Serial Repitching Research Articles

Systematic profiling of ale yeast protein dynamics across fermentation and repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is among the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout 2 fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

Functional diversity and plasticity in the sugar preferences of Saccharomyces MALT transporters in domesticated yeasts.

Maltose and maltotriose, together with glucose, are the major carbohydrates found in malts. Thus, brewing yeasts grown in malt-based brewing processes with serial re-pitching have likely increased their ability to uptake these sugars during domestication by modulating the expression and copy number of maltose transporter genes (MALT, also known as Malx1). However, the molecular basis for and structural insights into the sugar preferences of MALT proteins remain to be elucidated. Here we report the functional evaluation of two novel Saccharomyces cerevisiae MALT proteins, ScMalt#2p and ScMalt#5p, from industrial brewing yeasts, focusing on their maltose and maltotriose preferences. Structural models of the MALT proteins generated by AlphaFold2 and functional analyses of substitution mutants revealed that a very small number of amino acid residues in two spatially adjacent transmembrane helixes, TMH7 and TMH11, appear to be crucial for sugar preference. Thus, subtle conformational alterations conferred by a small number of amino acid polymorphisms within MALTs would contribute to the adaptation of domesticated brewing yeasts to the constrained carbohydrate environment of industrial wort during beer brewing.

Comparing the efficiency of applying yeast of different generations in high gravity brewing

When fermenting beer, in addition to the formation of basic, secondary, and by-products, which largely determine the taste and aroma of the beverage, yeast is accumulated. Such yeasts can be reused in subsequent serial repitching, which allows cost savings compared to yeast cultivated for each fermentation. This paper investigates the influence of yeast generation on the progress of the wort fermentation process and the quality of beer obtained, specifically in high gravity brewing. It was established that the increase in the number of yeast generation in the range from the first to the fourth generation causes a slight acceleration of the fermentation process of high-gravity wort, while at higher values of the fermentation generation number it slows down. Yeasts secreted after six or four serial wort fermentations with a gravity of 15 °P/18 °P, respectively, have a good physiological state and can be reused. The yeast generation number does not significantly affect ethanol content and sensory beer quality indicators. However, the use of yeast of the seventh and eighth generation causes a slight increase in the pH and color of high gravity beer, the appearance in the aroma of noticeable extraneous shades ‒ yeast tone, tone of young beer. As wort gravity increases, the number of yeast re-use cycles decreases, which is a consequence of the influence of various stress factors, specifically high osmotic pressure, and increased ethanol levels. Therefore, for the fermentation of wort with a gravity of 15 °P, it is recommended to use yeast up to five generations, for 18 °P wort ‒ to four generations. To obtain beer with appropriate physicochemical parameters and high sensory evaluation in high gravity brewing technology, it is advisable to ferment wort with a gravity of 15 °P with yeast for up to five generations.

​Laboratory Scale Study of the Effect of Wort Gravity International Bitterness Unit Levels and Serial Repitching on Viability of an Ale Yeast Strain

​Laboratory Scale Study of the Effect of Wort Gravity International Bitterness Unit Levels and Serial Repitching on Viability of an Ale Yeast Strain

Serial Repitching Method for New England IPAs with Midfermentation Dry Hopping

Serial Repitching Method for New England IPAs with Midfermentation Dry Hopping

Does Inter-Organellar Proteostasis Impact Yeast Quality and Performance During Beer Fermentation?

