Application of Plackett–Burman experimental design for investigating the effect of wort amino acids on flavour‐active compounds production during lager yeast fermentation

Aroma‐active higher alcohols and esters are produced intracellularly in the cytosol by fermenting lager yeast cells, which are of major industrial interest because they determine aroma and taste characteristics of the fermented beer. Wort amino acid composition and their utilization by yeast during brewer's wort fermentation influence both the yeast fermentation performance and the flavour profile of the finished product. To better understand the relationship between the yeast cell and wort amino acid composition, Plackett–Burman screening design was applied to measure the changes in nitrogen composition associated with yeast amino acids uptake and flavour formation during fermentation. Here, using an industrial lager brewing strain of Saccharomyces pastorianus , we investigated the effect of amino acid composition on the accumulation of higher alcohols and volatile esters. The objective of this study was to identify the significant amino acids involved in the flavour production during beer fermentation. Our results showed that even though different flavour substances were produced with different amino acid composition in the fermentation experiments, the discrepancies were not related to the total amount of amino acids in the synthetic medium. The most significant effect on higher alcohol production was exercised by the content of glutamic acid, aromatic amino acids and branch chain amino acids. Leucine, valine, glutamic acid, phenylalanine, serine and lysine were identified as important determinants for the formation of esters. The future applications of this information could drastically improve the current regime of selecting malt and adjunct or their formula with desired amino acids in wort. Copyright © 2017 The Institute of Brewing & Distilling     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

Impact of Dark Specialty Malts on Extract Composition and Wort Fermentation

Dark specialty malts are important ingredients for the production of several beer styles. These malts not only impart colour, flavour and antioxidative activity to wort and beer, they also affect the course of wort fermentations and the production of flavour‐active yeast metabolites. The application of considerable levels of dark malt was found to lower the attenuation, mainly as a result of lower levels of fermentable sugars and amino acids in dark wort samples. In fact, from the darkest caramel malts and from roasted malts, practically no fermentable material can be hydrolysed by pilsner malt enzymes during mashing. Compared to wort brewed with 50% pilsner malt and 50% dark caramel malt or roasted malt, wort brewed with 100% pilsner malt contained nearly twice as much fermentable sugars and amino acids. Reduced levels of yeast nutrients also lowered the fermentation rate, ranging from 1.7°P/day for the reference pilsner wort of 9 EBC to 1.1°P/day for the darkest wort (890 EBC units), brewed with 50% roasted malt. This additionally indicates that lower attenuation values for dark wort are partially due to the inhibitory effects of Maillard compounds on yeast metabolism. The application of dark caramel or roasted malts further led to elevated levels of the vicinal diketones diacetyl and 2,3‐pentanedione. Only large levels of roasted malt gave rise to two significant diacetyl peaks during fermentation. The level of ethyl acetate in beer was inversely related to colour, whereas the level of isoamyl acetate appeared to be affected by the use of roasted malt. With large levels of this malt type, negligible isoamyl acetate was generated during fermentation.     

浆水中产香酵母菌菌株的筛选及其增殖培养基优化

以甘肃传统酿制的浆水为材料,采用经典平板分离法对浆水中的酵母菌进行分离纯化,通过感官评定、性能测定及气相色谱-质谱法半定量法分析筛选产香性能优良的酵母菌菌株,对其进行生理生化及分子生物学鉴定,并采用单因素结合Design-Expert设计响应面试验优化了该菌株的增殖培养基。结果表明,从浆水中分离出16 株酵母菌菌株,并成功筛选出了1?株产香性能优良的酵母菌菌株Y16,经鉴定为异常汉逊酵母（Hansenula anomala）。优化的培养基增殖配方为：10?°Be′麦芽汁,其中最佳的营养物添加量分别为葡萄糖11.0?g/L、牛肉膏2.3?g/L、KH2PO4?0.8?g/L。在该条件下,细胞浓度可达到8.75×108?个/mL,是初始麦芽汁发酵培养基细胞浓度的4?倍多;乙酸乙酯的产量达到256.35?μg/mL,比初始发酵培养基增加了25.39%,柠檬烯的产量达到0.083?μg/mL,比优化前增加了31.75%,该菌株增香效果明显,可为浆水发酵直投式增香菌剂的开发提供菌株来源和技术参考。     

High Gravity Brewing by Continuous Process Using Immobilised Yeast: Effect of Wort Original Gravity on Fermentation Performance

