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     

Could non-Saccharomyces yeasts contribute on innovative brewing fermentations?

With the advances in the production of beer worldwide, more challenges arise each year in the search for new approaches to the development of distinctive beverages. Attempts to obtain products with more complex sensory characteristics have led experts and brewers to prospect for non-conventional yeasts, i.e., non-Saccharomyces yeasts that may provide a new range of perspectives in terms of techniques and approaches. Besides the widespread use of Dekkera/Brettanomyces for the production of sour beers, other species are emerging for presenting unusual metabolic features that include the production of fruity esters, and a distinctive enzymatic apparatus. Wickerhamomyces anomalus and Torulaspora delbrueckii stand out as the most promising yeasts in brewing processes. Such new tendencies in the use of non-Saccharomyces yeasts comprise the production of low-alcohol beers, functional beers, and bioflavoring approaches. This is a little explored field in brewing practice, still requiring extensive research with practical application. In this sense, this review aims to present the main points for the application of non-conventional yeasts in beer production, and thus contribute to future advances in the topic.     

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.     

LONG-CHAIN FATTY ACID COMPOSITION AS A CRITERION FOR YEAST DISTINCTION IN THE BREWING INDUSTRY

The diversity of yeasts isolated from brewing plants and its role on beer quality makes yeast distinction a major concern in industrial microbiological control. Several approaches have been tried to develop rapid and simple methods to perform such tasks. Among these, stands the utilization of long-chain fatty acid composition of total yeast biomass. In this paper results are reported showing the potential of this technique to characterize yeast flora isolated from industrial plants. Fatty acid profiles of brewing species are clearly differentiated from those of non-Saccharomyces strains using statistical data treatment by principal component analysis (PCA). Distinction between brewing and wild strains of Saccharomyces spp. was not apparent. In comparison, fatty acid profiling showed higher discriminating ability than growth on lysine medium for non-Saccharomyces strains. For distinction of S. cerevisiae var. diastaticus from other Saccharomyces strains, growth on starch medium showed to be necessary.     

CONTROL OF FERULIC ACID AND 4-VINYL GUAIACOL IN BREWING

Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pof^δ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.     

THE INCIDENCE AND SIGNIFICANCE OF WILD SACCHAROMYCES CONTAMINANTS IN THE BREWERY

The persistence of low levels of contamination by non-brewing Saccharomyces through several batch fermentations establishes the immuno-fluorescent method as a very sensitive procedure for estimating the microbiological purity of pitching yeasts. Trade return figures for draught beers show that in this brewery the principal cause for high rejection rates has, on several occasions, been contamination of pitching yeasts with “wild” Saccharomyces. The recommendation is made that pitching yeasts should be discarded when the level of infection achieves 100 cells of wild Saccharomyces per million cells of brewing yeast.     

Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery ‘Wild’ Yeast Contaminants

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known ‘wild’ yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA‐encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non‐Saccharomyces yeast species as well as for brewing and non‐brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non‐introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub‐grouped depending on restriction profiles generated with HinfI.     

FERMENTATION PROPERTIES OF BREWING YEAST WITH KILLER CHARACTER

The cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae has been transferred to three different commercially-used brewing yeasts; two ale strains and one lager strain. The ease with which the character can be transferred is very strain dependent. In addition to killer character, mitochondria from the brewing strain have been transferred into the new ‘killer’ brewing strains. Fermentations carried out with the manipulated strains produced beers which were very similar to those produced by the control brewing strains. The beers produced by killer brewing strains containing brewing yeast mitochondria were most like the control beers and could not be distinguished from them in three glass taste tests. In addition to producing good beers the genetically manipulated yeasts killed a range of contaminant yeasts and were themselves immune to the action of Kil-k₁ killer yeasts.     

