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     

Study on the buffering capacity of wort

The effect of the buffering capacity of wort on a beer fermentation and the contribution of pH, phosphate, amino acids and organic acids to the buffering capacity of wort were studied. The beer pH depended on the buffering capacity of the corresponding wort and an increase in the buffering capacity of the wort could prolong the time of diacetyl conversion. The higher the wort pH was, the larger the wort buffering capacity. Phosphate was not an effective buffer at the pH of wort. Glutamic acid, aspartic acid and histidine gave weak contributions to the buffering capacity of wort, and the total contribution of these three amino acids was <0.1. Organic acids contributed substantially to the buffering capacity of the wort and the total contribution of organic acids to the buffering capacity of the wort was estimated to be about 0.31. The buffering capacity of lactic acid, citric acid, succinic acid, fumaric acid and pyruvic acid was 30, 50, 77, 15 and 9% of that of acetic acid, respectively, at the same mass concentration. Copyright © 2015 The Institute of Brewing & Distilling     

Malted and unmalted oats in brewing

Using oats as a raw material in brewing has recently become the focus of increased interest. This is due to research findings that have shown that oats can be consumed safely by coeliac sufferers. It is also a response to consumer demand for products with novel sensory properties. In this study, beer was produced entirely from oat malt, from barley malt and from oat and barley malts mixed with various quantities of unmalted oats. Compared with barley wort, wort made from malted oats provided a lower extract content and had a higher protein content, but a lower free amino nitrogen content (FAN). The oat wort also showed increased viscosity and haze. The addition of unmalted oats during wort production produced significant changes in the physico‐chemical parameters of both oat and barley worts and beers. Unmalted oats caused an increase in wort viscosity and haze, and a reduction in total soluble nitrogen and FAN. Unmalted oats also contributed to lowering the concentration of higher alcohols and esters. Beer made from 100% oat and barley malts exhibited a similar alcohol content. The use of an oat adjunct in both cases resulted in a lower ethanol content. The introduction of enzyme preparations during the production of wort with oat adjunct had many benefits: increased extract content and FAN; a higher volume of wort; and a lower viscosity that led to faster wort filtration. This research suggests that the use of enzymes is necessary to make production using a high proportion of oats in the grist profitable. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

THE INFLUENCE OF FREE ALPHA-AMINO NITROGEN IN SORGHUM BEER FERMENTATIONS

The interactions of wort free α-amino nitrogen (FAN) and sugar in sorghum beer fermentations were quantified and a simple equation derived. This equation describes the wort FAN demand as a function of the sugar concentration necessary to produce a fully fermented beer within 48 hours. The influence of wort FAN on sorghum beer fermentations had not been quantified so research was undertaken to define sorghum beer yeasts' requirement for wort FAN and the interactions that occur between wort FAN and sugar. Laboratory, sorghum malt and adjunct, worts mashed to cover a wide range of FAN and wort sugar concentrations, were fermented and analysed. The initial wort FAN affects the ethanol production rate, FAN uptake and sugar utilisation rates.     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

Influence of malted barley and exogenous enzymes on the glucose/maltose balance of worts with sorghum or barley as an adjunct

The sugar profile of wort from laboratory malted barley, malted sorghum, unmalted barley and unmalted sorghum grains mashed with commercial enzyme preparations were studied. Similar levels of glucose to maltose (1:7) were observed in wort of malted barley and malted sorghum. Mashing barley or sorghum grains with commercial enzymes changed the glucose to maltose ratio in both worts, with a greater change in wort from sorghum grains. Although hydrolysis with commercial enzymes released more glucose from maltose in sorghum wort, the same treatment retained more maltose in barley wort. Adding malted barley to sorghum grains mashed with commercial enzymes restored the glucose to maltose ratio in sorghum mash. Fermentation of wort produced from all barley malt (ABM) mash and commercial enzyme/barley malt/sorghum adjunct (CEBMSA) mash of similar wort gravity was also studied. ABM and CEBMSA worts exhibited similar glucose to maltose ratios and similar amino acid spectra. However, ABM released more individual amino acids and five times more proline than wort from commercial enzyme/barley malt/sorghum adjunct. ABM produced 27% more glucose and 7% more maltose than CEBMSA. After fermentation, ABM mash produced 9.45% ABV whilst commercial enzyme/barley malt/sorghum adjunct mash produced 9.06% ABV. Restoration of the glucose/maltose ratio in the CEBMSA mash produced wort with a sugar balance required for high gravity brewing. © 2020 The Institute of Brewing & Distilling     

