The Beer Phenolic Profile Change During «Dry» Hopping in the Post-fermentation Stage Conditions

Статья посвящена вопросу изменения фенольного профиля пива в условиях «холодного» охмеления во время дображивания пива. Оценено влияние метаболизма дрожжевых клеток и типа хмеля во время дображивания на изменение общего количества полифенолов, изоксантогумола, изогумулона, кверцетина и рутина. Показано, что применение низовой расы дрожжей Rh и горького типа хмеля Mагнум позволяет добиться высокого (до 123,0 мг/дм3) содержания полифенолов в пиве к 1 сут «холодного» охмеления в отличие от тонко-ароматного хмеля Tетнангера, применение которого дает максимум количества полифенолов к 14 сут процесса. Применение верховой расы дрожжей Nottinghem приводит к замедлению увеличения концентрации полифенолов в среде, поскольку только к 7-14 сут вне зависимости от типа хмеля происходит их накопление. Показана зависимость в течение первых 14 сут изменения количества изоксантогумола от расы дрожжей и типа хмеля, а в последующем - только от типа хмеля. Авторами получены результаты, свидетельствующие о том, что кверцетин не вовлекается низовыми дрожжами в метаболический цикл, в отличие от верховых. Изменение концентрации рутина в пиве не зависит от расы дрожжей, и определяется своим содержанием в хмеле определенного типа. В работе показана взаимосвязь между процессами изменения содержания изогумулона и изоксантогумола при «холодном» охмелении в зависимости от ряда факторов. Проведение органолептического анализа позволило соотнести балловую оценку дескрипторов пива с основными показателями фенольного профиля. The article is devoted to the issue of beer phenolic profile changing in the conditions of «dry» hopping during the after-fermentation of beer. The influence of the yeast cells metabolism and the hop type during fermentation on the change in the polyphenol, isoxanthohumol, isogumulone, quercetin and rutin total amount, was evaluated. It has been shown that the Rh lager yeast race use and the Magnum bitter hop type makes it possible to achieve a high (up to 123.0 mg/dm3) polyphenol content in beer by 1 day of «dry» hopping, in contrast to the finely aromatic Tettnanger hops, the use of which gives the maximum polyphenol amount by 14 days of the process. The Nottinghem ale yeast race use leads to a slowdown in the polyphenol concentration increase in the medium, since their accumulation occurs only by 7-14 days, regardless of the hop type. The dependence during the first 14 days of the change in the isoxanthohumol amount on the yeast race and the hop type, and subsequently - only on the hop type was shown. The authors obtained results indicating that quercetin is not involved in the metabolic cycle by lager yeast, in contrast to ale yeast. The change in the rutin concentration in beer does not depend on the yeast race and is determined by its content in a certain type of hop. The paper shows the relationship between the processes of changing the isogumulone and isoxanthohumol content during «dry» hopping, depending on several factors. Organoleptic analysis made it possible to correlate the beer descriptors scoring with the phenolic profile main indicators.

A Novel Approach to Develop Lager Yeast with Higher NADH Availability to Improve the Flavor Stability of Industrial Beer

Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry.

Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

Improving the Utilization of Isomaltose and Panose by Lager Yeast Saccharomyces pastorianus

Approximately 25% of all carbohydrates in industrial worts are poorly, if at all, fermented by brewing yeast. This includes dextrins, β-glucans, arabinose, xylose, disaccharides such as isomaltose, nigerose, kojibiose, and trisaccharides such as panose and isopanose. As the efficient utilization of carbohydrates during the wort’s fermentation impacts the alcohol yield and the organoleptic traits of the product, developing brewing strains with enhanced abilities to ferment subsets of these sugars is highly desirable. In this study, we developed Saccharomyces pastorianus laboratory yeast strains with a superior capacity to grow on isomaltose and panose. First, we designed a plasmid toolbox for the stable integration of genes into lager strains. Next, we used the toolbox to elevate the levels of the α-glucoside transporter Agt1 and the major isomaltase Ima1. This was achieved by integrating synthetic AGT1 and IMA1 genes under the control of strong constitutive promoters into defined genomic sites. As a result, strains carrying both genes showed a superior capacity to grow on panose and isomaltose, indicating that Ima1 and Agt1 act in synergy to consume these sugars. Our study suggests that non-GMO strategies aiming to develop strains with improved isomaltose and panose utilization could include identifying strains that overexpress AGT1 and IMA1.

Lager yeast design through meiotic segregation of a fertile Saccharomyces cerevisiae x Saccharomyces eubayanus hybrid

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae x Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15 C, 15 Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.

