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     

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.     

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.     

SOME REASONS WHY HIGH GRAVITY BREWING HAS A NEGATIVE EFFECT ON HEAD RETENTION*

Our aim was to examine the effect of high gravity brewing on head retention with respect particularly to the effect of high gravity brewing on hydrophobic polypeptide levels. High gravity brewed beer had poorer head retention values when compared to a similarly brewed low gravity beer. Analysis of hydrophobic polypeptide levels in both high gravity wort (20° Plato) and low gravity wort (10° Plato) produced using a lauter tun, revealed that the high gravity wort contained 8% less hydrophobic polypeptide than the low gravity wort (undiluted basis). Analysis of hydrophobic polypeptides throughout the brewing process for these 10°P and 20°P brews demonstrated that the hydrophobic polypeptide content decreased, especially during the kettle boil and fermentation. Furthermore, the high gravity brewed beer suffered the greatest loss, leaving the final beer with approximately 40% less hydrophobic polypeptides than the low gravity beer. Brewing at 10°P and 20°P using a mash filter demonstrated that these filters can improve the head formation and stability of the resultant beers at sales gravity. However, the low gravity beer still produced a more stable foam (Rudin value 93 s) when compared to the high gravity beer (Rudin value 83 s). The mash filter slightly increased the hydrophobic polypeptide extraction. It is concluded that the mash filter produced higher hydrophobic polypeptide levels in the final beers, as well as having a positive effect on reducing the levels of foam negative compounds such as fatty acids in the wort, and therefore slightly improved head retention values .     

精酿啤酒酿造中存在的乙醇胁迫问题及对策

精酿啤酒麦汁浓度高、酒精度高、浓烈香郁,越来越受到啤酒爱好者的青睐,是我国啤酒酿造的新兴重要领域。但是较高浓度的乙醇对酵母的胁迫危害已经成为制约精酿啤酒高酒精度发酵的瓶颈。该文阐述了国内外精酿啤酒发展现状,指出了精酿啤酒的乙醇胁迫问题。结合酿酒酵母乙醇耐受机理研究进展从乙醇耐受菌株选育,发酵过程中增加海藻糖浓度、提高麦角甾醇含量、添加氮源4个方面探讨了解决精酿啤酒乙醇胁迫问题的对策,为精酿啤酒的高酒精度酿造提供了思路和方法。     

Acid Washing and Serial Repitching a Brewing Ale Strain of Saccharomyces cerevisiae in High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.     

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。     

Recent developments in high gravity beer-brewing

Beer-brewing under high gravity (HG, specific gravity of 13–18 °P) and very high gravity (VHG, specific gravity of >18 °P) wort conditions has a wide range of benefits, from improved process economics to reduced environmental impact. This review highlights the research developments in HG and VHG brewing over the last 10 years. New research emphasizes the importance of multifaceted strategies to achieve comprehensive development in HG brewing. As the brewing yeast is exposed to a variety of stresses (e.g., osmotic stress, ethanol stress) during HG conditions, stress-alleviating strategies have been the prime focus of researchers. Studies have focused on mitigating nitrogen limitation, promoting inexpensive brewing adjuncts, and realizing the potential of exogenous enzymes in HG brewing. In addition, recent studies have addressed the importance of hybrid and mutant yeasts as well as yeast flocculation and immobilization under high specific gravity conditions. Furthermore, studies to unravel yeast physiology under HG brewing and process modelling have received much attention.     

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP™, an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.     

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.     

Comparing the Impact of Environmental Factors During Very High Gravity Brewing Fermentations

The impact of the initial dissolved oxygen, fermentation temperature, wort concentration and yeast pitching rate on the major fermentation process responses were evaluated by full factorial design and statistical analysis by JMP 5.01 (SAS software) software. Fermentation trials were carried out in 2L‐EBC tall tubes using an industrial lager brewing yeast strain. The yeast viability, ethanol production, apparent extract and real degree of fermentation were monitored. The results obtained demonstrate that very high gravity worts at 22°P can be fermented in the same period of time as a 15°P wort, by raising the temperature to 18°C, the oxygen level to about 22 ppm, and increasing the pitching rate to 22 × 10⁶ cell/mL. When diluting to obtain an 11.5°P beer extract, the volumetric brewing capacity increased 91% for the 22°P wort fermentation and 30% using the 15°P wort. After dilution, the fermentation of the 22°P wort resulted in a beer with higher esters levels, primarily the compound ethyl acetate.     

