Continuous Primary Beer Fermentation with Brewing Yeast Immobilized on Spent Grains

This work demonstrated the technological feasibility of the three‐phase airlift bioreactor (ALR) with brewing yeast immobilized on spent grains (a brewing by‐product) for continuous beer production. The optimum fermentation performance of the one stage immobilized cell bioreactor was achieved at residence times between 18–25 h (dilution rate 0.04–0.055 h^?1) and was characterized by an apparent degree of attenuation in the range of 70–80%. The productivity of the system in terms of ethanol concentration in green beer (ca. 4.2%) was satisfactory. Although the diacetyl concentration in the young beer was high (0.32 mg L^?1 at D = 0.04 h^?1) it is speculated that the level could be reduced by cell growth control, aeration and temperature optimisation. The immobilized yeast fermentation in the ALR was shown to be robust in recovery after process upsets.     

三种不同酵母对啤酒发酵及风味的影响

本实验采用Y1110、安琪、模式三种不同的啤酒酵母,在同种工艺条件下测定发酵过程中α-氨基氮(α-AN)、pH、双乙酰、高级醇等指标,并比较三种不同啤酒的风味物质含量。结果表明:Y1110增殖最快,α-AN和pH值下降最快,双乙酰还原较快,后酵结束双乙酰含量最低,啤酒样品含醇量较高,适于醇厚型啤酒酿造;安琪酵母增殖最慢,α-AN和pH值下降最慢,啤酒样品含酯量较高,适于淡爽型啤酒酿造;模式酵母酯类与醇类含量都很高,不适于实际生产。     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

PRODUCTION OF BEER USING IMMOBILIZED YEAST ENCODING α-ACETOLACTATE DECARBOXYLASE

Genetically modified brewer's yeast encoding α-acetolactate decarboxylase (α-ALDC) was tested in immobilized yeast bioreactors for main fermentation of beer. The α-ALDC enzyme produced by the transformant catalyzes the direct conversion of α-acetolactate to acetoin without formation of diacetyl. The long lagering period required for beer maturation in conventional brewing can thus be shortened or even omitted. Three different packed bed bioreactors were employed, with volumes of 1.6 dm³, 5 dm³ and 25 dm³. The 5 dm³ column had a slightly conical geometry in contrast to the others which had cylindrical shapes. Sintered glass beads were chosen as the carrier material on the basis of experiments with the parent strain. The brewing performance of the transformant compared well with that of the parent strain in the immobilized system. Fermentation, utilization of amino acids (including isoleucine, valine and leucine) and flavour formation were practically identical with both strains, the only difference being a marked decrease in the formation of diacetyl by the transformant. Small differences were, however, observed in the long-term biochemical stability. By using yeast encoding α-ALDC in the immobilized yeast system the total (primary and secondary) fermentation time could be reduced to approximately 2–6 days, compared with 3–6 weeks in a conventional batch process.     

Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity

The characteristic buttery taste of diacetyl has long been a major problem in the brewing industry, and the foam stability of unpasteurized beer is often influenced by proteinase A (PrA), which is encoded by PEP4 and released from yeast cells into beer during brewing. A recombinant industrial brewer’s yeast strain that reduces the diacetyl content of beer and improves foam stability was constructed. We constructed a PGK1p-ILV5-PGK1t expression cassette, which was introduced into one of the PEP4 alleles via PCR-mediated homologous recombination. Then, the second PEP4 allele was disrupted using the Cre-loxP recombination system, and the recombinant strain was designated as S-CSIK12. The results show that the diacetyl production of S-CSIK12 is always lower than that of the host strain at all stages of beer fermentation. In addition, brewing with S-CSIK12 reduced the PrA activity of the final beer by 44 % compared with that using the wild-type strain. The head retention of the beer brewed with S-CSIK12 (260 ± 2 s) was better than that of the host strain S-6 (212 ± 3 s). Considering that more PrA is released from yeast cells during the final stage of main fermentation and that the timing of yeast cropping is determined by diacetyl reduction, brewing with strains that have low diacetyl production also reduced the PrA activity of the beer and improved its head retention. The present study provides reference for the brewing industry as well as research on the diacetyl reduction and foam stability of beer.     