The physiological and biochemical properties regulation of lager brewer's yeast

Современные методы интенсификации производства напитков брожения часто основаны на активации жизнедеятельности дрожжей под воздействием различных технологических факторов. Одним из путей интенсификации является применение повышенной температуры в процессе накопления биомассы посевных дрожжей, что значительно влияет на удельную скорость размножения микроорганизмов. Исследование направлено на изучение влияния температурного режима накопления биомассы на морфологические и биохимические признаки дрожжей низового брожения. Изучение поставленной задачи проводилось с применением дрожжей низового брожения Saccharomyces cerevisiae расы 8аМ, f, 44 при трех температурных режимах: (6±2) °С, (20±2) °С и (30±2) °С. Исследования показали, что бродильная активность всех исследуемых рас дрожжей, биомасса которых накапливалась при (6±2) °С, выше, чем при (20±2) °С и при более (30±2) °С. Установлено, что в процессе накопления биомассы дрожжей S. cerevisiae расы 44 при температуре (30±2) °С происходит вытеснение исходной культуры другими по форме и размерам клеток. Выявлено пять отличных от исходной культуры колоний, изменения в которых устойчиво сохранялись при последующих многократных пересевах. Пять полученных штаммов обладали более низкой бродильной активностью по сравнению с исходной культурой, а три штамма полностью утратили флокуляционную способность. Modern methods of intensifying the fermentation beverages production are often based on the yeast vital activity activation under the various technological factors influence. One of the ways of intensification is the high temperature use in the process of seed yeast biomass accumulation, which significantly affects the specific rate of microorganisms reproduction. The study is aimed at studying the biomass accumulation temperature regime influence on the lager yeast morphological and biochemical characteristics. The task study was carried out with the use of lager yeast Saccharomyces cerevisiae race 8aM, f, 44 at three temperature conditions: (6±2) °C, (20±2) °C and (30±2) °C. Studies have shown that the fermentation activity of all the studied yeast races, the biomass of which was accumulated at (6±2) °C, is higher than at (20±2) °C and at more than (30±2) °C. It was found that during the S. cerevisiae race 44 yeast biomass accumulation at a temperature of (30±2) °C, the original culture is replaced by other cells in shape and size. Five colonies different from the original culture were identified, the changes in which were steadily preserved during subsequent repeated replanting. Five of the resulting strains had a lower fermentation activity compared to the original culture, and three strains completely lost their flocculation ability.

Screening lager yeast with higher ethyl-acetate production by adaptive laboratory evolution in high concentration of acetic acid

Ethyl-acetate is important for the flavor and aroma of the alcoholic beverages, therefore, there have been extensive efforts toward increasing its production by engineering yeast strains. In this study, we reported a new approach to breed non-genetic modified producing yeast strain with higher ethyl-acetate production for beer brewing. First, we demonstrated the positive effect of higher acetic acid concentration on inducing the expression of ACS. Then, we applied adaptive laboratory evolution method to evolve strain with higher expression level of ACS. As a result, we obtained several strains with increased ACS expression level as well as ethyl-acetate production. In 3 L scale fermentation, the optimal strain EA60 synthesized more ethyl-acetate than M14 at the same time point. At the end of fermentation, the ethyl-acetate production in EA60 was 21.4% higher than M14, while the other flavor components except for acetic acid were changed in a moderate degree, indicating this strain had a bright prospect in industrial application. Moreover, this study also indicated that ACS1 played a more important role in increasing the acetic acid tolerance of yeast, while ACS2 contributed to the synthesis of cytosol acetyl-CoA, thereby facilitating the production of ethyl-acetate during fermentation.

Long insert clone experimental evidence for assembly improvement and chimeric chromosomes detection in an allopentaploid beer yeast

Lager beer is made with the hybrid Saccharomyces pastorianus. Many publicly available S. pastorianus genome assemblies are highly fragmented due to the difficulties of assembling hybrid genomes, such as the presence of homeologous chromosomes from both parental types, and translocations between them. To improve the assembly of a previously sequenced lager yeast hybrid Saccharomyces sp. 790, and elucidate its genome structure, we proposed the use of alternative experimental evidence. We determined the phylogenetic position of Saccharomyces sp. 790 and established it as S. pastorianus 790. Then, we obtained from this yeast a bacterial artificial chromosome (BAC) genomic library with its BAC-end sequences (BESs). To analyze this data, we developed a pipeline (applicable to other assemblies) that classifies BESs pairs alignments according to their orientation. For the case of S. pastorianus 790, paired-end BESs alignments validated parts of the assembly and unpaired-end ones suggested contig joins or misassemblies. Importantly, the BACs library was preserved and used for verification experiments. Unpaired-end alignments were used to upgrade the previous assembly, and provided an improved detection of translocations. With this, we proposed a genome structure of S. pastorianus 790, which was similar to that of other lager yeasts; however, when we estimated chromosome copy number and experimentally measured its genome size, we discovered that one key difference is the outstanding S. pastorianus 790 ploidy level (allopentaploid). Altogether, our results show the value of combining bioinformatic analyses with experimental data such as long-insert clone information to improve a short-read assembly of a hybrid genome.

Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

The search for novel brewing strains from non-brewing environments represents an emerging trend to increase genetic and phenotypic diversities in brewing yeast culture collections. Another valuable tool is hybridization, where beneficial traits of individual strains are combined in a single organism. This has been used successfully to create de novo hybrids from parental brewing strains by mimicking natural Saccharomycescerevisiae ale × Saccharomyceseubayanus lager yeast hybrids. Here, we integrated both these approaches to create synthetic hybrids for lager fermentation using parental strains from niches other than beer. Using a phenotype-centered strategy, S. cerevisiae sourdough strains and the S. eubayanus × Saccharomyces uvarum strain NBRC1948 (also referred to as Saccharomyces bayanus) were chosen for their brewing aptitudes. We demonstrated that, in contrast to S. cerevisiae × S. uvarum crosses, hybridization yield was positively affected by time of exposure to starvation, but not by staggered mating. In laboratory-scale fermentation trials at 20 °C, one triple S. cerevisiae × S. eubayanus × S. uvarum hybrid showed a heterotic phenotype compared with the parents. In 2 L wort fermentation trials at 12 °C, this hybrid inherited the ability to consume efficiently maltotriose from NBRC1948 and, like the sourdough S. cerevisiae parent, produced appreciable levels of the positive aroma compounds 3-methylbutyl acetate (banana/pear), ethyl acetate (general fruit aroma) and ethyl hexanoate (green apple, aniseed, and cherry aroma). Based on these evidences, the phenotype-centered approach appears promising for designing de novo lager beer hybrids and may help to diversify aroma profiles in lager beer.