During beer production, yeast generate ethanol that is exported to the extracellular environment where it accumulates. Depending on the initial carbohydrate concentration in the wort, the amount of yeast biomass inoculated, the fermentation temperature, and the yeast attenuation capacity, a high concentration of ethanol can be achieved in beer. The increase in ethanol concentration as a consequence of the fermentation of high gravity (HG) or very high gravity (VHG) worts promotes deleterious pleiotropic effects on the yeast cells. Moderate concentrations of ethanol (5% v/v) change the enzymatic kinetics of proteins and affect biological processes, such as the cell cycle and metabolism, impacting the reuse of yeast for subsequent fermentation. However, high concentrations of ethanol (> 5% v/v) dramatically alter protein structure, leading to unfolded proteins as well as amorphous protein aggregates. It is noteworthy that the effects of elevated ethanol concentrations generated during beer fermentation resemble those of heat shock stress, with similar responses observed in both situations, such as the activation of proteostasis and protein quality control mechanisms in different cell compartments, including endoplasmic reticulum (ER), mitochondria, and cytosol. Despite the extensive published molecular and biochemical data regarding the roles of proteostasis in different organelles of yeast cells, little is known about how this mechanism impacts beer fermentation and how different proteostasis mechanisms found in ER, mitochondria, and cytosol communicate with each other during ethanol/fermentative stress. Supporting this integrative view, transcriptome data analysis was applied using publicly available information for a lager yeast strain grown under beer production conditions. The transcriptome data indicated upregulation of genes that encode chaperones, co-chaperones, unfolded protein response elements in ER and mitochondria, ubiquitin ligases, proteasome components, N-glycosylation quality control pathway proteins, and components of processing bodies (p-bodies) and stress granules (SGs) during lager beer fermentation. Thus, the main purpose of this hypothesis and theory manuscript is to provide a concise picture of how inter-organellar proteostasis mechanisms are connected with one another and with biological processes that may modulate the viability and/or vitality of yeast populations during HG/VHG beer fermentation and serial repitching.

Serial re-pitching: its effect on yeast physiology, fermentation performance, and product quality

Serial re-pitching is a term given to a practice whereby yeast harvested at the end of fermentation is re-used in subsequent fermentations. The purpose of this paper was to review and summarize existing literatures, research data, and case studies to illustrate the effect of re-pitching on the physiology and fermentation performance of brewing yeast and the resulting quality of beer. Data related to biomarkers used to assess yeast physiology and fermentation performance and quality of beer were compared for various articles. And comparison of the results was done with caution as many of the studies were conducted using different yeast strains, wort gravity, pitching rate, and other fermentation conditions. This study confirms that serial re-pitchings aggravate the effect of pitching rate, wort gravity, cell age, yeast oxygenation, and yeast strain on yeast cell physiology, fermentation performance, and quality of final beer. However, further empirical research at molecular level is crucial.

Physiological Changes of Beer Brewer's Yeast During Serial Beer Fermentation

Serial repitching of beer brewer’s yeast plays an important role in the beer industry as an ineluctable economic factor. In this study, the viability and vitality changes, as well as strains’ anti-autolytic abilities during serial beer fermentation with typical ale and lager yeast strains, were investigated. While measuring the survival rate of yeast cells is not sufficient for evaluating and predicting the yeast fermentation capacity, physiological status determination of brewer’s yeast reflects the vitality and quality of yeast in serial beer fermentations. Accumulation of reactive oxygen species in yeast causes cell damage, leading to a decline in cell vitality and viability. Aged yeast cells, after several repitchings, can result in leakage of intracellular compounds into the fermented liquor. The lager yeast (Pilsner) examined, which harbors the partial genome from S. eubayanus, showed better robustness and higher activity than the ale yeast strain M79 examined, during serial fermentation. A holistic approach, including more indicators, should be applied in evaluating the fermentation performance of brewer’s yeast.

Physiological changes of Saccharomyces cerevisiae during serial re-pitching: a review

The morphological structure and physiological indexes of Saccharomyces cerevisiae have changed during serial re-pitching due to the stress conditions in serial handlings and the cells become aging. It is of great significance to study the physiological changes of S. cerevisiae during serial re-pitching to understand the anti-aging effect of S. cerevisiae. In this paper, the changes of the physiological indexes during re-pitching of yeast are summarized, and based on the analysis of the previous works further research directions are proposed.

