Stability of high cell density brewery fermentations during serial repitching

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the decreased yeast net growth observed in these high cell density brewery fermentations can adversely affect the physiological stability throughout subsequent yeast generations. Therefore, different O₂ conditions (wort aeration and yeast preoxygenation) were applied to high cell density fermentation and eight generations of fermentations were evaluated together with conventional fermentations. Freshly propagated high cell density populations adapted faster to the fermentative conditions than normal cell density populations. Preoxygenating the yeast was essential for the yeast physiological and beer flavor compound stability of high cell density fermentations during serial repitching. In contrast, the use of non-preoxygenated yeast resulted in inadequate growth which caused (1) insufficient yield of biomass to repitch all eight generations, (2) a 10% decrease in viability, (3) a moderate increase of yeast age, (4) and a dramatic increase of the unwanted flavor compounds acetaldehyde and total diacetyl during the sequence of fermentations. Therefore, to achieve sustainable high cell density fermentations throughout the economical valuable process of serial repitching, adequate yeast growth is essential.     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

Revealing the beneficial effect of protease supplementation to high gravity beer fermentations using "-omics" techniques

Background  

Addition of sugar syrups to the basic wort is a popular technique to achieve higher gravity in beer fermentations, but it results in dilution of the free amino nitrogen (FAN) content in the medium. The multicomponent protease enzyme Flavourzyme has beneficial effect on the brewer's yeast fermentation performance during high gravity fermentations as it increases the initial FAN value and results in higher FAN uptake, higher specific growth rate, higher ethanol yield and improved flavour profile.     

Accumulation and cellular distribution of zinc by brewing yeast

This study focused on the interactions between yeast and zinc in relation to beer fermentations. Yeast accumulation of zinc from growth media, including malt wort, was found to be rapid following inoculation with a brewing strain of Saccharomyces carlsbergensis. In contrast, at the onset of the fermentation, the uptake of other divalent cations such as magnesium and calcium was not as pronounced compared with zinc. At the end of fermentation, both growth media and yeast cells became zinc-depleted, the latter due to dilution of zinc to daughter cells following growth and cell division. In addition, in brewing fermenters, the levels of intracellular zinc were much higher in suspended yeast cells compared with cells that sedimented in the yeast cone at the end of fermentation. This may result in impaired yeast performance in subsequent fermentations if yeast is recycled into low zinc media and if the sub-population is composed by zinc-depleted daughter cells. Cellular uptake of zinc was mediated by a metabolism-dependent mechanism as evidenced by impaired uptake following heat shock. Zinc was thereafter localised in the yeast cell vacuole. As industrial fermentation processes may occasionally be suppressed due to zinc deficiencies, the findings of this study are pertinent for several yeast-based industries, especially beer production.     

Rapid ethanol fermentations using vacuum and cell recycle

Cell recycle and vacuum fermentation systems were developed for continuous ethanol production. Cell recycle was employed in both atmospheric pressure and vacuum fermentations to achieve high cell densities and rapid ethanol fermentation rates. Studies were conducted with Saccharomyces cerevisiae (ATCC No. 4126) at a fermentation temperature of 35°C. Employing a 10% glucose feed, a cell density of 50 g dry wt/liter was obtained in atmospheric-cell recycle fermentations which produced a fermentor ethanol productivity of 29.0 g/liter-hr. The vacuum fermentor eliminated ethanol inhibition by boiling away ethanol from the fermenting beer as it was formed. This permitted the rapid and complete fermentation of concentrated sugar solutions. At a total pressure of 50 mmHg and using a 33.4% glucose feed, ethanol productivities of 82 and 40 g/liter-hr were achieved with the vacuum system with and without cell recycle, respectively. Fermentor ethanol productivities were thus increased as much as twelvefold over conventional continuous fermentations. In order to maintain a viable yeast culture in the vacuum fermentor, a bleed of fermented broth had to be continuously withdrawn to remove nonvolatile compounds. It was also necessary to sparge the vacuum fermentor with pure oxygen to satisfy the trace oxygen requirement of the fermenting yeast.     

An expert system applied to the physiological analysis of early stage of beer fermentation

A fuzzy expert system was applied to the knowledge analysis of yeast physiology in the early stage of beer fermentation, when the wort was aerated. We used ergosterol and glycogen concentration in the wort as a suitable marker of physiological state of the cell population. The amount of both compounds influences the rate of fermentation, cell growth and the final taste of beer. The concentrations of ergosterol and glycogen including the number of cells can not be measured immediately during the relatively short aeration period, and incomplete experimental data are therefore found in laboratory logbooks. We therefore suggested that the fuzzy relation between the directly measurable dissolved oxygen concentration and the rate of ergosterol or glycogen formation should be identified and a fuzzy expert system should be used to analyze the behavior of the yeast.     

