THE EFFECT OF CARBOHYDRATE ADJUNCTS ON BREWER'S WORT FERMENTATION BY SACCHAROMYCES UVARUM (CARLSBERGENSIS)*

The use of carbohydrate adjuncts such as sucrose, fructose and glucose in brewer's wort significantly modifies the initial wort sugar spectrum and also the pattern of sugar uptake during fermentation by a strain of Saccharomyces uvarum (carlsbergensis). Under these conditions, the concentration of glucose and fructose in the wort was observed to increase when compared to worts in which corn starch was employed as an adjunct and glucose was taken up at a faster rate than fructose. The increase in glucose concentration in the wort also resulted in severe repression of maltose and maltotriose utilization with significant levels of these sugars remaining in the beer produced.     

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.     

FERMENTATIONS IN THE PRESENCE OF AMYLOGLUCOSIDASE

The addition of amyloglucosidase to fermenting wort produced changes in the spectrum of carbohydrates. Some oligosaccharides were broken down to a larger extent than others, and maltotriose and maltose were depleted at a higher rate than under normal conditions. It was also noted that the glucose concentrations increased in fermentations to which amyloglucosidase had been added. In order to determine whether the reduction in maltotriose and maltose was caused by the enzyme alone or by assimilation as well as enzymic degradation, maltotriose depletion rates were compared in fermentations with Sacch. cerevisiae and with Sacch. uvarum. The results indicated assimilation as well as degradation. Further evidence of maltotriose fermentation was obtained when glucose was continuously pumped into a fermentor at a rate approximating to the glucose formation by the enzyme. Also, under these experimental conditions, it was found that glucose levels increased, while maltose and maltotriose concentrations decreased. However, the observed increase in glucose during the fermentation was lower than would be expected from the mechanical addition, indicating a simultaneous uptake of glucose, maltose and maltotriose.     

Lachancea thermotolerans as an alternative yeast for the production of beer

The ability of Lachancea thermotolerans strains to ferment brewer's wort has been investigated. Initial fermentations with three L. thermotolerans strains compared the use of maltose and maltotriose, as well as production of glycerol and lactic acid and pH evolution over the course of the fermentation. The most promising strain was subsequently tested for additional traits important for beer production, including pitching rate, generational capacity, foam stability, hop tolerance, vicinal diketone production, oxygen requirement and flocculation. These tests suggest that L. thermotolerans may be a good choice for producing sour beers in a single fermentation step without the use of lactic acid bacteria. Copyright © 2016 The Institute of Brewing & Distilling     

SOME QUANTITATIVE ASPECTS OF THE FERMENTATION OF MALTOTRIOSE BY YEASTS

The comparative fermentation velocities with glucose and maltotriose of a series of different strains of top ale yeast show marked variation in the ratio of this function. There is a tendency for more flocculent yeasts to show a relatively lower maltotriose fermentation rate, although this is not absolute. The maltotriose fermentation rate is influenced by the nitrogen content of the cell in a similar manner to that demonstrated by Thorne (this Journal 1954, 227) for glucose. Certain factors affecting the rate of maltotriose fermentation have been investigated. Media and conditions have been elaborated to yield a reproducible determination of the maltotriose fermenation efficiency and this characteristic strain parameter has been shown to be of value in strain evaluation.     

THE EFFECTS OF GLUCOSE ADJUNCT IN HIGH GRAVITY FERMENTATION BY SACCHAROMYCES CEREVISIAE 2036

Laboratory fermentations of 16°Plato glucose adjunct worts by Saccharomyces cerevisiae 2036 demonstrated the absence of “glucose repression” of maltose and maltotriose uptake. However, when compared to worts in which maltose syrup was employed as an adjunct, residual glucose was present at the end of fermentation, maltose and maltotriose uptake rates were enhanced, fructose uptake was blocked and the sequence of sugar uptake was changed. These findings partially explain residual glucose and fructose that sporadically appear in commercial beers. Further research suggests that the physiological quality of the yeast is of prime importance in carbohydrate metabolism, and that critical concentrations of glucose vary with different physiological conditions for this brewing strain in 16°P wort .     

