Brewing Characteristics Research Articles

Isolation of sake yeast strains from Ariake Sea tidal flats and evaluation of their brewing characteristics.

Screening for new sake yeasts can expand the sensory diversity of sake, due to their production of metabolites that characterize sake's aroma and taste. In this study, mud from tidal flats in the Ariake Sea was screened for Saccharomyces cerevisiae strains with ethanol productivity suitable for sake brewing, and the brewing characteristics of isolated strains were evaluated. Five strains (H1-1, H1-2, H1-3, H3-1, and H3-2) classified as S. cerevisiae were isolated. Karyotype analysis by pulsed-field gel electrophoresis showed that five isolated strains were closely related to sake yeast strains (K7, K701, K9, K901, and Y52) instead of laboratory yeast strain. Results of small-scale brewing tests including sake yeast strains K701, K901, and Y52 showed that the five isolated strains have fermentation activity comparable to sake yeast strains. Principal component analysis (PCA) revealed that the five isolated strains produce higher levels of ethyl caproate and lower levels of acidic compounds than sake yeasts. In addition, isolated strains H3-1 and H3-2 produce higher levels of isoamyl acetate and lower levels of acetic acid than other isolated strains. Consequently, five S. cerevisiae strains that have high fermentation activity and differ from common sake yeast strains in terms of brewing characteristics were successfully isolated from the Ariake Sea.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Comparative Study on Phenolic Content and Antioxidant Activity of Different Malt Types.

Malt is the main raw material for beer production, which determines not only its taste and aroma profile, but to a large extent its biological value, as well. The aim of the present research was to determine the antioxidant profile of different malt types as a basis for the development of new types of beer with increased antioxidant activity. In the present study the main brewing characteristics, the phenolic profile and the antioxidant potential of 20 malt types used in craft breweries in Bulgaria have been examined. The main brewing characteristics have been determined by the standardized methods of the European Brewing Convention. Malt phenolic content was determined by two methods, and antioxidant potential by five different methods. Based on a statistical factor analysis performed by the principal component analysis, it was confirmed that there was a relationship between malt color and phenolic compounds content. The principal component analysis confirmed that there was a link between the content of the Maillard reaction products and malt biological activity. Malts with the highest degree of heat treatment were characterized by the highest antioxidant activity, which was due to the content of Maillard reaction products with antioxidant capacity.

Breeding sake yeast and identification of mutation patterns by synchrotron light irradiation

Sake yeast is one of the important factors that characterize the aroma and taste of sake. To obtain sake yeast strains with different metabolic capabilities from other strains, breeding of a sake yeast is an effective way. In this study, sake yeast strain Y5201 was mutagenized by synchrotron light irradiation to obtain the mutant strains showing different brewing characteristics from parental strain Y5201, and comparative genome analysis between strain Y5201 and mutant strains was performed to identify mutation points and patterns induced by synchrotron light irradiation. Screening with the drug-resistant and fermentation tests selected the nine mutants (C18, C19, C29, C50, C51, C52, C54, T25, and T49) from the mutagenized Y5201 cells. Principal component analysis results based on the analysis of the small-scale brewing test metabolites showed that the mutant strain C19 was different from other strains, which had higher productivity of ethyl caproate and isoamyl acetate than those of the Y5201. Comparative genome analysis revealed that mutants by synchrotron light irradiation had a higher diversity of single nucleotide substitutions and a higher frequency of Indel (insertion/deletion) in these DNA than ethyl methanesulfonate and UV irradiation. These results suggest that synchrotron light irradiation is an effective and unique mutagen for yeast breeding.

Genome Editing to Generate Sake Yeast Strains with Eight Mutations That Confer Excellent Brewing Characteristics.

Sake yeast is mostly diploid, so the introduction of recessive mutations to improve brewing characteristics requires considerable effort. To construct sake yeast with multiple excellent brewing characteristics, we used an evidence-based approach that exploits genome editing technology. Our breeding targeted the AWA1, CAR1, MDE1, and FAS2 genes. We introduced eight mutations into standard sake yeast to construct a non-foam-forming strain that makes sake without producing carcinogens or an unpleasant odor, while producing a sweet ginjo aroma. Small-scale fermentation tests showed that the desired sake could be brewed with our genome-edited strains. The existence of a few unexpected genetic perturbations introduced during breeding proved that genome editing technology is extremely effective for the serial breeding of sake yeast.

Industrially Applicable De Novo Lager Yeast Hybrids with a Unique Genomic Architecture: Creation and Characterization.