A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions

High gravity (HG) or very high gravity (VHG) brewing has become popular in modern breweries due to its economic and product quality advantages. However, there are the negative impacts such as the fermentation performance of brewer??s yeast in HG or VHG wort, which are closely related to changes in cell physiological activity. In the present study, 3 kinds of worts, with different gravities, were used to examine the systematic effects on fermentation performance and physiological activity of lager yeast FBY009505 (Saccharomyces pastorianus) and ale yeast FBY0099 (Saccharomyces cerevisiae), as well as the resulting beer flavor. Results showed that the responses of FBY009505 and FBY0099 to the HG or VHG worts were similar. The specific fermentation rate and viability of cropped yeast of FBY009505 and FBY0099 were decreased with increasing wort gravity. The increased wort gravity resulted in the increase of energy charge and the decrease of ??-glucosides transport rate and glycolytic enzyme activities. Moreover, the environmental stresses in the HG or VHG wort showed a higher inhibitory activity against ??-glucoside transport than glycolytic enzymes. The content of intracellular trehalose and glycerol of FBY009505 and FBY0099 increased with the increase in wort gravity. The results from this study provided a potential means to systematically understand the physiology of brewer??s yeast under HG or VHG conditions.     

Net effect of wort osmotic pressure on fermentation course, yeast vitality, beer flavor, and haze

The net effect of increased wort osmolarity on fermentation time, bottom yeast vitality and sedimentation, beer flavor compounds, and haze was determined in fermentations with 12° all-malt wort supplemented with sorbitol to reach osmolarity equal to 16° and 20°. Three pitchings were performed in 12°/12°/12°, 16°/16°/12°, and 20°/20°/12° worts. Fermentations in 16° and 20° worts decreased yeast vitality measured as acidification power (AP) by a maximum of 10%, lowered yeast proliferation, and increased fermentation time. Repitching aggravated these effects. The 3rd “back to normal” pitching into 12° wort restored the yeast AP and reproductive abilities while the extended fermentation time remained. Yeast sedimentation in 16° and 20° worts was delayed but increased about two times at fermentation end relative to that in 12° wort. Third “back-to-normal” pitching abolished the delay in sedimentation and reduced its extent, which became nearly equal in all variants. Beer brewed at increased osmolarity was characterized by increased levels of diacetyl and pentanedione and lower levels of dimethylsulfide and acetaldehyde. Esters and higher alcohols displayed small variations irrespective of wort osmolarity or repitching. Increased wort osmolarity had no appreciable effect on the haze of green beer and accelerated beer clarification during maturation. In all variants, chill haze increased with repitching.     

Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast

During fermentation, the yeast Saccharomyces cerevisiae produces a broad range of aroma-active substances, which are vital for the complex flavour of beer. In order to obtain insight into the influence of high-gravity brewing and fermentation temperature on flavour formation, we analysed flavour production and the expression level of ten genes (ADH1, BAP2, BAT1, BAT2, ILV5, ATF1, ATF2, IAH1, EHT1 and EEB1) during fermentation of a lager and an ale yeast. Higher initial wort gravity increased acetate ester production, while the influence of higher fermentation temperature on aroma compound production was rather limited. In addition, there is a good correlation between flavour production and the expression level of specific genes involved in the biosynthesis of aroma compounds. We conclude that yeasts with desired amounts of esters and higher alcohols, in accordance with specific consumer preferences, may be identified based on the expression level of flavour biosynthesis genes. Moreover, these results demonstrate that the initial wort density can determine the final concentration of important volatile aroma compounds, thereby allowing beneficial adaptation of the flavour of beer.     

Isolation and utilization of indigenous yeast strain for brewing of millet beer (OYOKPO)

Three yeast strains were isolated from a spontaneously fermented native millet (Pennisetum typhoideum) malt beer (Oyokpo). One of the yeast isolates found to have the most highly fermenting capacity was characterised and identified as a strain of Saccharomyces cerevisiae. The yeast was then utilised as the pitching yeast in a subsequent controlled fermentation of millet wort at 20°C for 120 hours. Bitter leaf (Vernonia amagdalina) extract was used as the bittering and flavouring agent. The Oyokpo beer sample produced under these conditions was found to possess both chemical and organoleptic qualities comparable to some extent, to the conventional barley malt beer. At the end of fermentation, the pH, specific gravity, alcohol content, reducing sugar content and protein content of the beer were 4.11, 1.0308, 2.81% (v/v), 4.00 (mg/ml) and 0.84 (mg/ml) respectively.     