使用路德类酵母生产无醇啤酒

研究路德类酵母菌落和细胞形态特征及其利用麦汁中糖的特点,发现其菌落为奶油色,半透明,有光泽,表面光滑,湿润,质软,中间凸起,边缘整齐;细胞为柠檬形,较大,两端出芽生殖。路德类酵母发酵89.9%的果糖和91.9%的葡萄糖,而对于蔗糖的利用率达到100%,但是不发酵麦芽糖和麦芽三糖。将路德类酵母应用于发酵麦汁制得无醇啤酒,并对其产酒精及风味物质的特点予以研究,结果发现：路德类酵母发酵麦汁产酒精量较低,约只相当于同条件下普通啤酒酵母的1/6,当原麦汁浓度低于8°P时,乙醇含量低于0.5%;风味物质的种类与普通啤酒基本相同,但含量相对较低,而与市售无醇啤酒相比,酯类含量较少,高级醇含量没有明显差异,口感较接近。     

Effect of Amyloglucosidase on Wort Composition and Fermentable Carbohydrate Depletion in Sorghum Lager Beers

A three‐factorial experiment with a level of confidence of P < 0.05 was performed to study fermentable carbohydrate depletion and ethanol production during 144 h fermentations of lager beers produced with barley malt (BM), sorghum malt (SM), refined maize (MZ) or waxy sorghum (WXSOR) grits treated during mashing with or without amyloglucosidase (AMG). The percentage glucose, maltose and maltotriose, based on total fermentable carbohydrates for the BM wort was 20, 68 and 13% and for the SM wort 35, 48 and 17% respectively. Treatment with AMG increased wort glucose from 9.3 to 24.5 g/L wort and total fermentable sugar equivalents, expressed as g glucose/L, from 59.2 to 72.6 g/L wort. The SM worts had approximately 50% more glucose and 40% less initial maltose content respectively compared to the BM worts. The WXSOR grits produced worts and beers with similar properties to those produced from the MZ adjuncts. AMG addition led to a >2.5 fold increment in wort glucose and 23% in total fermentable carbohydrate content. Linear regression analysis determined that the consumption rate of fermentable carbohydrates during fermentation followed first order reaction kinetics. Depletion times to reach 50% of the initial concentrations of glucose, maltose and maltotriose were 49, 128 and 125 h, respectively, clearly indicating that the fermenting yeast preferred glucose. Maltose and maltotriose depletion times of the AMG treated worts were significantly faster and lower, respectively, when compared with the untreated worts. At the end of the fermentation, the BM beers contained higher ethanol levels (5.1% v/v) than the SM beers (3.9% v/v). For AMG treated beers, no significant differences in ethanol content were observed among samples mashed with BM and beers produced from SM and MZ grits. The results demonstrated that AMG could be used to increase the initial concentration of glucose and total fermentable carbohydrates thus decreasing dextrin levels, especially from sorghum mashes.     

The selection and modification of brewer's yeast strains

In order to achieve a beer of high quality, the yeast culture must be effective in removing the desired nutrients from the growth medium (i.e. the wort), it must impart the required flavour to the beer and finally, the micro-organisms themselves must be effectively removed from the fermented wort after they have fulfilled their metabolic role. Brewer's wort contains the sugars sucrose, fructose, glucose, maltose and maltotriose, together with dextrin material. In the normal situation, brewer's yeast strains are incapable of fermenting the dextrin material; however, yeast strains capable of fermenting at least a part of this dextrin material and producing a palatable beer are now available.One of the important factors known to affect the fermentation rate is the intracellular yeast glycogen concentration which has been found to be influenced by storage conditions. The glycogen level at pitching significantly affects the fermentation rate of the yeast culture.     