The present work evaluated the influence of all‐malt wort original gravity on fermentative parameters and flavour‐active compound formation during primary fermentation of high gravity brewing by a continuous process using a lager yeast immobilised on a natural carrier obtained from brewer's spent grain (the main brewery by‐product). The all‐malt worts with original gravity (OG) ranging from 13.4 to 18.5°Plato were prepared by diluting a very‐high‐gravity wort (20°Plato) with sterile brewery water. The continuous assay was carried out in a bubble column bioreactor with a total working volume of 5.2 litres, at 15°C, using a constant gas flow rate of 250 mL/min (200 mL/min of CO₂ and 50 mL/min of air) and a dilution rate of 0.04 h^?1 (residence time of 25 h). The results indicated that as the wort OG was increased, the ethanol concentration of the outflowing beer increased. On the other hand, the continuous fermentation of the most concentrated worts (16.6 and 18.5°Plato) resulted in beers with unbalanced flavour profiles due to excessive ethyl acetate formation. The immobilised cell concentration appeared to be nearly independent from increasing wort OG.     

Linoleic Acid Supplementation of a Cropped Brewing Lager Strain: Effects on Subsequent Fermentation Performance with Serial Repitching

Most breweries collect yeast from a previous fermentation cycle for further use in a subsequent cycle. However, the cropped cells are deficient in membrane sterols and unsaturated fatty acids (UFAs) which are required for good fermentation performance in the next cycle. Consequently, the cellular levels of these compounds must be restored to obtain an optimal fermentation performance. There are currently three possibilities to satisfy this requirement. The common practice is aeration of the wort before pitching, thus providing oxygen needed for lipid synthesis during the first stages of fermentation. Oxygenation (aeration) of cropped yeast slurries is a second alternative. Finally, the addition of the required lipids to wort is sometimes suggested as an alternative to aeration. We examined a fourth possibility, namely the supplementation with UFA of cropped cells. Previously, we reported that the supplementation of stationary phase cropped brewer's yeast with linoleic acid is a good alternative to wort aeration. This conclusion resulted from results obtained with a well‐defined stirred synthetic fermentation medium. We also showed that cells cropped from non‐stirred tall‐tube fermented malt wort incorporated linoleic acid into different cellular lipid fractions, when suspended and supplemented in fermented wort. Now, we report that such yeast, pitched in malt wort in non‐stirred tall‐tubes, showed growth and attenuation profiles comparable to unsupplemented yeast in pre‐aerated wort. Moreover, the synthesis of acetate esters, which is known to be affected when UFAs are added directly to the wort, was not significantly affected. We hypothesize that the active uptake of linoleic acid during fermentation and its activation by coenzyme A (CoA) and phospholipid synthesis are responsible for the effects on ester synthesis, through repression of the alcohol acetyltransferase‐encoding gene, ATF1. In supplemented cropped yeast, these reactions occur prior to fermentation, thus avoiding interferences with acetate ester synthesis. In serial repitching experiments with repeated linoleic acid supplementation of the cropped yeast, the fermentation performances of the yeast remained comparable to those of non‐supplemented yeast in pre‐aerated wort. However, due to a progressive increase of cellular UFA, negative effects on acetate ester synthesis appeared. Nevertheless, the supplementation of cropped yeast with UFAs can be considered as an interesting alternative to wort oxygenation to restore optimal membrane functions.     

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     

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。     

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     

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。     

STRAIN SPECIFIC RESPONSE OF BREWER'S YEAST STRAINS TO ZINC CONCENTRATIONS IN CONVENTIONAL AND HIGH GRAVITY WORTS*

The effect of zinc on a variety of yeast strains is extensively documented in the literature. However, due to the varied experimental protocols employed in each study there is little opportunity to directly compare the strain specificity of this ion. In the present study, the response of six yeast strains (three Saccharomyces cerevisiae (ale type) and three S.cerevisiae (lager type)) to altered zinc concentrations, in both high (1080 OG) and conventional (1048 OG) gravity worts, was investigated. Varying the initial wort zinc concentration in both gravities had an effect on the ethanol production, rate of fermentation, cell number and sugar uptake of all six strains studied. The extent of the response was found to be dependent upon the zinc concentration, the strain employed and wort gravity employed.     

Degradation of 5-hydroxymethylfurfural during yeast fermentation

5-Hydroxymethyl furfural (HMF) may occur in malt in high quantities depending on roasting conditions. However, the HMF content of different types of beers is relatively low, indicating its potential for degradation during fermentation. This study investigates the degradation kinetics of HMF in wort during fermentation by Saccharomyces cerevisiae. The results indicated that HMF decreased exponentially as fermentation progressed. The first-order degradation rate of HMF was 0.693?×?10^?2 and 1.397?×?10^–2?min^?1 for wort and sweet wort, respectively, indicating that sugar enhances the activity of yeasts. In wort, HMF was converted into hydroxymethyl furfuryl alcohol by yeasts with a high yield (79–84% conversion). Glucose and fructose were utilised more rapidly by the yeasts in dark roasted malt than in pale malt (p?<?0.05). The conversion of HMF into hydroxymethyl furfuryl alcohol seems to be a primary activity of yeast cells, and presence of sugars in the fermentation medium increases this activity.     