啤酒酵母的基因改良研究动态

近年来 ,利用基因工程进行酵母的育种在发酵广谱碳水化合物、提高糖化效率 ,改良酵母凝聚特性和改善啤酒风味方面取得了很大成绩。基因重组菌株将逐步应用到生产实践中。     

INHIBITION OF BEER-SPOILAGE LACTIC ACID BACTERIA BY NISIN

149 strains of bacteria, mostly brewery contaminants able to spoil wort or beer, and 12 brewing strains of yeast (8 ale and 4 lager strains) have been screened using a well-test assay for sensitivity to the food preservative, Nisin (E234), Nisin inhibited growth of 92% of the gram-positive strains, predominantly lactic acid bacteria of the genera Lactobacillus and Pediococcus. In contrast, all 32 gram-negative strains tested, except 3 Flavobacter strains, were Nisin-resistant; in addition none of the brewing yeasts showed Nisin-sensitivity. Therefore. Nisin has potential applications in preventing spoilage of worts or beers by lactic acid bacteria.     

Brewer’s yeast in controlled and uncontrolled fermentations,with a focus on novel,nonconventional, and superior strains

Historically, brewing beer came from uncontrolled spontaneous fermentations; nowadays, spontaneous fermentation is still used in speciality products such as Belgian acid beers (e.g., lambic, gueze, and Rodenbach) or in various traditional beers worldwide. On the other hand, industrial fermentations sometimes require a combination of phenotypic traits that might not be commonly encountered in nature. In some cases, the demand for increased productivity, as well as changing consumer preferences, leads to a great interest towards improvement and/or design new strains or yeast variants. This review addresses the following topics: controlled and uncontrolled fermentations, and advances in the selection of novel, nonconventional, and superior strains for beer.     

Molecular and biochemical aspects of Brettanomyces in brewing

Brettanomyces is a semi‐domesticated yeast that is a crucial component of lambic beers and is increasingly attracting the attention of the brewing industry. Brettanomyces display Saccharomyces‐like features, such as a positive Crabtree effect, ethanol synthesis and tolerance to harsh environments. Additionally, Brettanomyces exhibit β‐glucosidase and esterase activities, the production of phenolic compounds and tetrahydropyridines, together with the ability to ferment dextrins and breakdown cellobiose from wooden casks. Although the importance of Brettanomyces species is documented in the production of different beer styles, the molecular and biochemical features of these species required for brewing are poorly understood. Therefore, this work reviews the current knowledge of the molecular biology and biochemistry underlying the performance of Brettanomyces in the brewing industry. © 2019 The Institute of Brewing & Distilling     

Investigation of beer‐spoilage ability of Dekkera/Brettanomyces yeasts and development of multiplex PCR method for beer‐spoilage yeasts

There have been many beer‐spoilage incidents caused by wild yeasts. Saccharomyces cerevisiae, Dekkera anomala and D. bruxellensis have been recognized as beer‐spoilage yeasts in the brewing industry. In contrast, the beer spoilage ability of Brettanomyces custersianus has not been well characterized, although this species was isolated from beer. In this study, the beer‐spoilage ability of currently described Dekkera/Brettanomyces yeast species was investigated. As a consequence, D. anomala, D. bruxellensis and B. custersianus were shown to grow in commercial beers. On the other hand, the remaining two Brettanomyces species, B. naardenensis and B. nanus, did not grow in beer. These results indicate that B. custersianus should be recognized as a beer‐spoilage species, in addition to S. cerevisiae, D. anomala, and D. bruxellensis. Therefore we developed multiplex polymerase chain reaction (PCR) for the simultaneous detection and identification of B. custersianus and the other beer‐spoilage yeast species. For this purpose, PCR primers were designed in the internal transcribed spacer region or 26S rDNA, and each PCR product was made in different sizes to easily discriminate the species from electrophoretic results. Specificity, reactivity and sensitivity of the designed primers were evaluated. As a result, the developed multiplex PCR method was shown to have high specificity and reactivity, and therefore was considered as an effective tool to identify beer‐spoilage yeast species. This tool can contribute to microbiological quality assurance in breweries. Copyright © 2015 The Institute of Brewing & Distilling     

