Optimization of the Fermentation Conditions for Brewing Yeast Biomass Production Using the Response Surface Methodology and Taguchi Technique

Yeast (including brewing yeast) and yeast-based preparations derived from bioprocesses or agroindustrial byproducts represent valuable feed additives and ingredients for ruminants. The optimization of brewing yeast biotechnological processing through fermentation mediated by the brewing yeast strain Saccharomyces pastorianus ssp. carlsbergensis W34/70 was investigated. The cultivation conditions (temperature, pH, carbon source, and nitrogen source) were selected and designed according to a Taguchi fractional experimental plan, with four factors on three levels, and their influence on the evolution of the bioprocess of obtaining the brewing yeast biomass was evaluated. The dependent variables were the yeast biomass amount in wet form, yeast biomass amount in dried form after lyophilization, dried yeast biomass wettability assayed through the contact angle (CA), protein content (PC), and dry matter content (DS). The effects that the experimental conditions had on the system responses were visualized in tridimensional space using the response surface methodology, and the combination of biotechnological parameters that ensured process quality and robustness was then determined using the Taguchi technique through its performance indicator, i.e., the signal-to-noise ratio. By optimizing the biotechnological parameters, this study provides a valuable contribution in the area of brewing yeast biomass processing, with the aim of producing probiotic yeast for ruminant nutrition.

Dynamic Optimisation of Beer Fermentation under Parametric Uncertainty

Fermentation is one of the most important stages in the entire brewing process. In fermentation, the sugars are converted by the brewing yeast into alcohol, carbon dioxide, and a variety of by-products which affect the flavour of the beer. Fermentation temperature profile plays an essential role in the progression of fermentation and heavily influences the flavour. In this paper, the fermentation temperature profile is optimised. As every process model contains experimentally determined parameters, uncertainty on these parameters is unavoidable. This paper presents approaches to consider the effect of uncertain parameters in optimisation. Three methods for uncertainty propagation (linearisation, sigma points, and polynomial chaos expansion) are used to determine the influence of parametric uncertainty on the process model. Using these methods, an optimisation formulation considering parametric uncertainty is presented. It is shown that for the non-linear beer fermentation model, the linearisation approach performed worst amongst the three methods, while second-order polynomial chaos worked the best. Using the techniques described below, a fermentation process can be optimised for ensuring high alcohol content or low fermentation time while ensuring the quality constraints. As we explicitly consider uncertainty in the process, the solution, even though conservative, will be more robust to parametric uncertainties in the model.

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase β-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high β-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for β-lyase activity. Selected phenolic off-flavour positive (POF +) hybrids were further sporulated to generate meiotic segregants with high β-lyase activity, efficient wort fermentation, and lack of POF, all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. Key points • CRISPR/Cas9-based mating-type switching was applied to industrial yeast strains. • Transformed strains could be readily mated to form intraspecific hybrids. • Hybrids exhibited heterosis for a number of brewing-relevant traits.

Migration of Hop Polyphenols in Beer Technology: Model Solution for Various Hopping Methods

Introduction. The research featured the effect of various hopping conditions on the content of polyphenolic compounds associated with the extraction and biotransformation of hop compounds. This mechanism is responsible for uncharacteristic beer flavor in the traditional production method. The research objective was to study the migration routes, influence factors, and changes in the content of hop polyphenols in model experiments in order to reduce various factors in the production process chain. The experiment was important from the point of view of identifying the polyphenols contribution to the beer colloidal system. Study objects and methods. The study involved granulated aromatic hop of Tetnanger variety harvested in 2019, aqueous and 4% aqueous-alcoholic solutions simulating the wort and young beer liquid phase, and brewing yeast Sacharomyces cerevisiae of races Rh (lager) and Nottingham (ale). The work used the generally accepted methods for assessing the content of polyphenolic compounds. Results and discussion. The research established various factors that affected the migration of hop polyphenolic groups. The acidity effect on the polyphenol was established as follows: pH 4.4 contributed to a 12% greater isoxanthohumol accumulation, while pH 5.2 promoted a six times greater accumulation of anthocyanogens than pH 4.4. The total content of polyphenols during boiling was constant and correlated with the phenolic compound in different groups. The conditions of “dry” hopping, simulating the wort clarification in Wirpool, increased the dissolution of anthocyanogens by six times in comparison with kettle hopping, which was associated with the turbulent flow. The isoxanthohumol sorption and formation rate during “dry” hopping was established when modeling the maturation conditions for different temperatures, oxygen levels, and yeast races. A lower temperature (5°C) had a negative effect on the isoxanthohumol sorption. The quercetin content was found to be in the range of 0.9–2.0 mg/dm³ at 5°C and 0.8–4.7 mg/dm³ at 20°C, which determined the temperature effect on extraction during “dry” hopping. The presence of yeast cells in the medium promoted the quercetin accumulation: the quercetin content doubled at 5°C and quadrupled at 20°C compared with the control. The rutin content in the control increased for two days, and minor fluctuations in the content of yeast cells were 5.0 ÷ 7.4 mg/dm3. A comparative analysis of the simple phenolic acids and aldehydes amounts under “dry” hopping conditions showed a greater decrease in their concentration because they were involved in the yeast consumption and biotransformation processes. Conclusion. The research made it possible to establish the phenolic compounds in various groups of migration routes under the conditions of classical (kettle) and “dry” methods of hopping, as well as their dependence on such factors as medium acidity, stirring intensity, temperature, oxygen content, and yeast race. The sorption rates of the polyphenolic compounds were established as follows: absorption of isoxanthohumol was at its highest during the first day of “dry” hopping, and that of rutin – within two days, while quercetin was not absorbed at all. Therefore, an additional fermentation stage can be considered as the most expedient method of “dry” hopping.