The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations

In recent years, Brettanomyces bruxellensis has found increasing application in brewery fermentations. Indeed, B. bruxellensis contributes to the flavour profile of many Belgian beers, typically during secondary or spontaneous fermentation. In North America, the yeast is used in primary fermentation to produce beers with ‘Brett’ characteristics with ‘fruity’ and/or ‘funky’ sensory profiles associated with the production of volatile esters and phenols. However, little is understood about the factors that influence flavour metabolite production or fermentation rate in this yeast. Here, the impact of temperature is reported on fermentation efficiency, flavour metabolite production and carbon utilisation of one commonly used and eight poorly characterised B. bruxellensis strains during wort fermentation. A high degree of strain and temperature‐dependent variability was found in fermentation efficiency and metabolite production amongst B. bruxellensis strains. Further, fermentation efficiency and carbon utilisation were temperature dependent, while ester production increased at higher temperature and phenol production was strain and temperature independent. These results indicate significant strain and temperature dependent variation, suggesting the potential application of strain variability as a tool to achieve product diversity in B. bruxellensis primary fermentations. © 2019 The Institute of Brewing & Distilling     

SOME EFFECTS OF WORT COMPOSITION ON THE RATE AND EXTENT OF FERMENTATION BY BREWING YEASTS

The time required to ferment worts of varied composition to a given extent is dependent upon the extent of exponential growth in the early stages of fermentation; in the worts studied this is determined by the concentration of assimilable nitrogen. When the concentration of all the non-carbohydrate nutrients in malt wort is halved by dilution with carbohydrate, the addition of appropriate quantities of serine or arginine restores the rate of fermentation to that of the malt wort. Minor nutrients, other than amino acids specifically required by the yeasts used, are thus present in at least two-fold excess in the malt wort. The yeast produced during exponential growth in malt wort (sp.gr. 1·040) is able to ferment rapidly much greater quantities of fermentable carbohydrate than are present in that wort. The majority of the strains of yeast examined ferment equally well when either glucose or maltose is added to malt wort and do so whether the sugar is added prior to fermentation or towards the end; however, one strain fails to ferment satisfactorily if a substantial quantity of glucose is added to wort prior to fermentation, because of the subsequent failure of the yeast to adapt to ferment maltose. It is suggested that most brewing strains do not require to adapt to maltose utilization during the fermentation of wort.     

Evaluation of a Brazilian Fuel Alcohol Yeast Strain for Scotch Whisky Fermentations

Traditionally, distilling companies in Scotland have employed a very limited number of yeast strains in the production of alcohol for Scotch whiskies. Recent changes such as the decline in availability of brewers' yeast as a secondary yeast strain and the availability of yeast in different formats (e.g., dried and cream yeast as alternatives to compressed yeast) have promoted interest in alternative Scotch whisky distilling yeasts. In previous work, we investigated different strains of yeasts, specifically Brazilian yeasts which had been isolated from and used in fuel alcohol distilleries. One of the Brazilian yeasts (CAT 1) showed a comparable fermentation performance and superior stress tolerance compared with a standard commercial Scotch whisky distilling yeast (M Type). The Brazilian CAT 1 yeast isolate was further assessed in laboratory scale fermentations and subsequent new make spirit was subjected to sensory analyses. The spirits produced using the Brazilian strain had acceptable flavour profiles and exhibited no sensory characteristics that were atypical of Scotch whisky new make spirit. This study highlights the potential of exploiting yeast biodiversity in traditional Scotch whisky distillery fermentation processes.     

凝固型火棘金樱子酸奶的研制

以火棘、金樱子与乳粉为主要原料,经酸奶发酵剂发酵,制成风味独特、营养丰富的保健酸奶。由对比试验选用直投式发酵剂为生产菌种,通过正交试验,优化出火棘金樱子酸奶最佳发酵工艺条件为:30 mL/100 mL果汁混合液中,添加量12 g/100 mL乳粉和7 g/100 mL白砂糖,配以0.20 g/100 mL复合稳定剂,接入3 mL/100 mL的酸乳发酵菌剂,在40℃下发酵8 h,后发酵时间为24 h。     