Lager beer is produced by Saccharomyces pastorianus, which is a natural allopolyploid hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus Lager strains are classified into two major groups based largely on genomic composition: group I and group II. Group I strains are allotriploid, whereas group II strains are allotetraploid. A lack of phenotypic diversity in commercial lager strains has led to substantial interest in the reconstitution of de novo allotetraploid lager strains by hybridization of S. cerevisiae and S. eubayanus strains. Such strategies rely on the hybridization of wild S. eubayanus isolates, which carry unacceptable traits for commercial lager beer such as phenolic off flavors and incomplete utilization of carbohydrates. Using an alternative breeding strategy, we have created de novo lager hybrids containing the domesticated S. eubayanus subgenome from an industrial S. pastorianus strain by hybridizing diploid meiotic segregants of this strain to a variety of S. cerevisiae ale strains. Five de novo hybrids were isolated which had fermentation characteristics similar to those of prototypical commercial lager strains but with unique phenotypic variation due to the contributions of the S. cerevisiae parents. Genomic analysis of these de novo lager hybrids identified novel allotetraploid genomes carrying three copies of the S. cerevisiae genome and one copy of the S. eubayanus genome. Most importantly, these hybrids do not possess the negative traits which result from breeding wild S. eubayanus The de novo lager strains produced using industrial S. pastorianus in this study are immediately suitable for industrial lager beer production.IMPORTANCE All lager beer is produced using two related lager yeast types: group I and group II, which are highly similar, resulting in a lack of strain diversity for lager beer production. To date, approaches for generating new lager yeasts have generated strains possessing undesirable brewing characteristics which render them commercially inviable. We have used an alternative approach that circumvents this issue and created new lager strains that are directly suitable for lager beer production. These novel lager strains also possess a unique genomic architecture, which may lead to a better understanding of industrial yeast hybrids. We propose that strains created using our approach be classified as a third group of lager strains (group III). We anticipate that these novel lager strains will be of great industrial relevance and that this technique will be applicable to the creation of additional novel lager strains that will help broaden the diversity in commercial lager beer strains.

Isolation of novel alcohol-tolerant spontaneous mutant strains from sake yeast Kyokai no. 6 and no. 7, and their brewing characteristics

Isolation of novel alcohol-tolerant spontaneous mutant strains from sake yeast Kyokai no. 6 and no. 7, and their brewing characteristics

Sake brewing characteristics of a novel strain of Saccharomyces cerevisiae isolated from flowers

Sake brewing characteristics of a novel strain of <i>Saccharomyces cerevisiae</i> isolated from flowers

Lachancea fermentati Strains Isolated From Kombucha: Fundamental Insights, and Practical Application in Low Alcohol Beer Brewing.

With a growing interest in non-alcoholic and low alcohol beer (NABLAB), researchers are looking into non-conventional yeasts to harness their special metabolic traits for their production. One of the investigated species is Lachancea fermentati, which possesses the uncommon ability to produce significant amounts of lactic acid during alcoholic fermentation, resulting in the accumulation of lactic acid while exhibiting reduced ethanol production. In this study, four Lachancea fermentati strains isolated from individual kombucha cultures were investigated. Whole genome sequencing was performed, and the strains were characterized for important brewing characteristics (e.g., sugar utilization) and sensitivities toward stress factors. A screening in wort extract was performed to elucidate strain-dependent differences, followed by fermentation optimization to enhance lactic acid production. Finally, a low alcohol beer was produced at 60 L pilot-scale. The genomes of the kombucha isolates were diverse and could be separated into two phylogenetic groups, which were related to their geographical origin. Compared to a Saccharomyces cerevisiae brewers’ yeast, the strains’ sensitivities to alcohol and acidic conditions were low, while their sensitivities toward osmotic stress were higher. In the screening, lactic acid production showed significant, strain-dependent differences. Fermentation optimization by means of response surface methodology (RSM) revealed an increased lactic acid production at a low pitching rate, high fermentation temperature, and high extract content. It was shown that a high initial glucose concentration led to the highest lactic acid production (max. 18.0 mM). The data indicated that simultaneous lactic acid production and ethanol production occurred as long as glucose was present. When glucose was depleted and/or lactic acid concentrations were high, the production shifted toward the ethanol pathway as the sole pathway. A low alcohol beer (<1.3% ABV) was produced at 60 L pilot-scale by means of stopped fermentation. The beer exhibited a balanced ratio of sweetness from residual sugars and acidity from the lactic acid produced (13.6 mM). However, due to the stopped fermentation, high levels of diacetyl were present, which could necessitate further process intervention to reduce concentrations to acceptable levels.