Influence of sorghum grain type on wort physico‐chemical and sensory quality in a whole‐grain and commercial enzyme mashing process

To determine the most suitable types of sorghum for whole‐grain adjunct in lager beer brewing, 14 cultivars of five different types: white tan‐plant, white non‐tan‐plant, red non‐tannin, white tannin (type II) and red tannin (type III) were evaluated. The effects of grain type on wort physico‐chemical and sensory quality with raw grain and malt plus commercial enzyme mashing were assessed. Tannin content correlated significantly and negatively with wort extract and fermentable sugars (p < 0.001) and free amino nitrogen (FAN; p < 0.1). This is attributable to inactivation of the exogenous enzymes by the tannins during the mashing process. However, the type II tannin sorghums had wort quality attributes closer to the non‐tannin sorghum types, probably owing to their relatively low tannin content (≤1%). Malting gave a great improvement in wort extract, fermentable sugars and FAN, but substantially influenced wort sensory properties in terms of higher sourness, bitterness and astringency, as well as the expected more malty flavour. Worts from raw red non‐tannin sorghums were similar to those of white tan‐plant sorghums in both physico‐chemical and sensory quality. Thus, red non‐tannin sorghums, in view of their better agronomic quality, have considerable potential as a whole‐grain adjunct in lager beer brewing. Copyright © 2013 The Institute of Brewing & Distilling     

Simultaneous Determination of Mono‐, Di‐, and Trihydroxyoctadecenoic Acids in Beer and Wort

Monohydroxy, dihydroxy‐, and trihydroxyoctadecenoic acids in beer and wort were simultaneously determined using gas chromatography after a solid extraction method. These three acids were detected at ppm levels in the wort. The monohydroxyoctadecenoic acids were not detected after wort boiling, but the dihydroxy‐ and trihydroxyoctadecenoic acids were transferred through wort boiling, fermentation and lagering into the finished beer. During the mashing using a laboratory mash bath, they gradually increased to about twice the levels those at mashing‐in. The amounts of dihydroxyoctadecenoic acid and trihydroxyoctadecenoic acid in commercial beer samples varied from 0.6 to 1.6 ppm and 6 to 15 ppm, respectively.     

SOME FACTORS AFFECTING THE CONCENTRATION OF DIACETYL IN BEER

Investigations of factors affecting the production of diacetyl during fermentation have shown large differences in the abilities of different strains of brewer's yeast to produce diacetyl. Studies on pilot scale and on small laboratory fermentations have established a connection between the concentration of amino acids in the wort and that of diacetyl in the fully fermented beer. Control of the ratio of amino acids to sugars in wort is thus an important factor in maintaining concentrations of diacetyl in beer at an acceptable level. The effects of increased pitching rate and of high-temperature fermentation in enhancing the production of diacetyl are demonstrated.     

Vitamins in brewing: presence and influence of thiamine and riboflavin on wort fermentation

Thiamine and riboflavin vitamers are present in a wide range of foods including beer. These vitamers play critical roles in a variety of enzymatic complexes and can promote and maintain metabolism. Currently, the presence and role of these vitamers in the malting and brewing industry have not been widely explored. This research investigated the effects of various fermentation conditions that may lead to the variations in the vitamin content in beer observed by previous researchers. The present research found that during fermentation, the thiamine content of wort is quickly utilized within the first 6 h of a standard fermentation and the uptake of this vitamin is not affected by increases in wort gravity. While no significant changes were observed in extracellular phosphorylated vitamers of thiamine, both free thiamine and thiamine diphosphate accumulated intracellularly during the wort fermentation. Meanwhile extracellular riboflavin vitamers were only poorly utilized during beer fermentations, however flavin mononucleotide rapidly accumulated intracellularly and more so under aerobic conditions. When yeast was exposed to an all‐malt high‐gravity wort, the thiamine or riboflavin utilization was not affected. However, thiamine utilization was reduced in adjunct‐driven high‐gravity worts. Notwithstanding the lowered thiamine uptake under high‐gravity conditions; there were some minor improvements in fermentation performance and yeast viability. The addition of thiamine to an all‐malt wort did appear to enhance yeast viability, both under normal and high‐gravity conditions. Copyright © 2016 The Institute of Brewing & Distilling     