啤酒中的双乙酰

双乙酰是啤酒中一个主要的风味物质。它是由前体物质α-乙酰乳酸在酵母细胞外经非酶氧化脱羧作用形成，然后渗透到细胞内经双乙酰还原酶的作用而消除。控制双乙酰含量的途径可以减少α-乙酰乳酸的形成或加速双乙酰的还原。生产控制措施，主要应加强酵母质量的管理并保证麦汁组成合理。     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

Optimization of alcohol‐free beer production by lager and cachaça yeast strains using response surface methodology

Alcohol‐free beers (AFBs) are an attractive segment of the beer market both for the brewing industry and for consumers. While AFBs produced by arrested/limited fermentation often suffer from a lack of volatile compounds, beer flavour can be improved by yeast selection and optimization of fermentation conditions. The yeast selection strategy was demonstrated by comparing traditional lager yeast with selected cachaça yeast strains. Correspondingly, response surface methodology was used to enhance the formation of the flavour‐active volatile compounds by optimization of the fermentation conditions (original wort extract, fermentation temperature, pitching rate). Statistical analysis of the experimental data revealed the relative significance of process variables and their interactions. The developed quadratic model describing the responses of total esters and higher alcohols to changes in process variables was used to predict the ideal fermentation conditions in terms of flavour formation. The predicted conditions were experimentally verified and alternative strategies of AFB production are suggested. Copyright © 2016 The Institute of Brewing & Distilling     

Enzymatic formation of styrene during wheat beer fermentation is dependent on pitching rate and cinnamic acid content

Styrene is formed by the thermal decarboxylation of cinnamic acid during wort boiling or by enzymatic decarboxylation during fermentation. The enzymatic reaction processes simultaneously to the decarboxylation of ferulic‐ and p‐cumaric acid to clove‐like 4‐vinylguaiacol and phenolic 4‐vinylphenol by the same PAD1 and FDC1 decarboxylase enzymes. However, the formation of styrene occurs much faster within the first hours of fermentation. In addition, the conversion of cinnamic acid starts immediately after pitching without an adaption of yeast on the new medium. Only after 120 min does the level of transposition decrease. Moreover, high cinnamic acid content in pitching wort, in combination with an open fermentation management, causes faster and higher styrene formation during this period. In contrast to the formation of 4‐vinylguaiacol, a correlation between pitching rate and styrene formation during open fermentation could be shown. The resulting time interval between styrene and 4‐vinylguaiacol formation provides scope for minimization strategies for styrene, while maintaining the typical wheat beer flavours. Copyright © 2012 The Institute of Brewing & Distilling     

Fed‐batch fermentation with glucose syrup as an adjunct for high‐ethanol beer brewing

To produce a beer with a high ethanol content, preliminary research on fed‐batch fermentation profiles with glucose syrup as an adjunct during the primary fermentation period was conducted. The ethanol concentration of the beer was elevated by feeding a glucose syrup into the fermentors at a later stage of primary fermentation. Fermentation trials were carried out using a typical lager strain, SC‐9, with a pitching rate at 7.0 × 10⁶ cells/mL. An all‐malt wort (12.5°P) was employed and the primary fermentation temperature was 14 °C. Glucose syrup was supplemented when the concentration of residual reducing sugars was decreased to ~10 g/L. Results showed that the supplemented glucose was consumed rapidly and that the ethanol concentration in the final beer was raised to 67.9 g/L. Additional growth of yeast was observed after feeding accompanied by a low yield of ethanol (~0.46 g/g). Formation of diacetyl was enhanced by yeast growth and two additional peaks were obtained after feeding. The peak value of the diacetyl concentration was 1.90 mg/L. The fed‐batch fermentation resulted in a beer with an overproduction of higher alcohols and esters, indicating that brewing under these experimental conditions led to an unbalanced flavour profile. Results of optimization demonstrated that the optimal conditions were found to be 15°P for initial wort extract, 10 °C for fermentation temperature and 20 × 10⁶ cells/mL for yeast pitching rate, leading to total higher alcohols of 173.8 mg/L, total esters of 22.8 mg/L and an acetaldehyde concentration of 40.5 mg/L. A 12 day maturation and fermentation temperature of 8 °C was needed to reduce the acetaldehyde to 14.3 mg/L. Copyright © 2014 The Institute of Brewing & Distilling     