Effect of hot trub and particle addition on fermentation performance of Saccharomyces cerevisiae

In this investigation, the effect of hot trub (a precipitation product of the wort boiling process in beer manufacturing) addition on fermentation performance was observed under variation of yeast vitality, and origin and the amount of hot trub. Its addition improved suspended cell concentrations for all yeast vitalities tested, and the more trub was added, the greater the effect. Further, pilot-scale fermentations showed significantly lower pH values and an accelerated extract degradation, thus, advancing fermentation by roughly 1 day for hot trub addition versus the fermentation of extremely bright wort. Since the positive effect of trub has often been associated with its particulate characteristics, fermentations with fractionated model particles, such as poly(vinylpyrrolidones) and kieselguhr, of different particle sizes were carried out under variation of yeast vitality and particle amounts. The addition of both particle types also improved fermentation performance, however, the effect was not as great as that of hot trub. Particulate material may improve the development of CO₂ from the fermenting medium, thus reducing its concentration and inhibitory effect on yeast metabolism. The most effective fraction of kieselguhr had a 40 μm peak which also occurred in particle size distributions of all hot trubs investigated. This could be of particular interest when discussing particle effects.     

Proteases supplementation to high gravity worts enhances fermentation performance of brewer's yeast

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, results in poor yeast viability and even stuck or sluggish fermentations. Although wort contains abundant proteins and longer chain peptides, brewer's yeast does not assimilate them due to the fact that cells hardly secrete proteases during fermentation. The objective of this study was to investigate the possibility for utilizing unavailable nitrogen from two types of high gravity worts (20 °P and 24 °P) by adding three food-grade commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of fermentations, respectively. Results showed that proteases supplementation significantly increased the FAN level and thus the amount of cell suspension in the later stages of fermentations (ca. 10 days later for 20 °P and 25 days later for 24 °P) (p < 0.05). Among the studied three proteases, we found that fermentations with Flavorzyme supplementation exhibited the best fermentation performance in terms of significantly improved wort fermentability, higher ethanol yield and flavor volatiles formation (p < 0.05). Furthermore, the foam of final beers produced by adding proteases was as stable as that of the control at each of the corresponding gravities.     

Immobilization of yeast cells in PVA particles for beer fermentation

In this study, the potential of application of non-aggressive LentiKat^® technique for brewer's yeast immobilization on polyvinyl alcohol was assessed. High cell loads of about 10⁹ cells/ml were achieved by this procedure and immobilization procedure had no adverse effect on cell viability. The stability and activity of obtained immobilized biocatalyst was tested in the growth studies and fermentations. Immobilized cells exhibited high fermentation activity in both, laboratory and pilot-scale fermentations. In three successive gas-lift reactor fermentations the apparent attenuation of around 80% was reached after only 2 days, indicating good potential of immobilized cells for development of continuous primary beer fermentation. LentiKat^® particles showed high mechanical and fermentative stability, since they endured 30 days of operating time during 6-month period without significant change of cell activity, particle shape and particle size.     

Analysis of the effect of inoculum characteristics on the first stages of a growing yeast population in beer fermentations by means of an individual-based model

The yeast Saccharomyces cerevisiae has a limited replicative lifespan. The cell mass at division is partitioned unequally between a larger, old parent cell and a smaller, new daughter cell. Industrial beer fermentations maintain and reuse yeast. At the end of fermentation a portion of the yeast is ‘cropped’ from the vessel for ‘serial repitching’. Harvesting yeast may select a population with an imbalance of young and aged individuals, but the output of any bioprocess is dependent on the physiology of each single cell in the population. Unlike continuous models, individual-based modelling is an approach that considers each microbe as an individual, a unique and discrete entity, with characteristics that change throughout its life. The aim of this contribution is to explore, by means of individual-based simulations, the effects of inoculum size and cell genealogical age on the dynamics of virtual yeast fermentation, focussing on: (1) the first stages of population growth, (2) the mean biomass evolution of the population, (3) the rate of glucose uptake and ethanol production, and (4) the biomass and genealogical age distributions. The ultimate goal is to integrate these results in order to make progress in the understanding of the composition of yeast populations and their temporal evolution in beer fermentations. Simulation results show that there is a clear influence of these initial features of the inocula on the subsequent growth dynamics. By contrasting both the individual and global properties of yeast cells and populations, we gain insight into the interrelation between these two types of data, which helps us to deal with the macroscopic behaviour observed in experimental research.     