Development of sake yeast haploid set with diverse brewing properties using sake yeast strain Hiroshima no. 6 exhibiting sexual reproduction

Among sake yeast strains, Kyokai no. 7 (K7) and its closely related strains (K7 group) are predominantly used because of their excellent brewing properties. In the sake industrial sector, the need for various types of yeast strains is high. Although crossbreeding is an effective method for generating genetic diversity that should result in diverse characteristics, most K7 group strains lack normal sporulation ability, including the ability to undergo meiotic chromosomal recombination, which leads to difficulties in crossbreeding. Accordingly, the improvement of sake yeast strains primarily depends on mutagenesis and suitable selection in a stepwise manner. Our recent study revealed that the long-preserved sake yeast strain Hiroshima no. 6 (H6) does not belong to the K7 group despite genetically being extremely similar. In addition, H6 exhibited normal sporulation. Thus, we isolated haploid cells from H6 and mated them with previously isolated haploid cells of K7 group strains. The crossbred diploid strains had normal sporulation ability; hence, we performed tetrad analysis. The brewing characteristics of the obtained haploid set were extremely diverse. Principal component analysis based on the volatile and organic acid components measured using small-scale sake brewing tests revealed that the haploid strains derived from each diploid strain displayed a characteristic distribution. Thus, we demonstrated the availability of genetic crossbreeding using H6 with sporulation ability to facilitate both the development of novel sake yeast strains with many desirable characteristics and analyses of the function of sake yeast.

Draft Genome Sequence of Saccharomyces cerevisiae Strain Pf-1, Isolated from Prunus mume.

Saccharomyces cerevisiae strain Pf-1 is a yeast isolated from Prunus mume; it potentially can be used to produce wine and traditional Japanese sake. Here, we report the draft genome sequence of this strain. The genomic information will provide a deeper understanding of the brewing characteristics of this strain.

Kazbek – The First Czech Aroma “Flavor Hops” Variety: Characteristics and Utilization

The world market of craft beer, especially dry hopped beers, has been constantly growing in the last few years. That is why new varieties of hops are still being bred. This article gives the genetic, agronomic, chemotaxonomic and brewing characteristics of Kazbek, the first “flavor hops” variety bred in the Czech Republic. Kazbek genetically belongs to a group of bitter American hops resulting from Brewers Gold variety, but the alpha acids content is relatively low, 5.0–8.0% by weight. The content of a homologous series of geranyl esters (acetate – propionate – isobutyrate) in an amount 2–4% of total essential oils (1–1.5 g/100g) is considered to be the originator of the specific, mainly citrus-like aroma of Kazbek. Moreover, the essential oil fraction contains about 25 sulfurous substances, predominantly thioesters. In the pilot scale brewing tests, the quality of Kazbek hops was proved both in kettle and dry hopping of Pilsner lagers. The overall sensory impression of kettle hopped lagers was comparable to Saaz hops, the differences were in the character of hop-derived flavor and bitterness. The best results of dry hopped beers were achieved for a 2.5 g/l hop dose and a contact time of 3 days.

Aspergillus oryzae을 이용한 pellet 누룩 제조 및 약주의 품질특성

Aspergillus oryzae 83-10 (AO) was inoculated in a wheat pellet with different concentrations of raw materials to produce Nuruk. The sterilized raw material (S) was found to be stable in the production of pellet-Nuruk and was suitable for the production of high enzyme power; high enzyme activity was obtained after fermentation for 32 h. The characteristics of pellet-Nuruk were investigated and the brewing characteristics were compared with those of the commercial SongHak (SH) Nuruk. The enzyme activities (dry base, unit/g) of S-AO (sterilized material and inoculated with A. oryzae) pellet-Nuruk (32 h culture) and the commercial SH Nuruk were compared. The activities of glucoamylase, α-amylase, and acidic carboxypeptidase were significantly higher in S-AO Nuruk. In terms of brewing characteristics, the soluble solid (°Brix) and total acid (%) were significantly higher in the SH mesh, and the amino acid content and alcohol content (%) were significantly higher in the S-AO mesh. The amount of Nuruk added for yakju fermentation was set at 30 unit/g × g of rice weight based on the glucoamylase activity. The enzymatic activity immediately after preparation was 26.69 unit/mL and 17.58 unit/mL for S-AO and SH, respectively. Based on these results, preparing pellet-type Nuruk should considerably save fermentation time compared with traditional Nuruk products.

Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast.

In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungus Aspergillus oryzae. During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy, A. oryzae, and Grifola frondosa glycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition of A. oryzae and G. frondosa glycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast.

Brewing characteristics of piezosensitive sake yeasts

ABSTRACTApplication of high hydrostatic pressure (HHP) treatment to food processing is expected as a non-thermal fermentation regulation technology that supresses over fermentation. However, the yeast Saccharomyces cerevisiae used for Japanese rice wine (sake) brewing shows high tolerance to HHP. Therefore, we aimed to generate pressure-sensitive (piezosensitive) sake yeast strains by mating sake with piezosensitive yeast strains to establish an HHP fermentation regulation technology and extend the shelf life of fermented foods. The results of phenotypic analyses showed that the generated yeast strains were piezosensitive and exhibited similar fermentation ability compared with the original sake yeast strain. In addition, primary properties of sake brewed using these strains, such as ethanol concentration, sake meter value and sake flavor compounds, were almost equivalent to those obtained using the sake yeast strain. These results suggest that the piezosensitive strains exhibit brewing characteristics essentially equivalent to those of the sake yeast strain.

Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast.

The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms.

재래누룩에서 분리한 N4와 N9 효모의 증류식 소주 양조특성

Soju is a Korean traditional distilled alcoholic beverage produced from mashes various crops and Nuruk which is cultured with wild microorganisms. This study was conducted to investigate rice-Soju brewing characteristics of yeasts isolated from Korean traditional Nuruk. The general components of rice (Hanarumbyeo) raw materials were 14.7 g of water, 6.8 g of crude protein, 0.9 g of crude lipid, 0.4 g of crude ash, and 76.5 g of carbohydrate in 100 g. Saccharifying and proteolytic activities in Hanarumbyeo ipguk (solid-state culture of Aspergillus luchuensis) were also determined. The alcohol content of the fermented wash from isolates was 15.37-16.58% (v/v), which is 16.7-36.0% higher than that of industrial yeasts (12.33-13.19%). Reducing sugar contents were 2.04-3.92 and 7.92-8.78 g/100 mL in the isolates and industrial yeasts, respectively. The isolated yeasts showed 25.2-52.7% higher yield of distillates (41% alcohol) compared to industrial yeasts. Forty-one components were detected in the rice distillated Soju (25% alcohol) and principal component analysis revealed differences between the isolated and industrial yeasts with respect to the contents of i-BuOH, isobutanal diethyl acetal, ethyl caprate, and tetradecanoic acid.

Characterization of brewing microorganisms isolated from Korean traditional nuruk for Cheongju production.

The objective of this study was to select and identify yeasts and molds isolated from traditional nuruk and to investigate their brewing characteristics for Cheongju production. The yeast strains Y190 (Accession ID-KACC 93251P), Y263 (Accession ID-KACC 93252P), and Y270 (Accession ID-KACC 93253P) showing high alcohol and flavor productivity were isolated and identified by phylogenetic inference based on an internal transcribed spacer 2 region sequence analysis. In addition, Aspergillus oryzae 83-10 (Accession ID-KACC 93254P) showing the highest enzyme activity was isolated. This study provides basic data for the production of Korean Cheongju and assesses the applicability of these three yeast strains and A. oryzae 83-10 isolated from traditional nuruk.

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Phenotypes and brewing characteristics of sake yeast Kyokai no. 7 mutants resistant to valproate

Restoration of Traditional Korean Nuruk and Analysis of the Brewing Characteristics.

In this study, a total of 58 different kinds of nuruk (a traditional Korean fermentation starter) were prepared, including 46 kinds of restored nuruk from ancient documents. Each nuruk was evaluated by analysis of its saccharification power, and the enzyme activities of glucoamylase, α-amylase, β-amylase, protease, and β-glucanase. The range of saccharification power (sp) of the restored nuruk ranged between 85 and 565 sp. The diastatic enzymes, α-amylase, β-amylase, and glucoamylase, were significantly correlated to the saccharification power value; conversely, β-glucanase and protease did not have a correlative relationship with saccarification power. In addition, their brewing properties on chemical and organoleptic aspects of traditional alcoholic beverage production were compared. Each raw and supplementary material contained in nuruk showed its own unique characteristics on Korean alcoholic beverage brewing. For the first time, in this study, the traditional Korean nuruk types mentioned in ancient documents were restored using modernized production methods, and also characterized based on their brewing properties. Our results could be utilized as a basis for further study of traditional alcoholic beverages and their valuable microorganisms.