酿造水中的锌离子对啤酒酿造的影响

啤酒中的锌离子来源于麦芽、大米、酿造用水、酒花。实验表明在啤酒酿造过程中,锌离子可激活酶的活性、提高酶的催化作用,促进糖化、发酵;提高麦汁中糖、氨基酸的含量;促进双乙酰的还原,降低双乙酰的含量;激活乙醇脱氢酶,降低乙醛,提高酵母活力,降低酵母死亡率;提高发酵度,缩短发酵时间。     

锌离子在啤酒酿造中的作用与控制

啤酒中锌离子来源于麦芽、大米、酿造用水、酒花。Zn^2 在啤酒酿造过程中可起到催化荆作用，与氨基酸结合形成Zn-氨基酸螯合物。在啤酒酿造过程中，可激活酶提高酶的作用；促进糖化、发酵；促进蛋白质合成及其稳定性；缓解某金属离子的毒性作用，促进挥发物质的产生和双乙酰的还原，缩短发酵时间，提高啤酒质量；但含量过量会使啤酒非生物稳定性降低，影响啤酒质量。通过对糖化过程和发酵过程的控制，可降低醪液pH值。加入少量小麦芽，加入适量ZnCl2，ZnSO4及酵母营养盐等，可实现对Zn^2 的有效控制，达到最佳酿造浓度。     

DIACETYL—A REVIEW: PART I—ANALYTICAL AND BIOCHEMICAL CONSIDERATIONS: PART II—BREWING EXPERIENCE

Diacetyl and 2,3-pentanedione are normal products of yeast metabolism and are formed in every brewery fermentation. The desired level in the final beer depends on the particular flavour aimed for but, in all types of beer, flavour defects are caused by excessive concentrations of diacetyl and many brewers might be happy to have no diacetyl in the beer. Recent improvements in analytical techniques show that many of the problems associated with diacetyl are due to the occurrence of compounds which can give rise to diacetyl in the finished beer. These compounds include the so-called “precursor,” acetolactic acid, but possibly other compounds such as the bisulphite addition compound of diacetyl are also involved. Study of the factors affecting diacetyl formation and removal by yeast shows how the concentration of diacetyl in beer can be controlled, and the processes at present used to regulate the diacetyl concentration in beer are described. The yeast strain used, the condition of the pitching yeast, the wort composition, the detailed management of the fermentation and the treatment of the beer during packaging and storage can all affect the diacetyl content of the beer.     

泡沫陶瓷固定化酵母细胞啤酒连续主发酵工艺

采用泡沫陶瓷固定化酵母细胞,并对其应用于啤酒连续主发酵进行了实验研究,获取了优化的工艺条件:发酵温度16℃,稀释率0.044h-1。在此工艺条件下,实际测得浓度降低为7.09%,嫩啤酒双乙酰含量为0.144mg/L。嫩啤酒主要理化指标的测定结果表明:采用固定化酵母细胞啤酒主发酵工艺不会对啤酒质量造成影响。     

Overview of craft brewing specificities and potentially associated microbiota

The brewing process differs slightly in craft breweries as compared to industrial breweries, as there are fewer control points. This affects the microbiota of the final product. Beer contains several antimicrobial properties that protect it from pathogens, such as low pH, low oxygen and high carbon dioxide content, and the addition of hops. However, these hurdles have limited power controlling spoilage organisms. Contamination by these organisms can originate in the raw materials, persist in the environment, and be introduced by using flavoring ingredients later in the process. Spoilage is a prominent issue in brewing, and can cause quality degradation resulting in consumer rejection and product waste. For example, lactic acid bacteria are predominately associated with producing a ropy texture and haze, along with producing diacetyl which gives the beer butter flavor notes. Other microorganisms may not affect flavor or aroma, but can retard fermentation by consuming nutrients needed by fermentation yeast. Quality control in craft breweries today relies on culturing methods to detect specific spoilage organisms. Using media can be beneficial for detecting the most common beer spoilers, such as Lactobacillus and Pediococci. However, these methods are time consuming with long incubation periods. Molecular methods such as community profiling or high throughput sequencing are better used for identifying entire populations of beer. These methods allow for detection, differentiation, and identification of taxa.     