APPLICATION OF PULSED FIELD CHROMOSOME ELECTROPHORESIS IN THE STUDY OF CHROMOSOME XIII AND THE ELECTROPHORETIC KARYOTYPE OF INDUSTRIAL STRAINS OF SACCHAROMYCES YEASTS

Pulsed field chromosome electrophoresis is a powerful new technique in yeast genetics which permits the resolution of intact yeast chromosomes in an agarose gel matrix. We utilized contour-clamped homogeneous electric field electrophoresis (CHEF) to survey representative strains of Saccharomyces yeasts from the brewing, baking, distilling, sake and wine industries for their electrophoretic karyotypes. All of the strains tested were found to have a unique chromosomal profile, indicating the potential of this technology for “fingerprinting” prototrophic strains of Saccharomyces yeasts. By employing an ILV2 gene probe specific for chromosome XIII, we determined that all of the industrial strains of Saccharomyces yeasts possessed a chromosome XIII which migrated in an identical fashion to chromosome XIII from a reference haploid strain of Saccharomyces cerevisiae. While one lager yeast strain, Saccharomyces carlsbergensis M244, was found to contain two alleles of ILV2 when digested genomic DNA was probed with ILV2, the presence of a novel independently migrating chromosome XIII could not be detected. A homeologous chromosome XIII in this yeast will therefore have to be determined by genetic analysis. Pulsed field chromosome electrophoresis is concluded to be a technology with immediate application to Quality Control and Research and Development programs in industries using Saccharomyces yeasts.     

FATTY ACIDS AND ESTERS PRODUCED DURING THE SPONTANEOUS FERMENTATION OF LAMBIC AND GUEUZE

Lambic and gueuze are Belgian beers obtained by spontaneous fermentation of wort. During previous studies it was found that they result from the successive development of enterobacteria, Kloeckera and Saccharomyces yeasts, bacteria of the genus Pediococcus, and Brettanomyces yeasts. The beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate. This study of the content of the higher fatty acids during a 20 month fermentation period confirms the succession of the different micro-organisms. Pure cultures of isolated yeasts and bacteria produced fatty acids which were also found in the fermenting wort at periods when these organisms were active. Lambic and Gueuze are especially rich in caprylic (C₈) and capric (C₁₀) acids. These are probably produced by Saccharomyces and Brettanomyces. Important amounts of ethyl caprylate and ethyl caprate were also found. As ethyl caprate is almost absent in other beers, it might be considered as another typical aroma component of lambic and gueuze.     

Indian black rice: A brewing raw material with novel functionality

Indian black rice (Chakhao Poireiton) is a pigmented variety, rich in anthocyanins and other phytonutrients. With growing interest in the use of local raw materials in brewing, it was of interest to develop protocols for malting and brewing with Chakhao Poireiton to see whether the antioxidant capacity of anthocyanins could be delivered into finished beer. Protocols for brewing with 100% malted rice were developed and the performance of Indian black rice compared with that of an Italian white rice cultivar suited to brewing. The apparent fermentabilities of rice worts were 69.5% (black) and 67.3% (white), yielding beers of 3.28 and 3.19% ABV respectively. Black rice worts were deficient in free amino nitrogen (83.5 mg/L relative to 137 mg/L for white rice) and would need nitrogen supplementation to avoid issues with fermentation, e.g. elevated diacetyl. Black rice beer had an orange-red hue as a result of extraction of anthocyanin pigments (2.84 mg/L). The oxidative stability of 100% rice beers was measured using electron spin resonance spectroscopy and both samples were found to be unusually stable. Interestingly, when rice beers were blended with a control barley malt derived lager in varying proportions (10, 25, 50%), the oxidative stability was improved, relative to the control lager, particularly so in the case of black rice beer, which contained an antioxidant capacity over and above that of the white rice beer. Future studies are required to determine whether the noted oxidative stability of 100% rice malt beers results in a more flavour-stable beer. © 2019 The Institute of Brewing & Distilling     