改进酿酒酵母谷物发酵性能的诱变研究

酿酒酵母经诱变获得有效利用麦汁糖的优良性能。突变菌株经5d摇床培养使YNB基础培养基的麦芽三糖发酵度从32.4%提高到81.8%。薄层层析证明突变株麦芽三糖转运能力得到改善。模拟工业发酵实验证明突变菌株的麦汁糖发酵性能大大改善,而原有发酵优良性状未受到影响。说明该突变株益于工业啤酒生产和低热啤酒的生产。     

发泡酒酿造用贫氮酵母性质的研究

研究了贫氮酵母在发酵过程中麦汁各组成成分的利用情况及其生理特性，研究发现，贫氮酵母在贫氮麦汁中能利用少部分的脯氨酸和相当部分的麦芽三糖，发酵后的啤酒中总含氮量符合淡爽啤酒的要求；它是一株中等凝聚性的酵母，具有一定的实际应用价值。     

FAST AND SLOW FERMENTATION OF BREWER'S WORT BY STRAINS OF SACCHAROMYCES CEREVISIAE

The major biochemical factor determining whether a strain of yeast will ferment wort rapidly is the activity of the yeast in fermenting maltose. However, maltase is always present in sufficient quantity and consequently maltose permease determines the rate of utilization of this sugar by the yeast. Similarly the utilization of maltotriose is thought to be governed by the uptake system for this sugar.     

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     

A review of malting and malt processing for whisky distillation

This paper encompasses a re‐evaluation of published literature and data regarding wort attenuation in malt distilleries raising questions and discussing how the conventional wisdom has changed over time and what questions still need to be answered. Current knowledge is summarized in the following four points: (a) Under normal malting conditions, starch granules are partially degraded by a combination of α‐amylase and α‐glucosidase. This complex can open up the granule at specific sites on the surface and create characteristic ‘pin‐hole’ lesions, which may be widened by secondary hydrolysis by α‐ and β‐amylase, limit dextrinase and α‐glucosidase (maltase). (b) All of these diastatic enzymes can survive mild kilning, probably by forming heat stable complexes on and within the starch granules and can continue a complete degradation of starch when mashed at ambient temperatures with glucose as the end product. (c) At normal mashing temperatures, starch granules gelatinize and dissolve with a concomitant rapid degradation to glucose, maltose, maltotriose and dextrins ranging from degree of polymerization (DP) 4 to > DP20. If there is immediate wort boiling after run‐off, this is the final composition of starch derived carbohydrates according to the conventional paradigm. (d) All malt worts also contain a small amount of panose, isopanose as well as glucosyl maltodextrins, based on a core of 6₂‐α‐glucosyl maltose (panose) or 6‐α‐maltosyl glucose (isopanose), which are remnants of the α‐amylase/glucosidase degradation of granular starch. These dextrins are resistant to the action of debranching enzymes and their concentration may vary between 4 and 8% of the malt extract, depending on the degree of modification of the host starch granules. They may be created at the active sites of this enzyme complex when the granule is gelatinized. In a conventional mash of unboiled distilling wort, the spectrum of wort dextrins produced from gelatinized starch is reduced to true ‘limit’ dextrins of DP4–8 by continued α‐amylolysis during early fermentation. These dextrins will contain side chains of either maltose or maltotriose residues surrounding the α‐1,6‐glucosidic linkage and can be debranched by limit dextrinase during late fermentation, leaving only the above glucosyl maltodextrins dextrins in the spent wash. Copyright © 2016 The Institute of Brewing & Distilling     