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.

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase beta-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high beta-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for beta-lyase activity. Selected phenolic off-flavour positive (POF+) hybrids were further sporulated to generate meiotic segregants with high beta-lyase activity, efficient wort fermentation and lack of POF; all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast.

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.

Residual Brewing Yeast as Substrate for Co-Production of Cell Biomass and Biofilm Using Candida maltosa SM4

Candida maltosa was cultivated in the liquid phase of residual brewing yeast, a major brewery residue, to produce biomass and biofilm. Using response surface methodology, the effect of two variables at two different levels was investigated. The independent variables were agitation speed (at 100 and 200 rpm), and aeration (at 1 and 3 L min−1). Aeration was identified to be important for the production of both biomass and biofilm, while agitation was the only factor significantly affecting biofilm production. The maximal production of biofilm (2.33 g L−1) was achieved for agitation of 200 rpm and aeration of 1 L min−1, while the maximum for biomass (16.97 g L−1) was reached for 100 rpm agitation and 3 L min−1 air flow. A logistic model applied to predict the growth of C. maltosa in the exponential phase and the biofilm production, showed a high degree of agreement between the prediction and the actual biomass measured experimentally. The produced biofilms were further characterized using Fourier-transform infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and Thermogravimetric Analysis (TGA). FTIR allowed the identification of methyl, carbonyl ester and sulfate groups, and revealed the presence of uronic acid moieties and glycosidic bonds. Water-retention ability up to relatively high temperatures was revealed by TGA, and that makes the produced biofilm suitable for production of hydrogels. SEM also gave indications on the hydrogel-forming potential of the biofilm.

INVESTIGATION OF THE FERMENTATION PROCESS OF BEER WORT BASED ON ALTERNATIVE RAW MATERIALS

At present, brewing is one of the most dynamically developing branches of the processing industry. To increase the economic efficiency of production and give beer a varied taste in production, it is proposed to use non-traditional plant raw materials. The replacement of expensive brewing malt with unmalted carbohydrate-containing materials is one of the most important and urgent tasks of the brewing industry. This article examines the fermentation process of beer wort with the addition of apple juice and honey base. The lack of nitrogen-containing and phosphorus-containing compounds in honey and fruit wort, consumed by yeast, leads to a slowdown in the fermentation process, a decrease in the rate of reproduction of yeast, and their fermentation activity. In brewing, the fermentation process is the main one, as a result of which the organoleptic characteristics of the finished beer are formed, therefore, great importance is attached to the optimal composition of the medium for fermentation with brewing yeast. To optimize the composition of the wort, it is proposed to use natural sources of assimilable nitrogen and phosphorus, in particular, milk whey up to 20% by volume. In samples of fermented bases with the addition of milk whey, the concentration of alcohol is higher, and the deeper fermentation of the extract is deeper. The dynamics of the fermentation of apple and honey wort with different amounts of milk whey has been investigated, as a result of which it has been established that the introduction of 20% milk whey into the apple wort makes it possible to obtain a drink with the best organoleptic characteristics, as well as to reduce the duration of fermentation to 5 days. When fermenting honey-based wort, you can limit the addition of whey to 20%, which will reduce the fermentation time by 2 days compared to the control sample.