Control and prediction of the course of brewery fermentations by gravimetric analysis

A simple, fast and cheap test suitable for predicting the course of brewery fermentations based on mass analysis is described and its efficiency is evaluated. Compared to commonly used yeast vitality tests, this analysis takes into account wort composition and other factors that influence fermentation performance. It can be used to predict the shape of the fermentation curve in brewery fermentations and in research and development projects concerning yeast vitality, fermentation conditions and wort composition. It can also be a useful tool for homebrewers to control their fermentations.     

Continuous production of non-alcohol beer by immobilized yeast at low temperature

Summary A system for production of non-alcohol beer is described. A limited fermentation is carried out with immobilized cells ofSaccharomyces cerevisiae in a packed bed reactor. In the reactor, combined stress factors such as low temperature (2–4°C) and anaerobic conditions limit cell metabolism. Of the available sugars only a small amount of glucose is metabolized, resulting in low concentrations of ethanol (<0.08%). The absence of oxygen affects the redox balance of the yeast cell, and thus stimulates formation of esters and higher alcohols. Products are formed by reduction of wort aldehydes, as well as reduction of intracellular metabolites. Despite the stress conditions, biomass increases during prolonged production periods. In batch experiments,S. cerevisiae strain W34 grows at low temperatures and a mininum growth temperature of –2 °C was found, indicating that a further reduction of temperature during production will not inhibit growth. The characteristics of the system allow its use in very different applications. Potential applications of the immobilized system are discussed.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Isolation and Characterization of Brewer's Yeast Variants with Improved Fermentation Performance under High-Gravity Conditions

To save energy, space, and time, today's breweries make use of high-gravity brewing in which concentrated medium (wort) is fermented, resulting in a product with higher ethanol content. After fermentation, the product is diluted to obtain beer with the desired alcohol content. While economically desirable, the use of wort with an even higher sugar concentration is limited by the inability of brewer's yeast (Saccharomyces pastorianus) to efficiently ferment such concentrated medium. Here, we describe a successful strategy to obtain yeast variants with significantly improved fermentation capacity under high-gravity conditions. We isolated better-performing variants of the industrial lager strain CMBS33 by subjecting a pool of UV-induced variants to consecutive rounds of fermentation in very-high-gravity wort (>22° Plato). Two variants (GT336 and GT344) showing faster fermentation rates and/or more-complete attenuation as well as improved viability under high ethanol conditions were identified. The variants displayed the same advantages in a pilot-scale stirred fermenter under high-gravity conditions at 11°C. Microarray analysis identified several genes whose altered expression may be responsible for the superior performance of the variants. The role of some of these candidate genes was confirmed by genetic transformation. Our study shows that proper selection conditions allow the isolation of variants of commercial brewer's yeast with superior fermentation characteristics. Moreover, it is the first study to identify genes that affect fermentation performance under high-gravity conditions. The results are of interest to the beer and bioethanol industries, where the use of more-concentrated medium is economically advantageous.     

Effects of Lys and His supplementations on the regulation of nitrogen metabolism in lager yeast

Significant positive correlations between wort fermentability and the assimilation of Lys and His under normal-gravity and high-gravity conditions indicated that Lys and His were the key amino acids for lager yeast during beer brewing. In order to obtain insight into the roles of Lys and His in nitrogen regulation, the influences of Lys, His and their mixture supplementations on the fermentation performance and nitrogen metabolism in lager yeast during high-gravity fermentation were further investigated in the present study. Results showed that Lys and His supplementations improved yeast growth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lys supplementation up-regulated Ssy1p–Ptr3p–Ssy5p (SPS)-regulated genes (LYP1, HIP1, BAP2 and AGP1) dramatically compared to nitrogen catabolite repression (NCR)-sensitive genes (GAP1 and MEP2), whereas His supplementation activated SPS-regulated genes slightly in exponential phase, and repressed NCR-sensitive genes significantly throughout the fermentation. Lys and His supplementations increased the consumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover, Lys and His supplementations exhibited similar effects on the fermentation performance, and were more effective than their mixture supplementation when the same dose was kept. These results demonstrate that both Lys and His are important amino acids for yeast nitrogen metabolism and fermentation performance.     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

Influence of aeration during propagation of pitching yeast on fermentation and beer flavor

The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.