PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Differentiation of Brewing and Related Yeasts Based on PCR Amplification and Restriction Fragment Length Polymorphism of Ribosomal DNA

A study to differentiate commercially applied brewing yeasts selected from the culture collection of the University of the Free State from related yeasts of the genus Saccharomyces using PCR amplification and RFLP of the internal transcribed spacers region was conducted. Differentiation was dependent on the restriction enzymes used to digest the amplified rDNA. Digestion with Hae III, Cfo I, Sau 3AI and Msp I divided representatives of the genus Saccharomyces into several unique groups. With Msp I the DNA patterns for the two brewing strains were similar, but could be differentiated from Sacch. cerevisiae and other species tested. It was also possible to distinguish some members of the Saccharomyces sensu stricto group i.e. Sacch. bayanus and Sacch. pastorianus from Sacch. cerevisiae and Sacch. paradoxus using Hae III as well as Sacch. paradoxus from the other sensu stricto members using Msp I digestion.     

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     

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.     

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.     

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.     

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.     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.     

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)‐generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR‐RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR‐RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR‐RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR‐RFLP coupled with pre‐selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants. Copyright © 2011 Society of Chemical Industry     

Association between polymorphisms of the heart fatty acid binding protein gene and intramuscular fat content,fatty acid composition,and meat quality in Berkshire breed

The objective of this study was to examine the relationship between polymorphisms of the heart fatty acid binding protein (H-FABP) gene and variation in intramuscular fat (IMF) content, fatty acid composition, and meat quality in Berkshire breed. We detected an association between IMF content and H-FABP HinfI restriction fragment length polymorphism (RFLP) genotype. The HH genotype showed the greatest IMF content among the genotype classes (P < 0.05). Regarding meat quality traits, only drip loss was significantly different among the H-FABP HinfI RFLP genotype classes (P < 0.01). In the case of fatty acid composition, the degree of saturation of fatty acids was greater in hh-genotype pigs than in HH and Hh genotypes. The H-FABP MspI RFLP genotype affected unsaturated fatty acid content, and the ratio of polyunsaturated fatty acid to saturated fatty acid (P < 0.05), whereas the H-FABP HaeIII RFLP genotype had no effect on fatty acid characteristics.     

RESTRICTION MAPPING OF THE POF I GENE

The gene (POFI) which imparts to certain yeasts the ability to decarboxylate phenolic acids to corresponding phenolic compounds has been analysed by restriction mapping. New restriction sites have been used to examine differences between Pof⁺ and Pof^? Saccharomyces cerevisiae strains. Southern Blot analysis of selected yeast strains has demonstrated that the POFI gene sequence is highly conserved between the Pof⁺ strain from which the gene was cloned, two Pof^? lager brewing strains and one Pof⁺ Saccharomyces brewery isolate. However, sequence differences have been found between the original Pof⁺ strain, a Pof^?laboratory strain and a Pof^? ale brewing strain.     

Long Term Serial Repitching and the Genetic and Phenotypic Stability of Brewer's Yeast

Two brewing yeast strains, one employed for the production of an ale type product and the other used solely for bottle conditioning, were serially repitched over a period of one year. Subsequently each yeast culture was compared to the original stocks for a variety of phenotypic and genotypic characteristics. Fermentation performance was assessed in terms of flocculation capacity and the time required to achieve attenuation in 150 barrel fermentation vessels, while the propensity for the population to accumulate variants was assessed by analysing giant colony morphology. Changes to the genome were monitored by DNA fingerprinting of each yeast culture using RAPD‐PCR and RFLP. Although some colony morphology variation was observed between fresh and old ale yeast cultures, there were no detectable genetic changes or alterations in fermentation characteristics to either yeast strain over the course of serial repitching. It is suggested that although some brewing yeast strains are susceptible to genetic drift, others are more resilient and can remain stable over extended periods of time. The propensity to produce variants may therefore play a significant role in determining the number of times a strain may be serially repitched, or its suitability for beer fermentations.     

Genetic Identification of Lophius budegassa and L. piscatorius by PCR-RFLP Analysis of a Mitochondrial tRNAGlu/Cytochrome bSegment

Identification of Lophius budegassa(black‐bellied angler) and L. piscatorius(angler) (Lophiiformes) was carried out on the amplification of a 486 bp tRNA^Glu/cytochrome b segment using the polymerase chain reaction (PCR). Direct DNA sequencing of 6 PCR products was carried out. Six restriction endonucleases (AluI, CfoI, HaeIII, HinfI, Mae, and ScrFI) with different species‐specific restriction fragment length polymorphism (RFLP) were selected. Digestions of PCR products from 30 individuals showed no intraspecific polymorphism. Double digestions (CfoI and HinfI, and HaeIII and ScrFI) were simpler and more rapid than single digestions. This technique is suitable for distinguishing tails of both Lophius species.     

Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus

Two distinct genetic groups (Saaz and Frohberg) exist within the hybrid Saccharomyces pastorianus (S. cerevisiae × S. eubayanus) taxon. However, physiological/technological differences that exist between the two groups are not known. Fermentative capability of the parental S. eubayanus has likewise never been studied. Here, 58 lager strains were screened to determine which hybrid group they belonged to, and selected strains were characterized to determine salient characteristics. In 15 °P all‐malt wort fermentations at 22 °C, Frohberg strains showed greater growth and superior fermentation (80% apparent attenuation, 6.5% alcohol by volume in 3–4 days) compared to all other strains and maintained highest viability values (>93%). Fermentation with S. eubayanus was poor at the same temperature (33% apparent attenuation, 2.7% alcohol by volume at 6 days and viability reduced to 75%). Saaz strains and S. eubayanus were the least sensitive to cold (10 °C), though this did not translate to greater fermentation performance. Fermentation with S. eubayanus was poor at 10 °C but equal to or greater than that of the Saaz strains. Performance of Saaz yeast/S. eubayanus was limited by an inability to use wort maltotriose. [¹⁴C]‐Maltotriose transport assays also showed negligible activity in these strains (≤0.5 µmol min^?1 g^?1 dry yeast). Beers from Saaz fermentations were characterized by two‐ to sixfold lower production of the flavour compounds methyl butanol, ethyl acetate and 3‐methylbutyl acetate compared to Frohberg strains. Higher alcohol and ester production by S. eubayanus was similar to that of Frohberg strains. Copyright © 2013 John Wiley & Sons, Ltd.     

Differentiation of lactic acid bacteria based on RFLP analysis of the tuf gene

The tuf gene, which encodes the elongation factor Tu, is universally distributed in Gram-positive bacteria. A small region of the tuf gene was evaluated for the differentiation of Lactobacillus strains using restriction fragment length polymorphism (RFLP) analysis. The 803 bp portion of tuf genes from 9 closely-related Lactobacillus reference strains were successfully amplified by polymerase chain reaction (PCR), and restriction patterns of each were evaluated with a set of restriction enzymes, which included AluI, HaeIII, and Sau3AI. The results implied that restriction patterns obtained from 2 of these restriction enzymes (AluI and HaeIII) could effectively differentiate closely-related Lactobacillus species.     

The effect of hopping regime,cultivar and β‐glucosidase activity on monoterpene alcohol concentrations in wort and beer

Previous studies show that the complexity of hop aroma in beer can be partly attributed to the hydrolysis of glycosidically bound monoterpene alcohols extracted from hops during the brewing process to release volatile aglycones. However, fundamental studies that examine the extraction of glycosides during brewing and their subsequent hydrolysis by yeast have not been performed. Furthermore, extraction of other hop‐derived compounds into beer shows a strong dependency on the hop cultivar being used and the point at which it is added. This study focused on the extent of glycoside extraction owing to hopping regime and cultivar, and their hydrolysis by yeast β‐glucosidase activity. Glycoside concentrations of wort made with three different hopping regimes and three cultivars were measured by the difference in volatile aglycone concentrations between samples treated with purified β‐glucosidase and untreated samples. Aglycone concentrations were measured by solid‐phase microextraction gas chromatography–mass spectrometry. Additionally, β‐glucosidase activities for 80 different yeast strains and their effect on aglycone concentration in wort were determined. Results showed that yeast have a wide range of abilities to hydrolyse glycosides with a maximum hydrolysis occurring after 3 days of fermentation regardless of yeast activity. Although it was shown that yeast are capable of glycoside hydrolysis, glycoside concentrations in wort are low and make small contributions to hop aroma. These results help explain the extent to which different brewing yeasts and hopping regimes contribute to hoppy beer aroma through the hydrolysis of non‐volatile hop‐derived compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

沙城龙眼葡萄园土壤中酵母菌多样性研究

为分离筛选具有产区特色的菌种,本实验采用梯度稀释和划线分离技术,从沙城地区3 个龙眼葡萄园土壤中共分离得到210 株酵母菌。按照形态表型分为9 类,又利用核糖体5.8S rDNA-ITS 区域RFLP 方法将这9 类酵母在分子水平上进行了区分,其中两类酵母为常见酵母,分别为东方伊萨酵母(Issatchenkia orientalis)和热带假丝酵母(Candida tropicalis)。实验证明了该地区土壤中酵母菌的多样性。     

Screening of β‐Glucosidase and β‐Xylosidase Activities in Four Non‐Saccharomyces Yeast Isolates

The finding of new isolates of non‐Saccharomyces yeasts, showing beneficial enzymes (such as β‐glucosidase and β‐xylosidase), can contribute to the production of quality wines. In a selection and characterization program, we have studied 114 isolates of non‐Saccharomyces yeasts. Four isolates were selected because of their both high β‐glucosidase and β‐xylosidase activities. The ribosomal D1/D2 regions were sequenced to identify them as Pichia membranifaciens Pm7, Hanseniaspora vineae Hv3, H. uvarum Hu8, and Wickerhamomyces anomalus Wa1. The induction process was optimized to be carried on YNB‐medium supplemented with 4% xylan, inoculated with 106 cfu/mL and incubated 48 h at 28 °C without agitation. Most of the strains had a pH optimum of 5.0 to 6.0 for both the β‐glucosidase and β‐xylosidase activities. The effect of sugars was different for each isolate and activity. Each isolate showed a characteristic set of inhibition, enhancement or null effect for β‐glucosidase and β‐xylosidase. The volatile compounds liberated from wine incubated with each of the 4 yeasts were also studied, showing an overall terpene increase (1.1 to 1.3‐folds) when wines were treated with non‐Saccharomyces isolates. In detail, terpineol, 4‐vinyl‐phenol and 2‐methoxy‐4‐vinylphenol increased after the addition of Hanseniaspora isolates. Wines treated with Hanseniaspora, Wickerhamomyces, or Pichia produced more 2‐phenyl ethanol than those inoculated with other yeasts.     

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