Part II: the influence of the serial repitching ofSaccharomyces pastorianuson the uptake dynamics of amino acids during the fermentation of barley and gluten-free buckwheat and quinoa wort

The present paper is part of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of the barley, buckwheat and quinoa fermentation medium. In particular, it focuses on the uptake dynamics of amino acids during 11 successive fermentations. Samples were taken every 20 h after pitching, analysed for the particular amino acid content and statistically evaluated. The term ‘completion time’ (t95), here defined as the percentage attenuation time necessary for ~95% of the total assimilation, has been introduced. In addition, ‘the serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Amino acids that were essentially affected by serial repitching were glutamine, arginine, alanine and tryptophan in barley, aspartate, glutamate and tryptophan in buckwheat, and all in the quinoa wort fermentation. As opposed to buckwheat and quinoa, in barley the amino acids behaved more or less independently from each other, which for buckwheat and quinoa indicates a more general systemic change in the yeast. From the amino acids point of view, buckwheat can be fully regarded as a suitable gluten-free substitute for barley beer since the amino acid assimilation was very consistent and hardly influenced by the serial repitching, especially regarding the final amino acid assimilation. In the case of quinoa, the assimilation of all amino acids became significantly affected after the sixth fermentation and quinoa is probably unsuitable for the production of beer-like beverages. Results suggest no substitutional potential of quinoa for barley beer, but if a nutrient-rich beverage of choice from quinoa malt is intended to be prepared, it seems that the serial repitching is limited to six fermentations at most. Copyright © 2015 The Institute of Brewing & Distilling

Part III: the influence of serial repitching ofSaccharomyces pastorianuson the production dynamics of some important aroma compounds during the fermentation of barley and gluten-free buckwheat and quinoa wort

The present paper is the last report of a comprehensive study regarding the influence of the serial repitching of Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat or quinoa fermentation medium. In particular, it focuses on the production dynamics of important volatile compounds typically associated with the aroma of beer. Samples were taken every 24 h after 11 serial repitchings of a single starter culture, analysed for the particular aroma compound content by distillation followed by gas chromatography with flame ionization detection. The term ‘serial repitching factor’ is used for the first time to support the visual evaluation of the influence of serial repitching. Results showed that the levels of methanol in the quinoa wort fermentation were only slightly higher than in barley and in practical terms independent of successive fermentation. The behaviour of acetaldehyde in quinoa was similar to that in barley. However, there was a final 2-fold lower production of some important aroma compounds compared with barley and buckwheat and for this reason quinoa cannot be recommended as a gluten-free substitute to produce a bottom-fermented beer. Regarding the buckwheat wort fermentation, a 2- to 3-times lower final acetaldehyde content than in barley is desirable, whereas a relatively high methanol content is not desirable. Barley and buckwheat showed comparable sum concentrations and similar overall profiles of some important aroma compounds. From this perspective, buckwheat appears to be a promising substitute for barley as a brewing raw material. The overall conclusions of our comprehensive study (Parts I–III) are that buckwheat shows adequate brewing properties to substitute for barley in the commercial preparation of a bottom-fermented gluten-free beer-like beverage, and yeast can be repitched at least 11 times. In contrast, quinoa in practical terms shows no substitutional potential for barley in beer; however, it has many nutritious advantages, thus the commercial preparation of a unique, bottom-fermented gluten-free ‘non-beer-like’ beverage – where the yeast could be repitched six times at most – appears feasible. Copyright © 2015 The Institute of Brewing & Distilling

Part I: the influence of serial repitching ofSaccharomyces pastorianuson the uptake dynamics of metal ions and fermentable carbohydrates during the fermentation of barley and gluten-free buckwheat and quinoa wort

