Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

Continuous beer production was investigated in a high cell-density culture system which consisted of two stages for the fermentation and sedimentation of yeast cells. The continuous culture was carried out for a fermentation time of 5,500 h without contamination, at varying dilution rates and fermentation temperatures in the ranges of 0.017-0.033 h⁻¹ and 6.5–8.5°C, respectively. This process was found to be suitable for continuous and stable beer brewing. Under these conditions, the cell concentration in the first stage was about 80 times as high as that in the exit of the second stage. Concentrations of viable cells, sugar and ethanol were maintained at 1.3 × 10⁹ cells/ml, 25 and 36 g/l, respectively, and were hardly affected by fermentation temperature. Concentrations of ethyl acetate, isoamyl alcohol and isoamyl acetate were similar in the fermentation temperature ranges of 6.5–8.5°C, and the amounts at a fermentation temperature of 7°C were comparable to those of lager-type beer. Diacetyl flavor, which is known to be an effluent component that causes deterioration in the second stag e (young beer), was maintained at 1.2 ppm at a dilution rate and fermentation temperature of 0.022 h⁻¹ and 7°C, respectively. The diacetyl flavor was due to the accumulation of vicinal diketone, the precursor of which is acetohydroxy acid. The acetohydroxy acid was converted to vicinal diketone by pretreatment at 60°C for 30 min. The vicinal diketone was then consumed by the yeast during after-fermentation at a fermentation temperature of 3°C. Using this method, total vicinal diketone decreased below 0.3 ppm for an after-fermentation time of 6.8 h, which was 225 times as fast as that of after-fermentation without the pretreatment. This process may make it possible to achieve continuous beer fermentation from the fermentation stage to after-fermentation for diacetyl removal.     

Reliable estimation of the key variables and of their rates of change in alcoholic fermentation

The paper establishes a rigorous probabilistic framework for the reconciliation of apparently conflicting data from various physical and chemical measurements related to the key biological variables of alcoholic fermentation: the ethanol and the residual sugar concentrations. The analysis is carried out on a database consisting of 15 beer fermentation experiments, for which off-line determinations of ethanol concentration, fermentable sugar concentration, wort density and refractive index are available, as well as on-line records of evolved CO₂. The basic reconciliation method uses mass balance and monotonicity constraints derived from the biological knowledge of the fermentation process. In order to provide interpolated values and rate estimates, smoothness requirements are added. The reconciliation procedure gives more reliable estimates than any given measurement, detects outliers, helps fixing problems in the experimental setting and is also applicable on line.     

Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method

The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.     

几株啤酒酵母的筛选及发酵性能比较*

啤酒酵母是啤酒酿造的灵魂,可以直接影响啤酒品质。在啤酒酿造过程中,由于啤酒酵母被多次传代和保藏,造成优良菌种发酵性能衰退等问题,导致发酵不彻底,影响最后啤酒的风味质量。为此以8株Lager型啤酒酵母为出发菌株,通过平板分离纯化获得80株分离菌株,再经过三角瓶发酵初筛和复筛、发酵罐中试发酵实验最终获得了8株发酵性能优良的啤酒酵母。其中,6株酵母可应用于酿造双乙酰含量低于0.1 mg/L的啤酒;3株酵母发酵度高于70%,适合酿造干啤酒;1株酵母发酵度低于50%,适合酿造低醇啤酒。在风味方面:1株酵母酿造的啤酒醇酯比为3.3,啤酒酯香味较突出;另1株酵母酿造的啤酒醇酯比为4.5,啤酒高级醇含量较高。8株经过选育的啤酒酵母发酵特征明显,便于精酿啤酒厂实际应用。     

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.     

Simultaneous control of apparent extract and volatile compounds concentrations in low-malt beer fermentation

Volatile compounds cause undesirable flavor when their concentrations exceed threshold values in beer fermentation. The objective of this study is to develop a system for controlling apparent extract concentration, which indicates the fermentation degree and which should be decreased below a targeted value at a fixed time under a constraint of tolerable amounts of volatile compounds. In beer fermentation, even though the production of volatile compounds is suppressed by maintaining a low fermentation temperature, a low temperature causes a delay in the control of apparent extract concentration. Volatile compound concentration was estimated on-line, and the simulation of apparent extract consumption and volatile compound production was performed. To formulate various beer tastes and conserve energy for attemperation, optimal temperature profiles were determined using a genetic algorithm (GA). The developed feedback control of the brewing temperature profile was successfully applied, and apparent extract and volatile compound concentrations at a fixed time reached their target concentrations. Additionally, the control technique developed in this study enables us to brew a wide variety of beers with different tastes.     

Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling

The use of the enzyme alpha-acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate-limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated alpha-acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on alpha-acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated alpha-acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free alpha-acetolactate decarboxylase. The advantage of immobilized alpha-acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.     

Ethanol production from sugar cane segments in a high solids drum fermentor

Summary A successful yeast fermentation for the production of relatively high concentration of ethanol (9% w/v) was carried out using sugar cane segments. Extraction of sugar from segments occurred simultaneously with ethanol formation. The beer produced was transferred to a fresh batch of sugar cane segments and the fermentation cycle was repeated successively three times with the same beer. A high cane to water ratio was obtained in a rotating drum fermentor which allowed for a minimal amount of liquid to be used during the fermentation process.     

