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.     

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.     

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。     

THE INFLUENCE OF OXYGEN ON THE PRODUCTION OF DIACETYL DURING FERMENTATION AND CONDITIONING

Although varying concentrations of oxygen in wort at pitching do not affect the production of diacetyl, its availability during fermentation has a significant effect. The enhanced production of diacetyl resulting from access of oxygen during fermentation is caused by its effect in increasing yeast growth and in altering the balance of amino acids relative to sugars and not by a direct oxidation of acetoin. Oxygen has no effect upon the concentration of diacetyl during storage and conditioning, provided that the beer is free from contamination.     

125th Anniversary Review: Diacetyl and its control during brewery fermentation

Diacetyl is a butter‐tasting vicinal diketone produced as a by‐product of yeast valine metabolism during fermentation. Concentration is dependent on a number of factors including rate of formation of the precursor α‐acetolactate by yeast, spontaneous decarboxylation of this acetohydroxy acid to diacetyl and removal of diacetyl by yeast via the action of various reductase enzymes. Lowering concentrations of diacetyl in green beer represents an expensive and time‐consuming part of the brewing process and strategies to minimize diacetyl formation or hasten its reduction have potential for improving overall efficiency of the lager brewing system. Here we review the processes that determine diacetyl levels in green beer as well as the various ways in which diacetyl levels can be controlled. The amount of diacetyl produced during fermentation can be affected by modifying process conditions, wort composition or fermentation technique, or by yeast strain development through genetic engineering or adaptive evolution. The process of diacetyl reduction by yeast is not as well understood as the process of formation, but is dependent on factors such as physiological condition, cell membrane composition, temperature and pH. The process of diacetyl removal is typically rate‐limited by the reaction rate for the spontaneous decarboxylation of α‐acetolactate to diacetyl. Copyright © 2013 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     

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。     

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     

酵母形态指标百分数离散度与啤酒质量的关联性

通过跟踪不同发酵罐相同发酵时间的啤酒发酵液中的酵母形态指标百分数离散度,检测发酵结束后各发酵罐的啤酒的质量指标,得出啤酒酵母指标百分数离散度与啤酒质量的关联性,即酵母形态指标百分数离散度与酒精度、发酵度、总酸这三个啤酒质量指标成负相关,与原麦芽汁浓度、外观糖度、乙醛浓度、pH值、双乙酰这五个啤酒质量指标成正相关.     

酵母自溶的成因及其对啤酒质量的影响

酵母自溶由酵母胞内蛋白分解酶外泄引起，影响酵母自溶的因素有：(1)酵母菌种；(2)麦汁营养成分组成不合理；(3)酵母使用代数过高；(4)酵母添加量过多；(5)温度、压力、pH值等发酵工艺条件控制不当；(6)酵母回收时间、方法、压力、酵母贮存条件；(7)微生物污染。酵母自溶会影响啤酒风味稳定性，使啤酒苦味、涩味加重；啤酒双乙酰含量增加；啤酒的泡持性下降；啤酒总酸偏高；啤酒pH值升高；增加啤酒过滤成本。防止酵母自溶的方法有：(1)选择优良强壮的出发菌株；(2)控制酵母添加量和使用代数；(3)制备营养丰富、组成合理的麦汁；(4)严格发酵工艺奈件；(5)加强酵母质量管理；(6)加强卫生管理，保证纯种发酵。     

锌离子在啤酒酿造中的作用与控制

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

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

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

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.     

“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。     

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.     

THE EFFECT OF SUSPENDED SOLIDS ON THE FERMENTATION OF DISTILLER'S MALT WORT

Yeast growth is increased and the initial rate of fermentation is accelerated by the residue of insoluble material which is normally present in distiller's malt wort. The increase in yeast concentration is accompanied by a high glycerol content in the fermented liquor and the formation of higher alcohols, particularly isobutanol and 2-methyl butanol, is also increased. The stimulation of yeast growth is independent of the state of aeration of the wort, suggesting that the effect is not associated with entrainment of oxygen by the solids, and the chemical nature and particle size of the suspended material do not appear to be important. Insoluble solids were without effect when yeast growth was limited by carbohydrate rather than by amino-nitrogen. The results suggest that a combination of electrostatic and absorption forces concentrate yeast cells and amino acids at the solid-liquid interface, increasing the rate of uptake of nutrients by the cell and thus diverting carbohydrate to processes associated with yeast growth.     

A NOVEL AND PRACTICAL YEAST VITALITY METHOD BASED ON MAGNESIUM ION RELEASE

Using a commercial lager brewing yeast, the immediate release of magnesium, potassium and phosphate ions by cells when inoculated into wort was evaluated to be directly related to its subsequent fermentation performance. Yeast which released appreciable amounts of these ions immediately after inoculation mediated improved fermentations as evidenced by better growth, higher ethanol concentrations and lower diacetyl levels at the end of fermentation. Conversely, yeast that absorbed these ions or released them at very low concentrations performed poorly throughout fermentation producing beers with lower ethanol concentrations and higher diacetyl levels. These observations led to the identification and development of a rapid, practical and highly sensitive method to measure Mg⁺⁺ released or absorbed by yeast as an indicator of its vitality and a predictor of its subsequent fermentative performance. Full method details of the Magnesium Release Test (MRT) are given.     

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.     

啤酒连续发酵中酵母生理状态的初步研究

研究了长时间的啤酒连续发酵过程中酵母生理状态的变化情况。结果发现 ,酵母凝聚性先上升后逐渐回复至起始值。酵母的发酵性能、死亡率基本不变。A、B类氨基酸在麦汁中浓度越大 ,在同样降糖率的连续和分批主酵液中的含量差别越小 ;C组氨基酸在麦汁中的浓度越大 ,在连续和分批主酵液中的含量差别也越大。连续和分批主酵液的高级醇含量基本相同 ,乙酸乙酯含量的差异是连续发酵和分批发酵生产的 2种啤酒风味差异的主要原因。