Optimization of alcohol‐free beer production by lager and cachaça yeast strains using response surface methodology

Alcohol‐free beers (AFBs) are an attractive segment of the beer market both for the brewing industry and for consumers. While AFBs produced by arrested/limited fermentation often suffer from a lack of volatile compounds, beer flavour can be improved by yeast selection and optimization of fermentation conditions. The yeast selection strategy was demonstrated by comparing traditional lager yeast with selected cachaça yeast strains. Correspondingly, response surface methodology was used to enhance the formation of the flavour‐active volatile compounds by optimization of the fermentation conditions (original wort extract, fermentation temperature, pitching rate). Statistical analysis of the experimental data revealed the relative significance of process variables and their interactions. The developed quadratic model describing the responses of total esters and higher alcohols to changes in process variables was used to predict the ideal fermentation conditions in terms of flavour formation. The predicted conditions were experimentally verified and alternative strategies of AFB production are suggested. Copyright © 2016 The Institute of Brewing & Distilling     

Wort composition and its impact on the flavour‐active higher alcohol and ester formation of beer – a review

The most significant factors influencing beer quality are the variety of aroma flavours that stem from a complex system of interactions between many hundreds of compounds. With increasing demand for flavour control and enhanced productivity, the presence of consistent and balanced amounts of higher alcohols and esters are critical aspects of process control. Extensive research has focused on the formation of flavour compounds by the brewing yeast and the factors that influence their synthesis. Fermenting wort is a complex medium from which the brewing yeast utilizes nutrients for living and growth and to where it places its metabolic by‐products. Thus, changes in wort composition will greatly influence final beer aroma. The current paper reviews up‐to‐date knowledge on the contribution of wort composition to the flavour quality of the final product, in particular higher alcohols and esters. Different wort constituents involved in the biosynthesis of these aromatic substances, and which therefore require control during brewery fermentations, are reviewed. Copyright © 2014 The Institute of Brewing & Distilling     

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     

TRYPTOPHOL,TYROSOL AND PHENYLETHANOL—THE AROMATIC HIGHER ALCOHOLS IN BEER

A gas chromatographic method is presented for routine quantitative analysis of aromatic higher alcohols in beer. A satisfactory overall order of reproducibility, sensitivity and simplicity was achieved under the conditions described. This method has been used to compare finished Canadian ales and lagers. It was possible to differentiate an ale from a lager fermentation by the tryptophol content of the product. Experiments were conducted to investigate the influence of several factors on the concentration levels of aromatic higher alcohols. These compounds are produced by the yeast during fermentation and the levels achieved depend upon the type of yeast used, the fermentation conditions, and differences in the wort fermented.     

高浓高温对啤酒酵母发酵性能的影响

以啤酒酵母S-6为实验菌株,研究了主发酵温度和原麦汁浓度对啤酒发酵的残糖、酒精度、风味物质和絮凝性等性能指标的影响。结果表明,原麦汁浓度一定时,主发酵温度对高级醇和乙酸酯的含量影响较大,主发酵温度由10 ℃提高至16 ℃时,高级醇含量提高了10%～20%,乙酸酯含量提高了8%～16%,但CO2累积质量损失、残糖、酒精度和絮凝性基本不受温度的影响;主发酵温度一定时,原麦汁浓度对酵母絮凝性影响较大,原麦汁浓度越高,酵母絮凝性越低,将高浓（18 °Bx）发酵液稀释50%至常浓（12 °Bx）,残糖、酒精度和高级醇的含量与常浓发酵液基本相同。该研究为选育高温高浓发酵低产高级醇同时强絮凝性酵母菌株提供了重要依据。     

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.     