FERMENTATION OF WORTS MADE FROM 100% RAW SORGHUM AND ENZYMES

Worts made from raw sorghum and enzymes were successfully fermented even though the level of FAN present (51 mg/l) is well below that essential for fermentation of wort made from malted barley. Changes in typical fermentation parameters such as specific gravity, pH uptake of free amino nitrogen (FAN) and ammonium ions mirrored the increase in yeast cell concentration. Yeast viability remained high throughout the fermentation. Under identical fermentation conditions, malted barley worts showed typical fermentation profiles. However, malted barley worts with specific gravity maintained by the addition of D-glucose, but in which the FAN was diluted to a level similar to that found in a wort made from sorghum and enzymes, fermented more slowly and failed to attenuate fully. Five consecutive fermentations, using yeast cropped from the preceding to pitch the current fermentation were conducted. The specific gravity profiles were essentially the same in all five fermentations. Final values of pH, yeast in suspension, yeast viability and FAN were also indistinguishable. The yeast crop taken from fermentations of worts made from raw sorghum and enzymes represented a 5-fold increase over the initial pitching rate. When compared to commercial beers, the beers derived from fermentation of worts made from raw sorghum and enzymes contained lower levels of ethyl acetate, and higher levels of both 2- and 3-methyl butanol. In the beers derived from sorghum, isobutanol was always less than 20% of the total higher alcohol concentration.     

THE PROPERTIES,COMPOSITION AND FERMENTABILITIES OF WORTS MADE FROM 100% RAW SORGHUM AND COMMERCIAL ENZYMES

A mashing regime was developed using 100% raw sorghum which enabled commercially acceptable hot water extracts to be obtained in 85 minutes with minimal use of a heat stable α-amylase and proteolytic enzymes. This gave worts of HWE 295 1°/kg, with FAN levels of about 40 mg/l and ammonium ion concentration of about 60 mg/l. Higher, but commercially unacceptable, levels of proteolytic enzymes gave worts with FAN from 84.5 to 95 (mg/l). Addition of an amyloglucosidase as the commercial preparation Amylo300L, was required to convert the HWE to fermentable extract. The addition of Amylo300L, increased the DP1, DP2 and DP3 carbohydrate fractions of the worts from 22% to more than 90% of the total, compared to about 80% for a wort made from malted barley without the use of enzymes. Two different proteolytic enzymes gave different extracts and FAN contents presumably reflecting either differences in susceptibilities of the sorghum to the two enzymes or the presence of different additional enzyme activities in the different preparations. The level of ammonium ions in malted barley worts was 86 mg/l and up to 88 mg/l in worts produced from sorghum and enzymes. Enzyme addition produced increased levels of ammonia. The content of Group A (the most readily assimilated) amino acids was proportionally higher in sorghum worts compared to malted barley wort. Worts made from raw sorghum and enzymes, containing as little as 40 mg/l FAN, were fully attenuated. The yeast consumed about 35 mg/l FAN and 45 mg/l ammonium ions. Under identical fermentation conditions, the same yeast, fermenting a malted barley wort of comparable extract consumed 104 mg/l FAN and 37 mg/l ammonium ions.     

