillus oryzae and sake yeast Saccharomyces cerevisiae. Breeding research on sake yeasts was originally developed in Japan by incorporating microbiological and genetic research methodologies adopted in other scientific areas. Since the advent of a genetic paradigm, isolation of yeast mutants has been a dominant approach for the breeding of favorable sake yeasts. These sake yeasts include (a) those that do not form foams (produced by isolating a mutant that does not stick to foams, thus decreasing the cost of sake production); (b) those that do not produce urea, which leads to the formation of ethyl carbamate, a possible carcinogen (isolated by positive selection in a canavanine-, arginine-, and ornithine-containing medium); (c) those that produce an increased amount of ethyl caproate, an apple-like flavor (produced by isolating a mutant resistant to cerulenin, an inhibitor of fatty-acid synthesis); and (d) those that produce a decreased amount of pyruvate (produced by isolating a mutant resistant to an inhibitor of mitochondrial transport, thus decreasing the amount of diacetyl). Given that sake yeasts perform sexual reproduction, sporulation and mating are potent approaches for their breeding. Recently, the genome sequences of sake yeasts have been determined and made publicly accessible. By utilizing this information, the quantitative trait loci (QTLs) for the brewing characteristics of sake yeasts have been identified, which paves a way to DNA marker-assisted selection of the mated strains. Genetic engineering technologies for experimental yeast strains have recently been established by academic groups, and these technologies have also been applied to the breeding of sake yeasts. Sake yeasts whose genomes have been modified with these technologies correspond to genetically modified organisms (GMOs). However, technologies that enable the elimination of extraneous DNA sequences from the genome of sake yeast have been developed. Sake yeasts genetically modified with these technologies are called self-cloning yeasts and do not contain extraneous DNA sequences. These yeasts were exempted from the Japanese government's guidelines for genetically modified food. Protoplast fusion has also been utilized to breed favorable sake yeasts. Future directions for the breeding of sake yeasts are also proposed in this review. The reviewed research provides perspectives for the breeding of brewery yeasts in other fermentation industries.

Registration of ‘Dana’—A Bittering Hop Cultivar with a Pleasant Hoppy Aroma

Hop (Humulus lupulus L.) is a perennial plant used mainly in the brewing industry because of its bitter and aromatic substances. Consequently, hop varieties are divided into two groups: those with aroma and those with high α‐acid content. Since the Slovenian varieties are all of the aroma type, the development of a variety with a high α‐acid content was our objective. ‘Dana’ (Reg. No. CV‐29, PI 665033), which was developed at the Slovenian Institute of Hop Research and Brewing through screening and breeding techniques, expresses an aromatic hop character as well as a high α‐acid content. It has brewing characteristics that are comparable with other hop varieties that are well known worldwide, giving Dana the opportunity to be a very good substitute for them in the brewing industry. Dana is moderately resistant to primary downy mildew (caused by Pseudoperonospora humuli Miyabe and Takah. G.W. Wilson) infection, has resistance to secondary downy mildew infections of cones, and has established virus‐ and viroid‐free nuclear stock material. The release of Dana should increase the production of the α‐acid group in Slovenia, since it is the first high‐α‐acid‐content variety produced in Slovenia that is well adapted to Slovenian pedoclimatic conditions.

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.

清酒酵母の一倍体の取得と実験室酵母との交配による醸造特性の遺伝解析

清酒酵母の一倍体の取得と実験室酵母との交配による醸造特性の遺伝解析

QTL mapping of sake brewing characteristics of yeast

A haploid sake yeast strain derived from the commercial diploid sake yeast strain Kyokai no. 7 showed better characteristics for sake brewing compared to the haploid laboratory yeast strain X2180-1B, including higher production of ethanol and aromatic components. A hybrid of these two strains showed intermediate characteristics in most cases. After sporulation of the hybrid strain, we obtained 100 haploid segregants of the hybrid. Small-scale sake brewing tests of these segregants showed a smooth continuous distribution of the sake brewing characteristics, suggesting that these traits are determined by multiple quantitative trait loci (QTLs). To examine these sake brewing characteristics at the genomic level, we performed QTL analysis of sake brewing characteristics using 142 DNA markers that showed heterogeneity between the two parental strains. As a result, we identified 25 significant QTLs involved in the specification of sake brewing characteristics such as ethanol fermentation and the production of aromatic components.

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small-scale sake-brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour.