The effect of temperature on fermentation and beer volatiles at an industrial scale

The aim of this work was to determine the impact of temperature on fermentation and maturation, and the volatile composition of beer and yeast viability on an industrial scale. Fermentations were conducted at 8.5, 10 and 11.5°C with maturation at ?1°C. During fermentation and maturation, the changes in extract, yeast growth and esters, alcohols and carbonyl compounds were investigated. Experiments confirmed that the temperature of fermentation had a significant impact on the course of fermentation and maturation. As the wort fermentation temperature increased, the content of acetaldehyde and vicinal diketones decreased whilst the content of esters and higher alcohols increased. Copyright © 2018 The Institute of Brewing & Distilling     

THE CONTROL OF VOLATILE ESTER SYNTHESIS DURING THE FERMENTATION OF WORT OF HIGH SPECIFIC GRAVITY

Some effects have been studied of fermenting wort of unusually high specific gravity, followed by dilution with water to give beer of normal original gravity. This procedure permits increased overall rates of beer production, but matching of flavours requires control of the level of flavour determinants. If not controlled, the concentration of volatile esters may be disproportionately increased so that, after dilution, beer flavour is markedly different. Higher alcohol production is not affected in this way. The concentration of esters can be adjusted to appropriate levels by increasing the production of yeast mass during fermentation. Unsaturated fatty acids, which increase yeast dry matter production without altering the rate of fermentation, are particularly effective in reducing the extent of ester synthesis.     

关于控制啤酒风味稳定性的技术思考

啤酒是由麦汁经啤酒酵母发酵后产生的一类风味独特的含酒精饮料,其独特风味主爰由酵母在麦汁中的总体代谢产物所产生并决定的,啤酒中主要风味物质包括醇类、酯类,羰基化合物、有机酸、硫化物胺及酚类等,并对其物质来源、影响因素及控制措施加以研究与探讨。     

Intracellular metabolite profiling of industrial yeast and the synthesis of flavour compounds in beer

Beer quality is largely guaranteed by a rational ratio of different flavour compounds. In this study, we present intracellular metabolic profiles in the light of lager beer flavour compound synthesis on an industrial scale. A total of 62 intracellular metabolites were identified and quantitatively analysed. Among these, amino acid was identified as the most dominant category of metabolites. We also report gene expression profiling of relevant genes encoding six representing amino acids, including serine, alanine, lysine, valine, leucine and asparagine. Nine genes involved in the amino acid synthesis – ALT1 , ALT2 , ASN1 , ASN2 , BAT1 , SER2 , SHM1 , SHM2 and LYA1 – displayed significantly higher level of expression at various fermentation stages in an industrial lager fermentation. However, concentrations of the recovered amino acids decreased towards the end of fermentation. Our results suggested that the defined amino acids could be assimilated by the yeast to be utilized for synthesis of aroma‐active metabolites at different stages in fermentation. Copyright © 2017 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.     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Apple Aminoacid Profile and Yeast Strains in the Formation of Fusel Alcohols and Esters in Cider Production

The amino acid profile in dessert apple must and its effect on the synthesis of fusel alcohols and esters in cider were established by instrumental analysis. The amino acid profile was performed in nine apple musts. Two apple musts with high (>150 mg/L) and low (<75 mg/L) nitrogen content, and four enological yeast strains, were used in cider fermentation. The aspartic acid, asparagine and glutamic acid amino acids were the majority in all the apple juices, representing 57.10% to 81.95%. These three amino acids provided a high consumption (>90%) during fermentation in all the ciders. Principal component analysis (PCA) explained 81.42% of data variability and the separation of three groups for the analyzed samples was verified. The ciders manufactured with low nitrogen content showed sluggish fermentation and around 50% less content of volatile compounds (independent of the yeast strain used), which were mainly 3‐methyl‐1‐butanol (isoamyl alcohol) and esters. However, in the presence of amino acids (asparagine, aspartic acid, glutamic acid and alanine) there was a greater differentiation between the yeasts in the production of fusel alcohols and ethyl esters. High contents of these aminoacids in dessert apple musts are essential for the production of fusel alcohols and most of esters by aromatic yeasts during cider fermentation.     

Alpha Amino Nitrogen and Fusel Alcohols of Sorghum Worts Fermented into Lager Beer

The levels of alpha amino nitrogen (AAN) and fusel alcohols during fermentations of lager worts produced from waxy sorghum grits either inoculated with yeast cultured in wort or yeast-malt media were performed. Worts produced from waxy sorghum grits had comparable AAN to commercial wort. The oxygen concentration in the reactor headspace changed from 20% at the beginning of fermentation to less than 1% after 72 hrs fermentation indicating a gradual change from aerobic to anaerobic conditions. The utilization of AAN for production of propanol, isobutanol and amyl-isoamyl alcohols from waxy sorghum grits was comparable to a control wort. Production of propanol, isobutanol and amyl-isoamyl alcohols followed the same trend over 144 hr fermentation. Isobutanol was produced in the lowest concentration. The initiation of propanol production occurred after 24 and 36 hr fermentation for worts inoculated with yeast cultured in wort and yeast-malt media, respectively. The final concentration of ethanol and fusel alcohols were within the expected range found in commercial beers. Worts produced from barley malt and waxy sorghum grits were an adequate substrate for Saccharomyces cerevisiae, and were comparable to a commercial wort. The utilisation of refined waxy sorghum grits as brewing adjuncts for lager beers was demonstrated.     

The Effect of Pitching Rate on Fermentation and Flavour Compounds in High Gravity Brewing

The effect of pitching rate on fermentation and production of flavour compounds was studied in high gravity wort using a lager yeast. Fermentation performance was followed by monitoring the total sugar content and yeast growth. Volatile compounds were evaluated by analysing higher alcohols, esters and carbonyl compounds at the end of fermentation. Faster fermentation rates and higher yeast counts were observed with the higher pitching levels. Lower amounts of 2‐ and 3‐methyl‐1‐butanols and higher levels of 2‐methyl‐1‐propanol were found at the increased pitching rates. The concentration of isoamyl acetate was reduced with an increased pitching rate. Higher amounts of diacetyl and 2,3‐pentanedione were obtained at the lower pitching levels.     

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 Contribution of Geraniol Metabolism to the Citrus Flavour of Beer: Synergy of Geraniol and β‐Citronellol Under Coexistence with Excess Linalool

The behaviour of hop‐derived monoterpene alcohols during fermentation by lager yeast was previously investigated in this laboratory. It was suggested that the concentration of geraniol and β‐citronellol in the finished beer increased depending on the initial concentration of geraniol in the wort. In addition, an additive effect among linalool, geraniol and β‐citronellol was found and 5 ug/L of geraniol and β‐citronellol was enough for this effect. In this paper, conditions regarding the enrichment of the initial concentration of geraniol in the wort were investigated. From the screening of various hop cultivars, Citra hop was selected as a geraniol‐rich cultivar. In addition, it was observed that coriander seed, which can be used in beer production as a flavourant, contained not only linalool but also geraniol at high levels. The use of Citra hop or coriander seed was effective for enriching the concentration of geraniol and β‐citronellol in the finished beers. In the Citra beer and in the coriander beer, the content of linalool was excess in comparison with the content of geraniol and β‐citronellol. Therefore, the synergy of geraniol and β‐citronellol, under coexistence of excess linalool, was examined. It was found that the flavour impression of excess linalool became more fruity and citrus by coexistence with geraniol and β‐citronellol and that the coexistence of all three monoterpene alcohols was effective for this synergy. The flavour characteristics of the Citra and coriander beer and the importance of geraniol metabolism for a citrus flavour in beer are discussed.     

影响啤酒中高级醇形成的因素

高级醇是构成啤酒风味的重要物质之一,影响其形成的因素包括原料、麦汗组成、酵母菌种及接种量、发酵条件、溶解氧、发酵度和贮存期。     

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.