THE CONTENT OF SOME ORGANIC ACIDS IN BEER AND OTHER FERMENTED MEDIA

The acid content of a range of ales and lagers has been measured for some organic acids related to the Krebs cycle, and found to vary widely. Acetate, pyruvate, lactate, succinate, pyroglutamate, malate and citrate were present in all cases and α-ketoglutarate was usually detected. α-Hydroxyglutarate was recognized in a number of beers. The effect of the acids on the pH of beer is assessed. The strain of yeast which is used markedly influences the levels of all acids except pyroglutamate and the conditions of yeast propagation have a substantial influence on the extent of acid accumulation. During the fermentation of wort and synthetic media the extent of organic acid excretion is proportional to the extent of fermentation, but the nature of the acids which are excreted varies during the fermentation period. In synthetic media, nitrogen source is shown to have a substantial effect on the accumulation of organic acid. Pyruvate and acetate levels vary inversely towards the end of fermentation, suggesting that yeast converts pyruvate to acetate.     

麦汁组成对啤酒有机酸的影响

有机酸是啤酒中的主要呈味物质,其含量和麦汁组成有着较密切的关系。麦汁中糖组成及氨基酸组成的改变,都会引起有机酸含量的变化,通过试验发现不同糖组成含量影响着有机酸的含量,同时氨基酸含量的高低也影响着啤酒最终的pH和总酸。     

SIMPLE PEPTIDES OF WORT AND BEER

The nitrogenous constituents of wort and beer have been fractionated and peptides have been separated from amino acids by chromatography on DEAE cellulose. Examination of corresponding fractions by electrophoresis and chromatography reveals that whereas there is a selective uptake of wort peptides by yeast, other peptides in beer are not present in the wort and have been formed during fermentation. Approximately 40% of wort peptides are used during fermentation.     

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.     

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre‐aeration, de‐aerated medium and de‐aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre‐aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.     

啤酒高浓酿造中氨基酸对酵母发酵性能和啤酒色值的影响

探讨在24?°P高浓啤酒发酵过程中8?种氨基酸（Met、Phe、Trp、Arg、His、Ile、Leu、Lys）的不同添加量（分别为原麦汁中相应氨基酸含量的0.5、1?倍和2?倍）对酵母生理特性、发酵性能和啤酒色值的影响。结果表明：8?种氨基酸的补充可显著提高麦汁发酵度、乙醇产量,促进酵母生长,提高酵母活细胞率,改善啤酒色值。其中,补充1?倍氨基酸的高浓麦汁发酵性能较好,与对照组相比,发酵度、乙醇产量、最大悬浮酵母细胞数和发酵结束时的酵母活细胞率分别提高了6%、17%、11%和10%。添加氨基酸的高浓酿造啤酒经稀释后,啤酒色泽依然鲜亮,且添加1?倍氨基酸酿造而成的啤酒经稀释后色差（ΔE）最小,色泽最接近青岛纯生啤酒。     

Use of Response Surface Methodology to Investigate the Effectiveness of Commercial Enzymes on Buckwheat Malt for Brewing Purposes

The influence of two factors, total concentration and fraction of three pairs of commercial enzymes, which showed statistical significance (Biocellulase W with Hitempase 2XL, Biocellulase W with Amylo 300 and Amylo 300 with Hitempase 2XL), were studied for their overall effect on buckwheat wort quality using response surface methodology (RSM). This study revealed that the addition of increasing levels of Hitempase 2XL to the buckwheat mash increased colour, extract levels, wort filtration, fermentability and total fermentable extract (TFE), along with decreasing viscosity values. Results also determined a high level of fermentability when an enzyme combination of 30% Biocellulase and 70% Hitempase was added to the mash. The addition of increasing levels of Amylo 300 to buckwheat mashes resulted in increases in fermentability and total fermentable extract (TFE), along with increases in total soluble nitrogen (TSN), free amino nitrogen (FAN) and Kolbach index (KI). With regard to the proposed optimal regime, although no synergistic effect was found when the three enzymes were used together, the optimum conditions for the production of buckwheat wort with lowest viscosity, highest extract and optimal fermentability were achieved using a joint model. Overall, the findings of this study demonstrate the feasibility of producing wort suitable for the brewing of gluten‐free beer from 100% malted buckwheat with careful optimisation of enzyme types and dosage levels.