STUDIES ON THE BINDING BETWEEN YEAST AND A MALT POLYSACCHARIDE THAT INDUCES HEAVY YEAST FLOCCULATION

A factor which causes heavy yeast flocculation of Saccharomyces cerevisiae 2036 was found to be associated with the malt husk by Axcell et al.¹ In the present work it was established that the factor is a polysaccharide and immunological techniques were used to show that the factor binds to the yeast cell surface during fermentation. It was shown that the factor is present at significantly higher concentrations in wort causing premature yeast flocculation than in normal wort. A particular malt husk extract, obtained using a mild aqueous extraction procedure, induced premature flocculation when added to fermentations in normal wort. In sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the malt husk extract, 4 protein bands (42,600; 17,500, 15,100 and 13,100 daltons) and a high MW (molecular weight) polysaccharide were identified. Antibodies were raised against electroeluted proteins as well as against the homogenized polyacrylamide gel containing the polysaccharide band. Enzyme-linked immunosorbent assays (ELISA) showed that the protein components were present in similar concentrations in premature flocculent and normal wort. In contrast, the high MW polysaccharide occurred at a significantly higher concentration in wort inducing premature flocculation than in normal wort. Immunogold electron microscopy showed that the high MW polysaccharide bound extensively to the surface of flocculent cells grown in premature flocculent wort. There was markedly less labelling on yeast cells grown in normal wort. Negligible labelling occurred when the yeast cells were incubated with antibodies against the different protein components of malt husk extract.     

The Horace Brown Medal Lecture: Forty Years of Brewing Research

Horace Brown spent fifty years conducting brewing research in Burton‐on‐Trent, Dublin and London. His contributions were remarkable and his focus was to solve practical brewing problems by employing and developing fundamental scientific principles. He studied all aspects of the brewing process including raw materials, wort preparation, fermentation, yeast and beer stability. As a number of previous presenters of the Horace Brown Lecture have discussed Brown's achievements in detail, the focus of this paper is a review of the brewing research that has been conducted by the author and his colleagues during the past forty years. Similar to Horace Brown, fundamental research has been employed to solve brewing problems. Research studies that are discussed in this review paper include reasons for premature flocculation of ale strains resulting in wort underattenuation including mechanisms of co‐flocculation and pure strain flocculation, storage procedures for yeast cultures prior to propagation, studies on the genetic manipulation of brewer's yeast strains with an emphasis on the FLO1 gene, spheroplast fusion and the respiratory deficient (petite) mutation, the uptake and metabolism of wort sugars and amino acids, the influence of wort density on fermentation characteristics and beer flavour and stability, and finally, the contribution that high gravity brewing has on brewing capacity, fermentation efficiency and beer quality and stability.     

Sourdough derived strains of Saccharomyces cerevisiae and their potential for farmhouse ale brewing

The Finnish farmhouse ale sahti is unique in that it is fermented with baking, rather than brewing strains of Saccharomyces cerevisae. The custom of maintaining farmhouse yeast cultures is however no longer practiced in Finland, and much yeast derived diversity in sahti beers has presumably been lost as a consequence. Here, the brewing potential of a number of sourdough derived strains was tested with respect to a number of different fermentation traits. Seven strains originally isolated from Finnish or Italian sourdough cultures were used to ferment high gravity sahti wort (20°P), and fermentation performance together with production of volatile compounds were assessed and compared with a reference baking yeast. Strains differed in terms of fermentation rate, yield, yeast viability and beer flavour profile. All were maltotriose positive, but utilisation varied so that alcohol yield could be greater or lower than that of the reference strain, with values ranging from 6.6 to 7.9% (v/v). Production of aroma compounds was also variable so that it was possible to identify strains producing high levels of esters and those with lower production, which could be used to emphasise flavours originating from raw materials. All strains generated 4-vinyl guaiacol and so would be suitable for other beers where this is a part of the normal flavour profile. Results suggest that sourdough isolates of S. cerevisiae are suitable for sahti production, but could also be applied to other beer styles as a way to differentiate products. © 2020 The Authors. Journal of the Institute of Brewing published byJohn Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

氯化锂诱变蛹虫草菌株液体发酵富集微量元素锌

为更好地开发蛹虫草资源,以蛹虫草为富锌载体,进行发酵富锌培养条件优化,选择最佳质量分数的Li Cl诱变蛹虫草,通过火焰原子吸收法检测诱变前后菌丝体中锌元素的含量。结果表明:蛹虫草富锌的最优碳源为蔗糖,质量分数为3%,最优氮源为蛋白胨,质量分数为3%,最佳培养时间为6 d,最佳接种量为5%,最佳装液量为100 m L/250 m L,培养基中添加的最佳Zn SO4质量浓度为100μg/m L,此时,富锌率为18.54%。Li Cl的最佳诱变条件为:Li Cl质量分数0.15%。选育出富锌较高菌株5株,最高富锌率达24.68%,比出发菌株提高了近42%,具有广阔的应用前景。