THE CAMBRIDGE PRIZE LECTURE 1996 FROM FIELD TO FIRKIN: AN INTEGRATED APPROACH TO BEER CLARIFICATION AND QUALITY

A concerted and integrated research programme has considerably enhanced the current understanding of fining agents and their effect on beer clarification and quality. In addition to previously published data, much new information has been presented to help develop this understanding. The numerous complex and often inter-related factors that affect both isinglass and copper fining performance have been discussed both qualitatively and quantitatively. This information has been used to identify the following critical control parameters which will facilitate the optimisation of beer quality and brewing process efficiency : Pre- and post-fining beer temperature beer particle levels and NMP charge wort pH cold wort clarity beer pH pre-filtration beer clarity . The discrete unit operations of the brewing process have been integrated into a single continuous process, where the efficiency of each operation depends on the efficiency of all of the previous operations, and impacts directly on that of all subsequent operations. Using this approach, beer clarity problems can in certain circumstances be traced back to variations in the microflora present on barley in the farmer's field. Thus to truly control beer quality, the brewing process must be considered to start not with mashing or malting, but rather with barley growing, and the process must be managed right through “from field to firkin” .     

高浓酿造啤酒发酵周期的研究进展

高浓酿造是采用高浓麦汁发酵,之后进行稀释的啤酒酿造技术,该方法可显著提高生产效率,被广泛应用于啤酒生产中。虽然高浓酿造技术基本发展成熟,但仍存在发酵周期长以及后稀释造成风味不平衡的问题。由于高浓麦汁的特性,会导致发酵不彻底、发酵缓慢以至于延长发酵周期。该文分别从原料、发酵条件、菌株质量方面对高浓酿造啤酒发酵周期影响因素作了综合阐述,并对解决发酵周期长这一问题进行了讨论,提供可行思路。     

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.     

高浓酿造技术在啤酒工业中的应用

高浓酿造技术在啤酒工业中的应用越来越广泛，其主要特点是在不增加设备的基础上能大幅度提高产量，对高浓酿造技术在啤酒工业中的应用进行了较为详细的论述，总结了高浓酿造的特点、高浓麦汁的制备、啤酒酿造糖浆的选择等。最后，讨论了高浓酿造技术对酿造工艺过程、啤酒酵母及最终产品的影响。     

Protein changes during malting and brewing with focus on haze and foam formation: a review

Beer is a complex mixture of over 450 constituents and, in addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides, and lipids. In beer, several different protein groups, originating from barley, barley malt, and yeast, are known to influence beer quality. Some of them play a role in foam formation and mouthfeel, and others are known to form haze and have to be precipitated to guarantee haze stability, since turbidity gives a first visual impression of the quality of beer to the consumer. These proteins are derived from the malt used and are influenced, modified, and aggregated throughout the whole malting and brewing process. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality wort and beer. During mashing, proteins are solubilized and transferred into the produced wort. Throughout wort boiling proteins are glycated and coagulated being possible to separate those coagulated proteins from the wort as hot trub. In fermentation and maturation process, proteins aggregate as well, because of low pH, and can be separated. The understanding of beer protein also requires knowledge about the barley cultivar characteristics on barley/malt proteins, hordeins, protein Z, and LTP1. This review summarizes the protein composition and functions and the changes of malt proteins in beer during the malting and brewing process. Also methods for protein identification are described.     

“珠研”2~＃菌株缩短发酵周期的生产性试验研究（初报）

经诱变、筛选处理的“珠研”２~＃菌株经小试、中试证明其啤酒发酵性能比原引进的菌株有较大的优越性。为验证此菌株对麦汁及扩大生产的适应性，我们对此菌株进行生产性扩大试验。实践证明，“珠研”２~＃菌株对麦汁及扩大生产适应性较强。该菌株具有发酵温度高、降糖快、双乙酰还原能力强、酵母凝集性好、发酵周期短、生产的啤酒质量优越等优点。取得与小试、中试基本一致的结果。我们认为“珠研”２~＃菌株啤酒快速发酵新工艺，可望有良好的推广应用前景。     

ON THE DIFFERING RATES OF FRUCTOSE AND GLUCOSE UTILISATION IN SACCHAROMYCES CEREVISIAE

Fructose is utilised slower than glucose when the two sugars are fermented separately. This phenomenon occurs in a growth promoting medium as well as in brewers wort when using the brewing yeast Saccharomyces cerevisiae. The use of a fructose adjunct in wort at concentrations of 2% w/v and above, may result in residual concentrations of fructose to remain at the end of fermentation and consequently taint the beer with a sweet off-flavour. Glucose and fructose have no effect on maltose utilisation. Thus they do not exert catabolite repression on the maltose membrane transport system in the particular brewing strain of S. cerevisiae under investigation, when fermented in brewers wort.     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling