PCR‐TTGE and RAPD‐PCR Techniques to Analyze Saccharomyces cerevisiae and Saccharomyces carlsbergensis Isolated from Craft Beers

In the Friuli Venezia Giulia region (North East of Italy) the production of craft beers has been increasing constantly. Usually microbreweries use yeasts supplied by Italian or foreign industrial breweries for beer production. Yeast species are often not known, moreover the vitality, the viability, the physiological state and the number of generation are not known. To improve the quality of the final product it is important to evaluate the quality of the yeast strain used and the lactic acid bacteria contamination. Various molecular methods have been developed to compare genetic characteristics of yeast strains used in beer and wine production. The methods proposed in this work, PCR‐TTGE and RAPD‐PCR techniques, allow the comparison of specific DNA sequences to identify and/or characterize yeast strains. The molecular methods are faster than traditional methods and they allowed the identification of the strains analysed as S. cerevisiae and the intraspecies differentiation among yeast strains tested within 8 h after cell growth.     

Biotyping Saccharomyces cerevisiae strains using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), a powerful biotyping tool for the identification of bacteria and clinical yeast isolates, was investigated as a method to rapidly identify industrial S. cerevisiae strains. In this study, an optimized sample preparation protocol was devised for the biotyping of S. cerevisiae strains. The results demonstrate that ethanol/formic acid protein extraction, of a cell density of 1 × 10⁸ cells co-crystallized with α-cyano-4-hydroxycinnmaic acid, is required to yield mass spectral profiles that are diagnostic of each strain type. Forty-four S. cerevisiae strains commonly employed in South African fermentation-based industries were biotyped in an attempt to create a yeast reference database within a local context. The data revealed that MALDI-TOF MS can be used for the rapid and accurate identification of laboratory and industrial S. cerevisiae strains.     

Differentiation of contaminating yeasts in brewery by PCR-based techniques

Restriction fragment length polymorphism (RFLP) patterns of PCR-amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied to the analysis of 19 yeast strains isolated in Polish breweries from pitching yeast, fermenting wort and final beer. One five-base (ScrFI) and two four-base cutting (HaeIII, MspI) restriction enzymes were used. Primer 21 (5′-GCTCGTCGCT-3′) was selected for RAPD analysis. Among the isolates tested, high molecular diversity in the length of electrophoretically separated products was observed. The results show that it is possible to differentiate between brewing yeast and wild yeast isolates by their electrophoretic patterns.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

Production of Beer with a Genetically Engineered Strain of S. cerevisiae with Modified Beta Glucanase Expression

This study used a recombinant Saccharomyces cerevisiae strain, which expressed both β‐glucanase enzyme and reduced Pro‐teinase A expression during wort fermentations. The genetic stability and fermentation features of the strain were examined. The recombinant strain's proteinase A activity was reduced compared to the parent strain; β‐glucanase was produced throughout the fermentation. The fermentation with the recombinant S. cerevisiae strain exhibited a larger reduction in β‐glucan content than what was observed with the control strain, with β‐glucan degradation above 80%. The foam stability period was reduced when the beer produced by the recombinant S. cerevisiae was stored for 3 months. SDS‐PAGE analysis of the beer proteins indicated that lipid transfer protein 1 had disappeared. Fermentation studies indicated that based on the parameters examined, this recombinant strain was suitable for industrial beer production.     

Sucrose Fermentation by Brazilian Ethanol Production Yeasts in Media Containing Structurally Complex Nitrogen Sources

Four Saccharomyces cerevisiae Brazilian industrial ethanol production strains were grown, under shaken and static conditions, in media containing 22% (w/v) sucrose supplemented with nitrogen sources varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Sucrose fermentations by Brazilian industrial ethanol production yeasts strains were strongly affected by both the structural complexity of the nitrogen source and the availability of oxygen. Data suggest that yeast strains vary in their response to the nitrogen source's complex structure and to oxygen availability. In addition, the amount of trehalose produced could be correlated with the fermentation performance of the different yeasts, suggesting that efficient fuel ethanol production depends on finding conditions which are appropriate for a particular strain, considering demand and dependence on available nitrogen sources in the fermentation medium.     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

Evaluation of a Brazilian Fuel Alcohol Yeast Strain for Scotch Whisky Fermentations

Traditionally, distilling companies in Scotland have employed a very limited number of yeast strains in the production of alcohol for Scotch whiskies. Recent changes such as the decline in availability of brewers' yeast as a secondary yeast strain and the availability of yeast in different formats (e.g., dried and cream yeast as alternatives to compressed yeast) have promoted interest in alternative Scotch whisky distilling yeasts. In previous work, we investigated different strains of yeasts, specifically Brazilian yeasts which had been isolated from and used in fuel alcohol distilleries. One of the Brazilian yeasts (CAT 1) showed a comparable fermentation performance and superior stress tolerance compared with a standard commercial Scotch whisky distilling yeast (M Type). The Brazilian CAT 1 yeast isolate was further assessed in laboratory scale fermentations and subsequent new make spirit was subjected to sensory analyses. The spirits produced using the Brazilian strain had acceptable flavour profiles and exhibited no sensory characteristics that were atypical of Scotch whisky new make spirit. This study highlights the potential of exploiting yeast biodiversity in traditional Scotch whisky distillery fermentation processes.     

Genome comparison and evolutionary analysis of different industrial lager yeasts (Saccharomyces pastorianus)

Fermented beverages, especially beer, have accompanied human civilizations throughout our history. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. For hundreds of years, lager yeast (Saccharomyces pastorianus) has been subjected to multiple rounds of domestication owing to artificial selection during beer production. As a result, this species comprises a genetically diverse collection of strains that are used in different breweries. However, the scope of genetic diversity captured during the domesticated evolution of this species remains to be determined. To begin to address this, we collected the genome information of the only four lager strains that had been whole‐genome sequenced. For the first time, genome comparison was conducted between lager yeasts and clear signatures were found that defined each industrial yeast strain. The genetic variation comprises both single nucleotide polymorphisms and insertions and deletions. In addition, the core–pan genome was introduced for the first time to the genomic analysis of lager yeasts, detecting numerous strain‐specific and species‐shared genes. Furthermore, phylogenetic tree and synteny analysis results obtained in this study revealed information regarding the evolutionary relationship and group differentiation of studied strains. Genome comparison of the lager strains will, therefore, enable the characterization of the overall genetic diversity of this species, assist in the identification of genomic loci that play important roles in regulating key industrial phenotypes, and highlight the understanding of the hybrid nature and evolutionary details. Copyright © 2016 The Institute of Brewing & Distilling     

啤酒酵母突变株发酵性能比较及随机扩增多态DNA分析

对9株啤酒酵母菌种及经过诱变获得的突变株进行了发酵试验，比较了不同菌株的发酵能力、产高级醇能力、双乙酰还原能力以及菌株稳定性。同时利用随机引物对不同啤酒酵母株的基因组DNA进行了随机扩增多态DNA（RAPD）分析，比较不同突变株之间基因组的分子差异。结果表明：不同酵母发酵14d后外观发酵度在72．3％-76．8％，其中酵母YZB具有较高的发酵能力，最终发酵产物的高级醇含量最低，双乙酰峰值最低为0．36mg／L，而酵母Y1110最终发酵产物的高级醇含量最低为67．4mg／L，但双乙酰峰值达0．41mg／L，后酵结束后这2株酵母的双乙酰均可降至0．1mg／L以下。菌株稳定性实验结果表明，在传代7次以后和第1代的主要性能没有明显变化。利用随机引物OPG-5对不同酵母的基因组进行RAPD分析，酵母Y1110、YZB和YZD可以通过特异的扩增谱带区别于其它菌株，该结果为啤酒酵母特异的分子标记奠定了基础。     

Identification of yeast strains using the polymerase chain reaction

Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

啤酒废弃物在食品工业中的应用

啤酒废弃物主要是啤酒废酵母和啤酒糟,在食品工业中有广泛应用。啤酒废酵母可用于生产酵母浸膏、营养调味品、胞壁多糖及蛋白粉;啤酒糟可用于制作面包饼干,生产调味品、食品添加剂、复合氨基酸,用作食用菌栽培原料,发酵生产燃料乙醇、甘油及沼气。啤酒废弃物在食品工业中具有非常广阔的应用前景,具有较好的社会效益和经济效益。     

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.     

Identification of coarse (IR-8), fine (PR-106) and superfine (Basmati-386) rice cultivars

Physical, cooking and protein characteristics of coarse (IR-8), fine (PR-106) and superfine (Basmati-386) rice cultivars were studied for establishing criteria of identification. Paddy grains of Basmati-386 had the least thousand seed weight while milled rice had maximum kernel length and L/B ratio. Basmati-386 took minimum cooking time and exhibited the highest elongation ratio. The ΔL/ΔW ratio better distinguished the three cultivars as Basmati-386 had a significantly higher value. SDS–PAGE of globulin and glutelin could be used as an identification tool for differentiating the three cultivars by protein patterns.     

啤酒酵母改良途径

啤酒酵母的改良途径有：诱变选育，选择自发突变株，无外源遗传损伤，较稳定，但突变频率低，需富集，诱变菌株可提高乙醇耐受性和耐糖度，从而提高发酵度，杂交育种，可育成抗嗜杀酵母的啤酒酵母，原生质体融合可培育出糖化型啤酒酵母，选育提高酵母凝絮性的菌株，以及培育新菌株。通过基因工程可培育出降低双乙酰的酵母，提高生香物质的酵母，降解大分子蛋白质的酵母及分解葡聚糖的酵母等。     

Storage stability of pasteurized non‐filtered beer

The influence of pasteurization on non‐filtered beers was investigated during accelerated storage (40°C, 41 days). Two beers were produced from the same hopped wort, which was fermented with two different yeast strains. Half of the bottled beers were tunnel pasteurized resulting in four different beer samples. The pasteurization influenced the volatile profile of both fresh beers, but during storage the differences between the volatile profiles of pasteurized and non‐pasteurized beers disappeared. During the storage period, the pasteurized beers gave a lower rate of radical formation, as evaluated by electron spin resonance spectroscopy, indicating a better oxidative stability. The pasteurization had no effect on the levels of the pro‐oxidative metals iron and copper. Pasteurization slightly increased the protein content of the beers. SDS–PAGE analysis showed that the two beers had different protein profiles, which changed during storage; however, pasteurization of both beers did not affect their protein profiles. The level of thiols were lowered in one beer and raised in the other beer by pasteurization, but during storage the levels of thiols decreased at the same rate in all of the beers. It was concluded that pasteurization had a positive influence on the oxidative stability of non‐filtered beer. Copyright © 2013 The Institute of Brewing & Distilling     

Sourdough derived strains of Saccharomyces cerevisiae and their potential for farmhouse ale brewing

The Finnish farmhouse ale sahti is unique in that it is fermented with baking, rather than brewing strains of Saccharomyces cerevisae. The custom of maintaining farmhouse yeast cultures is however no longer practiced in Finland, and much yeast derived diversity in sahti beers has presumably been lost as a consequence. Here, the brewing potential of a number of sourdough derived strains was tested with respect to a number of different fermentation traits. Seven strains originally isolated from Finnish or Italian sourdough cultures were used to ferment high gravity sahti wort (20°P), and fermentation performance together with production of volatile compounds were assessed and compared with a reference baking yeast. Strains differed in terms of fermentation rate, yield, yeast viability and beer flavour profile. All were maltotriose positive, but utilisation varied so that alcohol yield could be greater or lower than that of the reference strain, with values ranging from 6.6 to 7.9% (v/v). Production of aroma compounds was also variable so that it was possible to identify strains producing high levels of esters and those with lower production, which could be used to emphasise flavours originating from raw materials. All strains generated 4-vinyl guaiacol and so would be suitable for other beers where this is a part of the normal flavour profile. Results suggest that sourdough isolates of S. cerevisiae are suitable for sahti production, but could also be applied to other beer styles as a way to differentiate products. © 2020 The Authors. Journal of the Institute of Brewing published byJohn Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

Probiotic potential of γ-aminobutyric acid (GABA)–producing yeast and its influence on the quality of cheese

Kazakh cheese is a traditional dairy product in Xinjiang, China. To study the function and potential probiotic characteristics of yeast in Kazakh cheese and its contribution to cheese fermentation, we screened the γ-aminobutyric acid (GABA)–producing yeasts Pichia kudriavzevii 1–21, Kluyveromyces marxianus B13–5, Saccharomyces cerevisiae DL6–20, and Kluyveromyces lactis DY1–10. We investigated the potential probiotic properties of these strains and their use in cheese fermentation (cheeses designated CSP, CSM, CSS, and CSI, respectively); a control with no added yeast was designated CS. The results showed that the 4 yeast strains all showed high self-polymerization (2- and 24-h autoaggregation capacity of >80 and 90%, respectively), hydrophobicity (40–92% variation, low hydrophobicity in xylene, but within the range of probiotics), and the ability to survive the gastrointestinal tract (survival rate >75% after simulation), indicating the probiotic ability of the strains in vitro. The GABA production capacity of the CSM cheese increased (to 95.6 mg/100 g), but its protein content did not change significantly, and amino acid degradation was obvious. The GABA production capacity of the CSS cheese decreased (to 450 mg/kg); its protein content declined, and its amino acid content increased. Except for water and protein, we found no obvious differences in most physical and chemical indicators. Kluyveromyces marxianus B13–5 helped to form the desired texture. Multivariate statistical analysis showed that fermentation of the cheese with the 4 yeasts improved the production of esters and alcohols. The CSS cheese had good aroma production performance, because S. cerevisiae DL6–20 produced high concentrations of isoamyl alcohol, hexanoic acid ethyl ester, benzyl alcohol, octanoic acid ethyl ester, 3-hydroxy-2-butanone, and hexanoic acid; the content of 2-methyl-propanoic acid was low. Compared with the CSP cheese, the CSI and CSM cheeses had a fruitier aroma and a milder odor, but the CSI and CSM cheeses had high concentrations of ethyl acetate, butanoic acid, ethyl ester, 3-methyl-1-butanol-acetate, ethyl hexanoate, ethyl octanoate, acetic acid 2-phenylethyl ester, and ethyl lactate; concentrations of 3-methyl-butanoic acid, propanoic acid, acetic acid, and butanoic acid were low. The CSP cheese had stronger acid-producing ability. The order of fragrance production performance was CSS > CSI, CSM > CSP > CS. Research into the fermentation mechanisms of GABA-producing yeast in cheese will provide a theoretical basis for the quality control and industrial production of Kazakh cheese.     

Genetic and physiological characterization of yeast isolated from ripe fruit and analysis of fermentation and brewing potential

‘Wild’ and spontaneously fermented beers are growing in popularity in the craft beer industry. Most of these beers are fermented by the use of either pure cultures of unconventional yeast and bacteria or spontaneous fermentation using mixed local microflora. This study examined the potential of using pure strains of new isolates of wild yeast in the fermentation of a unique beer. The microbial communities from the fruit of pindo palm, loquat, hackberry and blackberry were collected in liquid culture, then plated for isolation. Ten isolates were selected for further analysis. Strains were identified by restriction fragment length polymorphism (RFLP) analysis and analysed for growth in a simple liquid media, fermentation in a complex media, alcohol tolerance and acid tolerance. Despite identification of some strains as the same species, they displayed a wide range of physiological properties. All strains were tolerant of pH values as low as 2.4, but none were tolerant of pH 1.9. Alcohol tolerance of different strains varied from 6 to 12%. Several strains had properties that suggest potential as primary fermenters, including the alcohol fermentation of a beer wort. Organoleptic properties of beers fermented with several of the strains demonstrated potential for commercial brewing. Copyright © 2014 The Institute of Brewing & Distilling