CAMBRIDGE PRIZE LECTURE IMPROVING YEAST FERMENTATION PERFORMANCE

A number of factors affecting yeast fermentation performance have been investigated. These include the mode of substrate feeding, nutrient supplementation, temperature, osmotic pressure, oxygen, intracellular ethanol accumulation, and yeast ethanol tolerance. Nutrient supplementation was observed to play a key role in yeast fermentations employing high gravity media and at high temperatures. Furthermore, complete attenuation of high gravity wort (25°P) could be achieved by optimizing the yeast pitching rate, fermentation temperature, and level of wort oxygenation. Genetic manipulation techniques, such as spheroplast fusion, were successfully employed to obtain ethanol and osmotolerant yeast strains. In addition, a number of stable 2-deoxy-D-glucose resistant mutants, isolated from brewing and non-brewing yeast strains, were observed to rapidly utilize maltose and maltotriose in the presence of high concentrations of glucose. Fermentation and ethanol production rates were increased in these strains. Therefore, employing strategies of optimized fermentation conditions and strain development have resulted in improvements in yeast fermentation performance.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Mutagenizing brewing yeast strain for improving fermentation property of beer

A brewing yeast mutant with perfect sugar fermentation capacity was isolated by mutagenizing the Saccharomyces pastorianus transformant, which carries an integrated glucoamylase gene and has one copy of non-functional alpha-acetolactate synthase gene. The mutant was able to utilize maltotriose efficiently, and the maltotriose fermentability in YNB-2% maltotriose medium increased from 32.4% to 72.0% after 5 d in shaking culture. The wort fermentation test confirmed that the sugar fermentation property of the mutant was greatly improved, while its brewing performances were analogous to that of the wild-type strain and the characteristic trait of shortened beer maturation period was retained. Therefore, we believe that the brewing yeast mutant would benefit the beer industry and would be useful for low caloric beer production.     

Attachment of MAL32-encoded maltase on the outside of yeast cells improves maltotriose utilization

The fermentation of maltotriose, the second most abundant fermentable sugar in wort, is often incomplete during high-gravity brewing. Poor maltotriose consumption is due to environmental stress conditions during high-gravity fermentation and especially to a low uptake of this sugar by some industrial strains. In this study we investigated whether the use of strains with an alpha-glucosidase attached to the outside of the cell might be a possible way to reduce residual maltotriose. To this end, the N-terminal leader sequence of Kre1 and the carboxy-terminal anchoring domain of either Cwp2 or Flo1 were used to target maltase encoded by MAL32 to the cell surface. We showed that Mal32 displayed on the cell surface of Saccharomyces cerevisiae laboratory strains was capable of hydrolysis of alpha-1,4-linkages, and that it increased the ability of a strain lacking a functional maltose permease to grow on maltotriose. Moreover, the enzyme was also expressed and found to be active in an industrial strain. These data show that expressing a suitable maltase on the cell surface might provide a means of modifying yeast for more complete maltotriose utilization in brewing and other fermentation applications.     

Maltose Transport by Brewer's Yeasts in Brewer's Wort

The kinetics of maltose transport by two industrial yeasts were studied. The ale and lager strain each showed both high and low affinity transport. For the lager strain, maltose transport was only weakly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter is the maltose‐specific type coded by MALx1 genes. For the ale strain, maltose transport was strongly inhibited by maltotriose, sucrose and trehalose, suggesting that its dominant maltose transporter may be the AGT1‐encoded type that also carries these sugars. Also glucose inhibited transport by the ale strain more than that by the lager strain. Instantaneous inhibition by ethanol at concentrations met in brewery fermentations was moderate (about 25% at 50 g ethanol · L^?1). The apparent Vmax for high affinity transport increased about 100‐fold between 0 and 30°C, whereas the Km (3 ± 1 mM) was constant. Standard activities of maltose transport and maltase were followed through pilot fermentations of 11–24°P worts. Rapid (20 s) measurements of the zero‐trans‐rate of maltose uptake were also made with each day's yeast (rapidly harvested and washed) in reaction mixtures containing the same day's wort labelled with tracer ¹⁴C‐maltose. Results suggested that maltose uptake is the dominant factor controlling the rate of maltose utilization in these wort fermentations.     

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