Recently, research has been focusing on the use of alternative raw materials for brewing purposes and gluten-free beer-like beverages from malted buckwheat and quinoa are of commercial interest. A common commercial process involves the serial repitching of the yeast biomass, but this has not been described using buckwheat and quinoa wort fermentations. Our research studies (Parts I–III) explored the serial repitching of the yeast strain Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat and quinoa fermentation medium. The present paper focuses on the fermentation performance and the uptake dynamics of metal ions and fermentable carbohydrates. Both pseudocereals showed high variations in all of the attributes examined during successive fermentations. In buckwheat the differences between successive fermentations were similar to those observed with barley, whereas differences in quinoa varied quite significantly from those observed with barley and showed a directional trend, suggesting a general weakening of the yeast from the sixth successive fermentation onward. In particular, the assimilation of the fermentable carbohydrates lessened and metal ion uptake appeared poorly controlled. It was concluded that buckwheat showed good potential for serial repitching of S. pastorianus TUM 34/70, whereas serial repitching of a quinoa wort appeared to be limited to five or six fermentations. Copyright © 2015 The Institute of Brewing & Distilling

Recycling microbial lipid production wastes to cultivate oleaginous yeasts

To reduce wastes and the costs of microbial lipid production, it is imperative to recycle resources, including spent cell mass, mineral nutrients and water. In the present study, lipid production by the oleaginous yeast Rhodosporidium toruloides was used as a model system to demonstrate resources recycling. It was found that the hydrolysates of spent cell mass were good media to support cell growth of various oleaginous yeasts. When serial repitching experiments were performed using 70g/L glucose and the hydrolysates alone as nutrients, it produced 16.6, 14.6 and 12.9g/L lipids, for three successive cycles, while lipid titre remained almost constant when spent water was also recycled. The cell mass hydrolysates could be used as equivalents to the mixture of yeast extract and peptone to support lipid production from corn stalk hydrolysates. Our results showed efficient recycling of lipid production wastes and should be helpful to advance microbial lipid technology.

LIVE/DEAD YEAST VIABILITY STAINING AS A TOOL FOR IMPROVING ARTISANAL PILSNER BEER PRODUCTION

The production of an artisanal beer, made by brewers using traditional practices on a small scale, is founded on the empirical adjustment of parameters, including yeasts handling and serial repitching. The aim of this study was to monitor yeast viability during different stages of artisanal beer productions through the Live/Dead Yeast viability staining and to correlate it with fermentation dynamics in order to increase process standardization and to maintain the quality of final products. Yeast viability and fermentation activities were evaluated during seven fermentation cycles of an artisanal pilsner beer. Yeast inoculated with higher viability performed generally better in fermentation, resulting in faster sugar consumption, faster ethanol production and stability. Handling yeast and serial repitching based on Live/Dead viability measurements, could be the key way to ensure reliable manufacture of high quality beer and to improve process standardization particularly for microbreweries, where variability of production can be a challenging point.

Analysis of aging in lager brewing yeast during serial repitching

Serial repitching of brewing yeast inoculates is an important economic factor in the brewing industry, as their propagation is time and resource intensive. Here, we investigated whether replicative aging and/or the population distribution status changed during serial repitching in three different breweries with the same brewing yeast strain but different abiotic backgrounds and repitching regimes with varying numbers of reuses.Next to bud scar numbers the DNA content of the Saccharomyces pastorianus HEBRU cells was analyzed. Gene expression patterns were investigated using low-density microarrays with genes for aging, stress, storage compound metabolism and cell cycle.Two breweries showed a stable rejuvenation rate during serial repitching. In a third brewery the fraction of virgin cells varied, which could be explained with differing wort aeration rates. Furthermore, the number of bud scars per cell and cell size correlated in all 3 breweries throughout all runs. Transcriptome analyses revealed that from the 6th run on, mainly for the cells positive gene expression could be seen, for example up-regulation of trehalose and glycogen metabolism genes. Additionally, the cells’ settling in the cone was dependent on cell size, with the lowest and the uppermost cone layers showing the highest amount of dead cells.In general, cells do not progressively age during extended serial repitching.

