Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

Breeding of lipoxygenase‐1‐less malting barley variety ‘Satuiku 2 go’

The lipoxygenase‐1‐less (LOX‐less) trait has positive effects on beer quality, in particular, improvement of flavour stability related to the reduction of beer‐deteriorating substances such as trans‐2‐nonenal. ‘Ryohfu’ is the only spring‐sown malting barley variety grown in Hokkaido, located in the northern part of Japan, and has been used in the Japanese brewing industry for over 20 years. ‘Satuiku 2 go’ was developed as the first LOX‐less malting barley variety in Japan by successive back‐crossing with molecular marker‐assisted selection to introduce the LOX‐less trait into the recurrent parent ‘Ryohfu’. The agronomic performance and general malt quality of ‘Satuiku 2 go’ were almost equivalent to those of ‘Ryohfu’. Wort and beer analyses at the pilot‐scale brewing trial indicated that the LOX‐less trait had little effect on the general characteristics. In contrast, the beers made from ‘Satuiku 2 go’ malt exhibited reduced levels of trans‐2‐nonenal and trihydroxyoctadecenoic acid. The sensory evaluation demonstrated the superiority of ‘Satuiku 2 go’ beers stored under differing conditions in terms of staleness. It can be concluded that the LOX‐less trait was effective in different genetic backgrounds of the recurrent parents used for the development of LOX‐less malting barley varieties. Copyright © 2018 The Institute of Brewing & Distilling     

The influence of LOX‐less barley malt on the flavour stability of wort and beer

The aim of this study was to investigate the influence of lipoxygenase‐less (LOX‐less) barley malt on the quality of wort and beer, with the main focus on beer flavour stability. In the current study, pilot‐scale (1000 L) brewing trials were conducted with a control barley malt AC Metcalfe and a LOX‐less barley malt, PolarStar. The results clearly indicated that the LOX‐less barley malt showed less nonenal potential than the control, although LOX activities in both barley malts were relatively low. The beer brewed from the LOX‐less barley malt contained much lower concentrations of trans‐2‐nonenal (T2N) and gamma‐nonalactone, especially after the (forced or natural) aging of the beer, compared with the beer brewed under the same conditions using the control malt. The sensory panel evaluation indicated similar results in the general flavour profile. The freshness scores of beer brewed from the LOX‐less malt were higher than those from the control malt, and this was more pronounced after forced aging. In addition, the beer brewed from LOX‐less malt had a much better foam stability, almost 30 s (NIBEM test). These results confirm that the use of the LOX‐less barley malt was beneficial to beer flavour stability and foam stability. Copyright © 2014 The Institute of Brewing & Distilling     

The Contribution of 4‐Hydroxyfuranone Derivatives to the Aroma of Commercial Beers and Malts

Analysis of hot water extracts from a range of commercial malts showed that 2,5‐dimethyl‐4‐hydroxy‐3(2H)furanone (DMHF) varied from undetectable (lager malt) to 4.2 mg/litre (crystal malt), a concentration twenty‐six times the flavour threshold in water. 5‐Methyl‐4‐hydroxy‐3 (2H)furanone (MHF) was detected in all samples except one but was always well under its flavour threshold value. 2(or 5)‐Ethyl‐5(or 2)‐methyl‐4‐hydroxy‐3(2H)furanone (EMHF) was not detected in any of the samples. Fermentation of lager, ale and crystal malt extracts with an ale strain of yeast led to the appearance of EMHF in all cases as well as additional DMHF. The greatest increases in both compounds was with the ale malt. Both increases and decreases occurred in MHF concentration as a result of fermentation but final levels were always well below the flavour threshold value. Analysis of ten commercial beers found DMHF in all the samples and in five cases levels exceeded twice the flavour threshold value in beer with flavour units from 2.4 to 9.1. A flavour panel noted that in four of these cases the beer had a distinctly sweet/caramel aroma which is typical of DMHF. EMHF was undetectable in six samples, detectable, but unquantifiable, in three cases but at about 80% of the flavour threshold value in the remaining sample. MHF was found in all samples but at insignificant levels. The results show that DMHF is an important flavour compound in British ales and EMHF may make a contribution in a limited number of situations. The contribution of malt type, brewery processing and yeast strain in determining the concentration of the two 4‐hydroxyfuranones in beer remains uncertain.     

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.     

Influence of hop harvest date of the ‘Mandarina Bavaria’ hop variety on the sensory evaluation of dry‐hopped top‐fermented beer

To impart a special hop aroma to beer, dry‐hopping is a technique that is becoming more and more popular with commercial breweries. Nevertheless, until now little was known about the factors that influence the reproducibility (and consistent product quality) of dry‐hopping with flavour varieties. One factor that could influence the sensory impressions and aroma profile compositions of dry‐hopped beers is the hop harvest date. Therefore, to determine the effects of different harvest dates of the flavour variety ‘Mandarina Bavaria’ on the aroma of top‐fermented beer, laboratory‐scale dry‐hopping trials were performed. Besides tasting sessions of brewed beers, relative quantities of selected hop‐derived, as well as beer‐originated aroma compounds, were investigated by headspace–solid‐phase microextraction–gas chromatography–mass spectrometry. Duo–trio tests between the beers hopped with pellets of different harvest dates showed no significant differences (α = 0.05) between them. In addition, these beers had similar profiles in a five‐point profile tasting scheme. On the other hand, relative concentrations of some hop‐derived aroma compounds – especially myrcene, which is known to be able to contribute to beer flavour – increased corresponding to a later harvest date, while beer originated volatiles were not different between the beers. Analytical results combined with the results of sensory evaluations led to the conclusion that the harvest date of Mandarina Bavaria was not a dominant factor in the dry‐hopping aroma of top‐fermented beers. High amounts of fermentation by‐products are likely responsible for masking effects resulting in no sensory distinctness between the samples with different hop aroma compound concentrations. Copyright © 2016 The Institute of Brewing & Distilling     

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.     

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     

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     

Effects of wax coatings and postharvest storage on sensory quality and aroma volatile composition of ‘Mor’ mandarins

BACKGROUND: Mandarins suffer from accumulation of off‐flavours after harvest. In this study the sensory quality and aroma profile composition of homogenised segments of untreated (control) and wax‐coated ‘Mor’ mandarins after 7 days at 20 °C or 3 or 6 weeks of cold storage at 5 °C were examined. RESULTS: Fruit taste score decreased during storage and following wax coating, and this was attributed to decreases in sourness and mandarin flavour and accumulation of off‐flavours. Aroma profiling identified 31 volatiles that decreased by at least 50% during storage and after waxing and thus may be involved in the observed flavour loss. In contrast, 13 volatiles, mostly belonging to ethanol fermentation and fatty acid and amino acid catabolism pathways, significantly increased at least twofold and probably contributed to off‐flavour enhancement. CONCLUSION: The results showed that after harvest there was a progressive decrease in sensory quality of ‘Mor’ mandarins. It is proposed that observed decreases in contents of sesqui‐ and monoterpenes and short‐chain oxygenated fatty acids may contribute to the decrease in mandarin flavour, whereas increases in the contents of ethanol fermentation metabolites and derivates of fatty acid and amino acid catabolism are most likely involved in causing the enhanced sensation of off‐flavours. Copyright © 2010 Society of Chemical Industry     

Improvement of flavour compound synthesis in a mixed culture system at high temperature by solid‐state fermentation

In order to enhance the synthesis of flavour compounds in solid‐state fermentation (SSF) at a high temperature, Bacillus subtilis XJ‐013 and Saccharomyces cerevisiae Z‐06 were used as a mixed culture with Monascus HQ‐3. The culture temperature was enhanced from 37°C to 56°C by the synergetic effect, and the synthesis of esterase was enhanced from 85.43 U/g to 129.65 U/g in the mixed culture system (over 50% higher than that of the culture using a single strain). This resulted in the synthesis of favourable flavour compounds in the solid‐state fermentation. These results signified that a mixed culture of Monascus and S. cerevisiae was favourable for enzyme production. The mixed culture of Monascus and B. subtilis resulted in a high culture temperature that promoted flavour compound synthesis in the solid‐state fermentation system dramatically. These results present a model to explain the synergetic effects between the fungus and the Bacillus in the solid‐state fermentation. Copyright © 2015 The Institute of Brewing & Distilling     

Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

THE OCCURRENCE OF TWO GEOMETRICAL ISOMERS OF 2,4,5-TRIMETHYL-1,3-DIOXOLANE IN BEER

Two geometrical isomers of 2,4,5-trimethyl-1,3-dioxolane have been detected in a range of commercial and experimental beers at levels up to ca 0.1 ppm. These compounds, plus a number of other dioxolanes, are present in unboiled wort but are lost by evaporation during wort boiling. The trimethyldioxolanes are then reformed during subsequent fermentation. The flavour threshold of a mixture of the two trimethyldioxolanes was found to be ca 0.9 ppm, at which concentration it produced in beer ‘phenolic’ and ‘astringent/drying’ flavour notes. However, these compounds are not present in sufficiently high concentrations to make a significant contribution to the flavour of beer.     

DISCRIMINANT ANALYSIS OF SENSORY AND INSTRUMENTAL DATA ON BEER

Stepwise discriminant analysis has been used to examine sensory and instrumental data on forty six different brands of ale from five brewing companies. The beers were classified also according to four evenly spaced bands of original gravity within the range 1·030 to 1·050. Two samples of each beer from brews several months apart were analysed. Eighty of the beers were correctly assigned to brewing companies from data on four sensory and five instrumental parameters. The most important of these parameters were iso-amyl alcohol content and caprylic flavour, both of which relate to products of fermentation. Eighty six of the beers were correctly classified into gravity bands using data on thirteen sensory parameters relating to the beer flavours. In this case the three most important parameters were body (palate-fullness), viscous (thick) mouthfeel and aldehydic/estery character.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 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     

Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production

Self‐cloning strains of industrial brewing yeast were constructed, in which one allele of α‐acetohydroxyacid synthase (AHAS) gene (ILV2) was disrupted by integrating Saccharomyces cerevisiae genes, γ‐glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of ILV2. The self‐cloning strains were selected for their resistance to CuSO₄ and identified by PCR amplification. The results of AHAS and glutathione (GSH) assay from fermentation with the self‐cloning strains in 500‐mL conical flask showed that AHAS activity decreased and GSH content increased compared with that of host yeasts. The results of pilot scale brewing in 5‐L fermentation tank also indicated that GSH content in beer fermented with self‐cloning strains T5‐3 and T31‐2 was 1.3 fold and 1.5 fold of that of host QY5 and QY31, respectively; and diacetyl content decreased to 64% and 58% of their hosts, respectively. The self‐cloning strains do not contain any heterologous DNA, they may be more acceptable to the public.     

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     

A novel approach for the production of a non‐alcohol beer (≤0.5% abv) by a combination of limited fermentation and vacuum distillation

Despite the increasing demand, the production of non‐alcohol beers is still limited by unsatisfactory or artificial flavour and taste. In this study, a novel approach to producing non‐alcohol beer is presented, in which the alcohol‐reducing techniques, limited fermentation and vacuum distillation were combined. Starting from barley and wheat malts, wort with a low level of fermentable sugars was prepared by infusion mashing and lautering. Limited fermentation was carried out by Saccharomycodes ludwigii at 18°C. When the level of fermentable sugar was reduced by 25%, the fermented wort was quickly cooled from 18 to 0°C and held at that temperature for two days. The young beer was obtained after degassing and removal of yeast and was then subjected to vacuum distillation at 0.06 MPa to remove the alcohol. The concentrated extract is suitable for storage and transportation. The final product of non‐alcohol beer was obtained by dilution with deoxygenated water and carbonation with 6.0 g/L CO₂, followed by addition of 8–12% of regular beer and equilibration for 2–3 days to develop normal beer aroma. The results showed that the non‐alcohol beer had several favourable properties, including the alcohol level of <0.5% (v /v), colour 7.0 (EBC), thiobarbituric acid value of 1.05 and ratio of alcohols to esters of 1.08. Compared with other methods for the production of non‐alcohol beer, this novel approach produced a favourable alternative to regular beers with similar flavour characteristics and satisfactory stability. Copyright © 2017 The Institute of Brewing & Distilling