Physicochemical characterization of sahti,an ‘ancient’ beer style indigenous to Finland

Sahti, a strong, unhopped farmhouse beer flavoured with juniper, is still actively brewed in rural areas in Finland. Presented here is the first comprehensive analysis of the physical and chemical properties of this unique beer style. Twelve sahti samples from the southwest of Finland were analysed and, while properties varied, the beers generally had high levels of alcohol (mean = 7.9% ABV) and high residual extract (mean = 9.5°P). Foam stability was negligible, as is typical for the style, and glycerol concentrations at 3.1 – 4.7 g L^?1 were higher than in reference beers (commercial lager, wheat beer and porter). These features may be attributed to the very high gravity conditions employed in brewing sahti beers. Bitterness levels were relatively low (3–13 IBU) owing to the absence or moderate use of hops. All samples contained detectable levels of the clove‐like compound 4‐vinylguaiacol owing to the use of baker's rather than brewer's yeast for brewing. Concentrations of higher alcohols and esters were high, with many individual aroma compounds being above the normal flavour thresholds. Results have highlighted the uniqueness of this style of beer in comparison to commercially available beers and have contributed to our understanding of the reasons for the particular sensorial properties of this traditional beer style. Copyright © 2015 The Institute of Brewing & Distilling     

Faba bean as a novel brewing adjunct: Consumer evaluation

The starch in the grains of legumes, such as faba bean (Vicia faba L.), offers an environmentally sustainable raw material for the brewing industry as their entire nitrogen fertiliser requirement can be provided by the natural process of biological nitrogen fixation. Faba bean is, therefore, distinguished from species such as spring barley (Hordeum vulgare L.), which require large amounts of synthetic nitrogen fertiliser. Consumer analysis of beer produced with faba bean as an adjunct compared with barley malt beers has not previously been assessed. This study evaluated the potential of beers brewed using 30% (w/w) dehulled bean (kernel) flour as an adjunct to malted barley, using a series of quantitative sensory tests. The first, a blind acceptance test with inferred preference, found no statistically significant difference in the taste score of the bean kernel flour adjunct beer when compared with conventional beer. In the second acceptance test, the knowledge that the beer was produced using beans did not affect the overall consumer impression of the beer, regardless of how this information was presented. These results suggest that the use of faba beans in brewing does not impact negatively on the taste or acceptability of the resultant beer. © 2019 The Institute of Brewing & Distilling     

CAPRYLIC FLAVOUR AS A FEATURE OF BEER FLAVOUR

Caprylic flavour is part of the distinctive overall flavour of a large proportion of beers in trade. The flavour occurs in the majority of lager beers and in about 20% of ales. It is correlated with the levels of octanoic acid and decanoic acid present in beer. Lager yeasts (S. uvarum) tend to liberate larger amounts of these two fatty acids during fermentation than do ale yeasts (S. cerevisiae). The flavour significance of these acids has been determined by correlating the results of sensory and instrumental analyses and also by adding the purified acids to beer. Implications of these two methods for assessing the flavour significance of chemical constituents of a complex product are discussed.     

三种不同酵母对啤酒发酵及风味的影响

本实验采用Y1110、安琪、模式三种不同的啤酒酵母,在同种工艺条件下测定发酵过程中α-氨基氮(α-AN)、pH、双乙酰、高级醇等指标,并比较三种不同啤酒的风味物质含量。结果表明:Y1110增殖最快,α-AN和pH值下降最快,双乙酰还原较快,后酵结束双乙酰含量最低,啤酒样品含醇量较高,适于醇厚型啤酒酿造;安琪酵母增殖最慢,α-AN和pH值下降最慢,啤酒样品含酯量较高,适于淡爽型啤酒酿造;模式酵母酯类与醇类含量都很高,不适于实际生产。