The influence of serial repitching of Saccharomyces pastorianus on its karyotype and protein profile during the fermentation of gluten-free buckwheat and quinoa wort

Gluten-free beer-like beverages from malted buckwheat and quinoa are somehow close to their commercial production, but rather high expenses are expected due to the relatively high price of grain, some technological adaptations of process and the need for external enzyme supplementation during mashing. One of the common and efficient cost reduction measures in the industrial scale is serial repitching of the yeast biomass, which has not been studied for the buckwheat and quinoa wort fermentation before. In that manner we have monitored possible changes in yeast's proteins and chromosomal DNA during eleven serial repitchings of the yeast Saccharomyces pastorianus strain TUM 34/70 for fermentation of the barley, buckwheat and quinoa wort. Karyotypes showed changes in regard to the raw materials used and many responsible candidate proteins are suggested which could cause these differences. Different relative expressions of some protein bands were also linked to the proteins involved in yeast stress response and proteins involved in fermentation performance. Results suggest that serial repitching of the strain TUM 34/70 seems suitable for the production of gluten-free beer-like beverages from buckwheat and quinoa.

Sustainability of industrial yeast serial repitching practice studied by gene expression and correlation analysis

Bottom-fermenting Saccharomyces pastorianus strains driving brewing fermentation processes are usually reused several times. It is still unclear, whether the number of successions may have an impact on cell physiology prompting consequences for brewing quality. In this study, fermentation performance of up to twenty consecutive runs in a brewery was investigated. For each run mRNA expression levels of cellular marker molecules, which are known to correlate with metabolism, hexose transport, aging processes, stress response mechanisms and flocculation capability was estimated to obtain information on changes in cell physiology over the successive runs. Low-density microarrays were used for this purpose and the resulting gene expression profiles were finally correlated with changes in the abiotic micro-environments. A surprising stability of the marker molecule expression profiles within each specific serial repitching was stated. Loss of flocculation or an advanced aging could not be detected during serial repitching in the analyzed brewery. However, certain runs of the serial repitchings showed high variation in stress response which was found to be caused by perturbations of the abiotic conditions. Regardless, the study showed that S. pastorianus can be used repeatedly in serial repitching processes without loss of prominent physiological characteristics.

Evaluation of Brewer’s Spent Yeast To Produce Flavor Enhancer Nucleotides: Influence of Serial Repitching

The present work evaluates the influence of serial yeast repitching on nucleotide composition of brewer's spent yeast extracts produced without addition of exogenous enzymes. Two procedures for disrupting cell walls were compared, and the conditions for low-cost and efficient RNA hydrolysis were selected. A HILIC methodology was validated for the quantification of nucleotides and nucleosides in yeast extracts. Thirty-seven samples of brewer's spent yeast ( Saccharomyces pastorianus ) organized according to the number of serial repitchings were analyzed. Nucleotides accounted for 71.1-88.2% of the RNA products; 2'AMP was the most abundant (ranging between 0.08 and 2.89 g/100 g dry yeast). 5'GMP content ranged between 0.082 and 0.907 g/100 g dry yeast. The sum of 5'GMP, 5'IMP, and 5'AMP represented between 25 and 32% of total nucleotides. This works highlights for the first time that although serial repitching influences the content of monophosphate nucleotides and nucleosides, the profiles of these RNA hydrolysis products are not affected.

Does Generation Number Matter? The Impact of Repitching on Wort Utilization

Serial repitching is an accepted practice in brewing. The number of completed fermentations (generation number) is the metric applied to brewing yeast fitness for fermentation. Despite this, fermentations that use freshly propagated (generation 0) yeast are deemed to be inefficient when compared to yeast that has been repitched one or more times. The aim of this study was to investigate the impact of yeast generation number (equating to the number of fermentations completed) on wort utilization and fermentation progression. Wort composition and pH downshift were used to assess “fitness to ferment” of yeast generations within laboratory scale fermentations. Differences in pH, FAN, and amino acid assimilation were observed.

Incidence and Characterization of Petites Isolated from Lager Brewing Yeast Saccharomyces cerevisiae Populations

Repeated reuse or “serial repitching” of yeast between successive fermentations exposes brewing yeast cells to a number of biological, chemical, and physical stresses. It is generally accepted that...