Kilning Conditions for the Optimization of Enzyme Levels in Buckwheat

Buckwheat is a novel ingredient in malting and brewing, and therefore, requires optimization of the malting and brewing processes. In this study, three temperature regimens, 48 hr at 40°C (KR1), 5 hr at 40°C + 11 hr at 50°C (KR2), and 5 hr at 40°C + 3 hr at 50°C + 3 hr at 60°C (KR3) were used to kiln green buckwheat malt produced from grains steeped for 12 hr at 10°C and germinated for 96 hr at 15°C. Kilning regimens KR1, KR2, and KR3 produced malts with a moisture content of 6.8, 6.2, and 5.7%, respectively. Although an increased enzymatic activity level in buckwheat malt was noted with increased thermal exposure during the kilning regimen, it is not reflected in its brewing characteristics with regard to extract and apparent fermentability after using an optimized mashing system. However, the effect of treatment at higher temperatures with respect to enzymatic activity levels in buckwheat malt is reflected by rapid visco analysis where decreased peak viscosities correlate to an increased amylolytic activity level. The results of this study suggest that buckwheat, kilned using KR3, which was found to contain highest levels of amylolytic enzymes and produced the highest levels of total soluble nitrogen and free amino nitrogen when optimally mashed, be recommended for the kilning regimen of buckwheat green malt.

花から分離した酵母の醸造特性

花から分離した酵母の醸造特性

Molecular breeding of the Mureka-non-forming sake koji mold from Aspergillus oryzae by the disruption of the mreA gene

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant nonpasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20°C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20°C or 30°C for 63 d. In the sensory evaluation of the sake sto red at 20°C or 30°C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.

Effect of viruses on agronomic and brewing characteristics of four hop (Humulus lupulus) cultivars in Australia

SummaryThe effect of Hop latent virus (HpLV), Hop mosaic virus (HpMV), and Prunus necrotic ring spot virus [PNRSV (apple, A, and intermediate, I, serotypes)], on the survival of softwood cuttings, the vigour (height) of early season growth, cone yield, and the levels of brewing organic acids in mature plants, was assessed in four hop (Humulus lupulus) cultivars in Tasmania, Australia. Virus infections were associated with an increase in the mortality of softwood cuttings following propagation. In all cultivars, height of early growth was a poor indicator of the effect of viruses on cone yield and levels of brewing organic acids (alpha and beta acids). In cv. ‘Nugget’, infection by the virus combinations studied was not associated with reductions in cone yield, however plants infected by PNRSV‐I, in 2000, had 11% lower alpha acids and 7% lower beta acids. In ‘Opal’, infection by HpLV and HpMV were the most deleterious to cone yield, however the effect of HpMV was ameliorated when in combination with PNRSV‐I. Reductions in alpha and beta acid content were attributable only to mixed infections of HpLV + HpMV in combination with either serotype of PNRSV. In ‘Pride of Ringwood’, yield loss was mostly attributable to HpMV and to a lesser extent, HpLV. Some ameliorations in cone yield loss occurred in plants containing a mixed infection between HpMV and HpLV or either of the ilarvirus serotypes. Both of the ilarviruses and HpMV caused reductions in alpha acid content. In ‘Victoria’, cone yield loss was mostly attributable to combinations of viruses such as HpLV + PNRSV‐I and HpLV + HpMV. The deleterious effect of HpLV + HpMV was ameliorated by PNRSV‐A and to a lesser extent, PNRSV‐I. Infection by the virus combinations studied did not significantly affect alpha and beta acid levels in either year. Results suggested the effect of viruses and their combinations differed between cultivars and varied between seasons. This information, when combined with knowledge of the rates of virus re‐infection, can be used to recommend control strategies for the Australian hop industry.

Basic Studies on the Cultivation and Breeding of Brewers' Rice in Kagawa Prefecture. Effect of the selection for leaf color on the protein content and some other brewing characteristics in late generation.

オオセト×山田錦およびオオセト×松山三井の組み合わせにおける雑種初期世代からの葉色に対する選抜が後代のタンパク質含有率や他の酒米特性に及ぼす影響について検討した.F2集団の出穂期における葉色値(SPAD-502型による読み取り値)は正規分布に似た連続変異を示し,その分散から推定した広義の遺伝率は,オオセト×山田錦0.517,オオセト×松山三井0.397であった.F2からF5まで,出穂期の葉色が濃い方(H群)と淡い方(L群)へ選抜を続けた結果,F6系統のタンパク質含有率はH群の方がL群よりも有意に高くなった.平均値で比較した稈長+穂長はH群の方がL群より長く,1000粒重はH群の方が軽かった.また,H群の方が出穂期迄日数は短く,心白粒発現率は高かった.F6系統における葉色値とタンパク質含有率との間には有意な正の相関関係が認められた.ただし,H群内には葉色値とタンパク質含有率との関係が異なる二つのグループが存在した.葉色値のF5-F6親子相関は有意であった.これらより,酒米の品種育成においても雑種初期世代における出穂期の葉色はタンパク質含有率に対する選抜指標として有効と判断した.

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.