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.     

多级串联悬浮床反应器系统中自絮凝颗粒酵母乙醇连续发酵耦合废糟液直接全循环使用的研究

在一套四级串联悬浮床生物反应器系统中,以双酶法制备的玉米粉糖化液为底物,进行了废糟液全循环条件下自絮凝颗粒酵母乙醇连续发酵的实验研究。在实验中,每隔5 d将从末级反应器收集到的发酵液集中精馏处理,得到的废糟液直接用于玉米粉调浆制糖。系统连续运行了120d,共进行了24批次实验,数据分析表明系统达到了平衡状态。在平均发酵时间为20h条件下,发酵终点乙醇浓度平均为11.7%（V/V）,残还原糖浓度平均为7.9g/L,装置运行平稳。这些工作为自絮凝颗粒酵母乙醇发酵耦合废糟液直接全循环使用、实现污染物源头减废、清洁生产奠定了理论和实验基础。     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

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 fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor

Glutathione in beer works as the main antioxidant compounds which correlates with beer flavor stability. High residual sugars in beer contribute to major non-volatile components which correlate to high caloric content. In this work, Saccharomyces cerevisiae GSH1 gene encoding glutamylcysteine synthetase and Scharomycopsis fibuligera ALP1 gene encoding alpha-amylase were co-expressed in industrial brewing yeast strain Y31 targeting at alpha-acetolactate synthase (AHAS) gene (ILV2) and alcohol dehydrogenase gene (ADH2), and new recombinant strain TY3 was constructed. The glutathione content from the fermentation broth of TY3 increased to 43.83 mg/l compared to 33.34 mg/l from Y31. The recombinant strain showed high alpha-amylase activity and utilized more than 46% of starch after 5 days growing on starch as sole carbon source. European Brewery Convention tube fermentation tests comparing the fermentation broth of TY3 and Y31 showed that the flavor stability index increased to 1.3 fold and residual sugar concentration were reduced by 76.8%, respectively. Due to the interruption of ILV2 gene and ADH2 gene, the amounts of off-flavor compounds diacetyl and acetaldehyde were reduced by 56.93% and 31.25%, comparing with the amounts of these from Y31 fermentation broth. In addition, as no drug-resistance genes were introduced to new recombinant strain, consequently, it should be more suitable for use in beer industry because of its better flavor stability and other beneficial characteristics.     

Dynamic neural networks as a tool for the online optimization of industrial fermentation

A system for online optimization of industrial fermentation based on a model with dynamic neural networks is described. The developed dynamic neural network, consisting of adapted neurons to consider the process dynamics, can model the complex, non-linear fermentation of beer in order to predict the future process. The predicted trajectories of gravity, pH, and diacetyl are in agreement with the experimental data measured at an automated pilot fermenter. It was possible to predict the future course of the batch fermentation as soon as 12 h of process data were available. In combination with the variational principle, the process model was used to optimize productivity. The temperature trajectory is optimized using a cost functional, including technical and technological conditions of the brewery in order to reduce the process time by steady product quality. The results show a reduction of the process time of up to 20%, which leads to an increase in utilization capacity.     

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.     

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.     

不同波长Ar^＋激光对啤酒酵母菌的诱变效应

本文应用514.5nm,496.5nm、488.0nm和476.5nm等四种不同波长的氩离子辐照啤酒酵母菌,激光辐照的功率密度300mw/cm~2、辐照时间12min。辐照后进行细胞培养和啤酒发酵试验,并测定细胞有关生理性能的变化。实验表明,上述四种波长的Ar~+激光对啤酒酵母细胞的生长繁殖和代谢主产物乙醇的生物合成有明显的促进作用,并能提高双乙酰还原酶的活力,降低发酵付产物双乙酰的含量。这些变化,有利于缩短啤酒发酵的周期,提高啤酒产量相改善啤酒的风味。     

Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.     

Absence of fks1p in lager brewing yeast results in aberrant cell wall composition and improved beer flavor stability

The flavor stability during storage is very important to the freshness and shelf life of beer. However, beer fermented with a yeast strain which is prone to autolyze will significantly affect the flavor of product. In this study, the gene encoding β-1,3-glucan synthetase catalytic subunit (fks1) of the lager yeast was destroyed via self-clone strategy. β-1,3-glucan is the principle cell wall component, so fks1 disruption caused a decrease in β-1,3-glucan level and increase in chitin level in cell wall, resulting in the increased cell wall thickness. Comparing with wild-type strain, the mutant strain had 39.9 and 63.41 % less leakage of octanoic acid and decanoic acid which would significantly affect the flavor of beer during storage. Moreover, the results of European Brewery Convention tube fermentation test showed that the genetic manipulation to the industrial brewing yeast helped with the anti-staling ability, rather than affecting the fermentation ability. The thiobarbituric acid value reduced by 65.59 %, and the resistant staling value increased by 26.56 %. Moreover, the anti-staling index of the beer fermented with mutant strain increased by 2.64-fold than that from wild-type strain respectively. China has the most production and consumption of beer around the world, so the quality of beer has a significant impact on Chinese beer industry. The result of this study could help with the improvement of the quality of beer in China as well as around the world.     

Data model for the elimination of matrix effects in enzyme-based flow-injection systems

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.     

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.