Possibilities to improve the antioxidative capacity of beer by optimized hopping regimes

Different hopping regimes were evaluated to investigate the effect on the oxidative stability of wort and beer. Compared with a single hop dosage at the beginning of wort boil, it was possible to increase the concentration of α‐acids in pitching wort and beer by applying incremental hop dosage, dry hopping or the use of a pre‐isomerized hop product in combination with an α‐acid extract, which concomitantly resulted in lower iron concentrations and an enhanced flavour stability as indicated by standard wort and beer analyses, atomic absorption spectroscopy, electron spin resonance spectroscopy and sensory analysis of fresh and force‐aged beers. The functional principle of hop dosage variations is explained by saving of α‐acids throughout the wort production process, which yields an increased formation and precipitation of pro‐oxidative acting transition metal ions (e.g. Fe) in α‐acid‐complexes during the whirlpool rest and fermentation. Consequently, fewer reactive oxygen species are generated. Additional laboratory trials simulating wort cooling and beer storage in buffered model solutions proved that un‐isomerized α‐acids are strong iron chelators and confirmed the functional principle of the applied hopping regimes. Negative effects of higher α‐acid contents on fermentation performance and depletion of the zinc concentration, which is an essential nutrient for yeast, could be excluded. Copyright © 2014 The Institute of Brewing & Distilling     

Optimisation of High Gravity and Diet Beer Production in a German Brewery by Flow Cytometry

Increasing the quantity of beer production without diminishing the quality of the product is a key concern of the beer producing industry. Modifications to the brewery's equipment and settings are the most commonly used methods to improve the brewing process, while the supreme importance of the physiological state of the beer producing organisms, the yeast cells, for the productivity of the brewing process is often poorly recognised. The work described here was designed to optimise two processes: the inoculation regime used to produce high gravity bottom-fermenting beer, and the production of high quality diet beer. To achieve these aims, flow cytometry was used to follow changes in the distribution of DNA, neutral lipid and 3β-hydroxsterol contents in Saccharomyces carlsbergensis strains during inoculation, fermentation and storage. This allowed potential time-saving alterations in the process to be identified. Double staining techniques proved that vigorous fermentative activity and long-term survival capacity during main and secondary fermentation requires intense multiplication of the yeast cells during inoculation. The production of high gravity beer was then enhanced by altering the schedule of the wort additions, and thus increasing the yeast's activities related to multiplication. To produce diet beer, oligosaccharides that remain after the standard brewing process are degraded by adding small amounts of wort, usually during secondary fermentation. However, during this period of fermentation the physiological activity of the yeast cells is hampered by low carbon and high ethanol concentrations. Adding small batches of wort at carefully defined time points and in optimised amounts, even during the main fermentation, improves the physiological state of the yeast cells and rapidly decreases the carbon concentration within the fermentation tank. Both of these factors help to promote quick fermentation to a high quality diet beer. Thus, the flow cytometric investigations provided a reliable basis for identifying effective means of improving the process regime for brewing both of these products.     

固定化酵母啤酒连续后发酵工艺

将主发酵进行完毕后的嫩啤酒先离心除去酵母,再经加热处理,冷却后通入装有木片作为固定化酵母载体的固定床反应罐进行连续后酵。通过试验确定工艺条件,并将连续后酵与传统后酵工艺生产的啤酒进行对比。试验结果表明,室温下,固定化16 h为宜,热处理温度为80℃,时间为10 min;设定后发酵温度为10℃,滞留时间为3.1 h,双乙酰还原速度大为提高,缩短了酒龄;经过风味测定和感官品评得出,采用连续后发酵工艺生产的啤酒,品质上无明显缺陷。     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling