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     

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     

Isolation and Characterisation of Sri Lankan Yeast Germplasm and Its Evaluation for Alcohol Production

Use of inferior yeast cultures represents one of the reasons for low fermentation efficiencies in Sri Lankan alcohol distilleries that use sugarcane molasses. The present study isolated and characterised yeast strains found in natural environments in Sri Lanka and evaluated their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Yeasts were characterised based on morphological and physiological features such as sugar fermentation and nitrate assimilation. Ethanol production, alcohol tolerance and growth rate of the most promising strains were monitored following laboratory fermentations of molasses. Over a thousand yeast cultures were collected and screened for fermentative activity and a total of 83 yeast isolates were characterised as higher ethanol producers. Most of these belonged to the genus Saccharomyces. Certain strains produced over 10% (v/v) alcohol in molasses media during 72 h laboratory fermentations. Only two strains, SL‐SRI‐C‐102 and 111, showed an appreciable fermentation efficiency of about 90%. The latter strain produced the highest level of ethanol, 11% (v/v) within a 48 h fermentation and exhibited improved alcohol tolerance when compared with the baker's yeast strains currently used in Sri Lankan alcohol distilleries. This study highlights the benefits of exploiting indigenous yeasts for industrial fermentation processes.     

Brewing with Rice Malt — A Gluten‐free Alternative

The preparation of beer‐like beverages with rice malt as the only raw material is reported. Several tests were performed on a laboratory scale and in a 25 L‐capacity pilot plant. Both the decoction and the infusion procedure were tested; malt and water were mixed in a ratio 1:3.5 for both methods and the mash was brewed without adding exogenous enzymes. The obtained worts were fermented using bottom fermenting yeasts, while “beers” were re‐fermented utilizing top fermenting yeasts and adding either sterile wort or sugar. A maximum ethanol of 4.5% vol. was obtained after the primary fermentation from an initial wort with an original gravity of 11.8°Plato. All parameters of the beer were found to be acceptable using a standard beer analysis. Owing to a suitable hop addition, an aroma very similar to that of a normal beer was obtained.     

The selection and modification of brewer's yeast strains

In order to achieve a beer of high quality, the yeast culture must be effective in removing the desired nutrients from the growth medium (i.e. the wort), it must impart the required flavour to the beer and finally, the micro-organisms themselves must be effectively removed from the fermented wort after they have fulfilled their metabolic role. Brewer's wort contains the sugars sucrose, fructose, glucose, maltose and maltotriose, together with dextrin material. In the normal situation, brewer's yeast strains are incapable of fermenting the dextrin material; however, yeast strains capable of fermenting at least a part of this dextrin material and producing a palatable beer are now available.One of the important factors known to affect the fermentation rate is the intracellular yeast glycogen concentration which has been found to be influenced by storage conditions. The glycogen level at pitching significantly affects the fermentation rate of the yeast culture.     

2μm DNA PLASMID IN BREWERY YEASTS

Using an agarose gel screening procedure, 2 μm DNA plasmid was detected in all of 10 brewing strains of Saccharomyces yeast examined and in both of two non-brewing, dextrin-utilising strains. Plasmid DNA was identified in yeast grown with access to air in MYGP medium or in hopped wort, and in yeast harvested from 3-day wort fermentations. The yeast plasmid is a suitable self-cloning vector for the genetic manipulation of brewing yeasts by transformation.     

The Production of Low-Alcohol Wines by Aerobic Yeasts

Aerobic yeasts of the genera Pichia and Williopis are commonly regarded as spoilage yeasts of beer and wine by causing turbidity, a surface film of yeast growth and often an excessive estery flavour. However, their ability to utilise sugars oxidatively for cell growth with the production of estery and other flavours of wine with only minimal production of ethanol suggests a method for the production of low-alcohol wines of pleasant “fermented” flavour without the need for additional equipment to remove alcohol by dialysis, reverse osmosis or distillation, or without the excessive sweetness remaining from arrested fermentation. Three strains of Pichia and one of Williopsis were examined for their ability to produce approximately 3%(v/v) ethanol and a good estery and fruity flavour. With normal anaerobic fermentation conditions, or with gentle stirring to prevent formation of a surface film, excessive amounts of alcohol were produced from grape juice of 15% or 20% (w/v) initial sugar concentration. However, an acceptably flavoured wine of alcohol content < 3% was produced by agitation and aeration during fermentation. The ethanol formed in the early stages of culture was oxidised to a final level < 3%, with the production of cell mass and an acceptable flavour.     

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.     

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.     

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     

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.     

SOME EFFECTS OF WORT COMPOSITION ON THE RATE AND EXTENT OF FERMENTATION BY BREWING YEASTS

The time required to ferment worts of varied composition to a given extent is dependent upon the extent of exponential growth in the early stages of fermentation; in the worts studied this is determined by the concentration of assimilable nitrogen. When the concentration of all the non-carbohydrate nutrients in malt wort is halved by dilution with carbohydrate, the addition of appropriate quantities of serine or arginine restores the rate of fermentation to that of the malt wort. Minor nutrients, other than amino acids specifically required by the yeasts used, are thus present in at least two-fold excess in the malt wort. The yeast produced during exponential growth in malt wort (sp.gr. 1·040) is able to ferment rapidly much greater quantities of fermentable carbohydrate than are present in that wort. The majority of the strains of yeast examined ferment equally well when either glucose or maltose is added to malt wort and do so whether the sugar is added prior to fermentation or towards the end; however, one strain fails to ferment satisfactorily if a substantial quantity of glucose is added to wort prior to fermentation, because of the subsequent failure of the yeast to adapt to ferment maltose. It is suggested that most brewing strains do not require to adapt to maltose utilization during the fermentation of wort.     

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     

STRAINS OF YEAST LETHAL TO BREWERY YEASTS

Strains of yeast that are lethal to brewery ale and lager yeasts have been isolated from production-scale two-stage stirred continuous fermentors. These strains release a “killer” factor which is highly active in the pH range 3.8–4.2. When the level of infection reaches 2% the concentration of killer factor is sufficient to give a selective advantage in continuous fermentation, whereupon the proportion of killer yeasts rises and the brewery yeast is rapidly killed. The beer acquires a characteristic off-flavour which has been described as “herbal/phenolic”. Both flocculent and non-flocculent killer strains have been found and these show the characteristics of Saccharomyces cerevisiae but appear to ferment additional wort sugar(s), have an abormally small cell-size and are pleomorphic in mixed culture.     

Evaluation of the fermentative potential of Candida zemplinina yeasts for craft beer fermentation

The fermentative potential of Candida zemplinina Y.01667 and Y.01670 was evaluated to explore the potential use of these yeasts for craft beer fermentations. Fermentation experiments were carried out at different temperatures and soluble solid concentrations, using synthetic media with glucose syrup as a sugar source and with a laboratory malt wort plus different adjuncts. Results showed that both strains fermented well at 14 °C and had improved fermentative activity at 20 °C. The fermentative kinetics of C. zemplinina Y.01667 and Y.01670 were not affected when experiments at higher concentrations of soluble solids were conducted. Furthermore, C. zemplinina strains had better growth, higher viable cells counts, less free amino nitrogen consumption, lower sedimentation rates and slighter changes in pH values, when compared with results of the lager beer yeast Saccharomyces cerevisiae S‐23 in the synthetic medium tested. Fermentations in a malt wort with different adjuncts indicated that C. zemplinina Y.01670 could possibly be used as a yeast in craft beer production. Copyright © 2016 The Institute of Brewing & Distilling     

Effect of the respiro-fermentative balance during yeast propagation on fermentation and wort attenuation

Breweries use different yeast strains to create beers with different flavours and aromas. Yeast propagation must produce yeast that performs consistently from the first fermentation to harvesting and re-pitching in subsequent fermentations. Breweries propagate yeast in wort leading to low efficiency fermentative growth in Crabtree-positive yeast. There is limited knowledge on the impact on beer production when fermenting with yeast propagated in sugar limited and nutrient supplemented wort. It was hypothesised that propagating yeast in this way would have a positive impact on subsequent fermentation performance. Saccharomyces cerevisiae was propagated at the laboratory scale in standard wort with a high carbon to nitrogen (C:N) ratio (850) or in modified wort supplemented with yeast extract to achieve a low C:N ratio (100) and at varying sugar concentrations. Propagation in low C:N wort with 2°P sugar yielded a 27% decrease in fermentation efficiency and a 46% increase in cell production compared to 2°P high C:N wort. This suggests nitrogen is critical to the respiro-fermentative balance during growth. Yeast propagated in standard wort resulted in slower fermentations and significant under-attenuation compared to yeast grown in the modified wort with low sugar and high nitrogen. The results of this study suggest the nitrogen and sugar content drive the respiro-fermentative balance during yeast propagation. The metabolism of yeast during propagation induces significant downstream impacts on the subsequent fermentation performance and wort attenuation. © 2020 The Institute of Brewing & Distilling     

Evaluation of the potential of commercial non-Saccharomyces yeast strains of Torulaspora delbrueckii and Lachancea thermotolerans in beer fermentation

This work evaluated the potential of three commercial non-Saccharomyces yeast strains Torulaspora delbrueckii (Biodiva and Prelude) and Lachancea thermotolerans (Concerto) for beer fermentation. The fermentation performance, volatile and non-volatile profiles were compared. Growth behaviours of all three yeast strains exhibited similar trends during the initial fermentation phase although a marked population decline was detected in strains Prelude and Concerto, which also showed a rapid utilisation of maltose, while strain Biodiva was unable to consume maltose and consumed lesser amounts of amino acids. Additionally, terpenoids inherently absent in the wort such as β-caryophyllene and geranyl acetone were produced in all beers, significantly higher in beers fermented with strain Prelude. For volatile profiles, Prelude and Concerto produced more ethanol and significantly higher amounts of acetate esters and long-chained ethyl esters. Strain Biodiva, on the other hand, produced higher amounts of isoamyl alcohol and ethyl butanoate.     

Contribution by Saccharomyces cerevisiae Yeasts to Fermentation and Flavour Compounds in Wines from cv. Kalecik karasi Grape

The effect of indigenous and commercial S. cerevisiae yeasts on fermentation and flavour compounds of wines was examined in pasteurised grape juice. The flavour compounds were analysed and identified by GC‐FID and GC‐MS, respectively and in general, the amounts of these volatiles were increased by the use of both indigenous and commercial yeasts. The levels of isoamyl alcohol, isoamyl acetate, ethyl octanoate and ethyl deconoate exceeded flavour thresholds. All grape juices were fermented to dryness. Selected yeasts produced higher ethanol concentrations compared to spontaneous fermentations.     

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains of Saccharomyces cerevisiae and interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the production of sparkling wine or to restart stuck fermentations. In addition, interspecies hybrid strains, previously isolated at the end of spontaneous fermentations, revealed a common amino acid signature. The location and potential influence of the amino acids exchanges is discussed by means of a first modelled Hxt3p structure. In comparison, hexokinase genes were more conserved in different Saccharomyces strains and hybrids. Thus, molecular variants of the hexose carrier Hxt3p, but not of kinases, correlate with different fermentation performances of yeast. Copyright © 2015 John Wiley & Sons, Ltd.