THE GENETIC MANIPULATION OF KILLER CHARACTER INTO BREWING YEAST

A procedure is described whereby the cytoplasmically-inherited killer character of a laboratory strain of Saccharomyces cerevisiae is transferred to a brewing yeast strain. Neither preparation of protoplasts of the brewing yeast nor mutation of its nuclear genes are required for this process. The brewing yeast killer strains produced have the advantages over their parent brewing cell that they kill sensitive yeasts and are immune to the killing action of certain killer yeasts. The method described offers significant advantages over the process of transformation as a means of genetically manipulating commercial yeasts.     

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.     

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     

Bioprospecting for brewers: Exploiting natural diversity for naturally diverse beers

The burgeoning interest in archaic, traditional, and novel beer styles has coincided with a growing appreciation of the role of yeasts in determining beer character as well as a better understanding of the ecology and biogeography of yeasts. Multiple studies in recent years have highlighted the potential of wild Saccharomyces and non-Saccharomyces yeasts for production of beers with novel flavour profiles and other desirable properties. Yeasts isolated from spontaneously fermented beers as well as from other food systems (wine, bread, and kombucha) have shown promise for brewing application, and there is evidence that such cross-system transfers have occurred naturally in the past. We review here the available literature pertaining to the use of nonconventional yeasts in brewing, with a focus on the origins of these yeasts, including methods of isolation. Practical aspects of utilizing nondomesticated yeasts are discussed, and modern methods to facilitate discovery of yeasts with brewing potential are highlighted.     

利用细胞质导入法选育优良葡萄酒嗜杀酵母

将核融合缺陷菌株嗜杀酵母5045(Kar1-1)的嗜杀质粒导入葡萄酒酵母1450(Screujsjae)中,已获成功。利用这种方法,我们选育出了具有1450细胞核和5045细胞质基因的嗜杀酵母R-58。通过萄萄酒酿造实践表明,该融合嗜杀株不仅具有抗野生酵母的能力,而且能酿制出优质葡萄酒。     

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.     

BREWING YEAST IDENTIFICATION AND CHROMOSOME ANALYSIS USING HIGH RESOLUTION CHEF GEL ELECTROPHORESIS

Contour-clamped homogeneous electric field (CHEF) gel electrophoresis has been used to study the karyotypes of a range of Saccharomyces cerevisiae yeast strains. The time required from sampling yeast cultures to CHEF analysis was achieved within six hours, making this procedure very useful in reference and quality control work in the brewing industry. Regions of the chromosome profiles were closely studied by adjusting electrophoresis conditions to increase resolution between bands. Both ale and lager strains of brewing yeasts were studied alongside haploid laboratory strains. By comparing different regions of the profiles even very closely related strains of lager yeast could be distinguished. Brewing strains consistently had significantly more chromosome bands than haploid laboratory strains. The electrophoretic karyotypes of brewing yeasts were represented as groups of bands on CHEF gels which apparently comigrated with their haploid chromosomal counterparts.     

Genetically-modified brewing yeasts for the 21st century. Progress to date

Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.     

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。     

ESTER CONTROL IN HIGH GRAVITY BREWING

Pilot brewing trials have shown that beers of good flavour can be produced using a high gravity brewing system in which worts of 1080° gravity are fermented and the resulting beers diluted to give an O.G. of 1040°. The flavour can differ from that of the control beers in that the diluted beers retain more ester and strong ale character. Trials have been carried out to study possible methods for control of ester production to permit product matching. A promising approach is controlled contact with oxygen during fermentation.     

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.     

GROWTH OF SACCHAROMYCES CEREVISIAE AND SACCHAROMYCES UVARUM IN A TEMPERATURE GRADIENT INCUBATOR

A temperature gradient incubator has been used to determine the effect of temperature on the growth of strains of Saccharomyces cerevisiae and Saccharomyces uvarum (including lager brewing yeasts formerly classified as Saccharomyces carlsbergensis). The maximum temperatures for growth (T_max) for all strains of S. cerevisiae examined were in the range 37.5°C-39.8°C and the optimum temperatures for the most rapid initial growth (T_opt) were in the range 30.0°C-35.0°C. Strains of S. uvarum, however, formed two distinct groups: Group A (including all brewing strains of S. uvarum tested) had T_max values 31.6°C-34.0°C and T_opt values 26.8°C-30.4°C; Group B had T_max values 38.2°C-40.0°C and T_opt values 30.0°C-34.6°C. It is proposed, therefore, that the species name S. carlsbergensis should be re-introduced and applied to those strains of S. uvarum (Group A) which have the lower T_max values. Minimum temperatures for growth (T_min) of the yeasts were not investigated as initial studies had shown that they could not be measured satisfactorily. Measurements of the generation times for one brewing strain of S. cerevisiae and one brewing strain of S. uvarum (Group A) over the temperature range 6.0°C-22.0°C have shown that there are significant differences between the yeasts at the lower end of the temperature range and that the relationship between generation time (GT) and temperature (T) for both yeasts closely follows the mathematical expression:      

DISCRIMINANT ANALYSIS OF SENSORY AND INSTRUMENTAL DATA ON BEER

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

Altered Patterns of Maltose and Glucose Fermentation by Brewing and Wine Yeasts Influenced by the Complexity of Nitrogen Source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.     

Inhibitory Effect of Fusarium Mycotoxins on Growth of Brewing Yeasts. 1 Zearalenone and Fumonisin B1*

The Fusarium mycotoxins zearalenone (ZEA) and fumonisin B₁ (FB₁) added to the growth medium in low and high concentrations, were investigated as a possible cause of inhibition of growth of Saccharomyces cerevisiae lager and ale strains. Toxic effects were assessed by measurement of dry weight or growth relative to controls, cell number, viability and conductance changes of the growth medium using indirect and direct methods. The Fusarium mycotoxins studied affected growth on brewing yeasts, but the inhibitory effect was dependent on concentration. Low concentrations (0.1–2 μg/ml) had no significant effect on growth compared to controls. Although high concentrations of both mycotoxins strongly affected growth, the inhibitory effect depended on toxin concentration and type, yeast strain, length of incubation and method used to assess growth. The lowest concentrations of mycotoxin causing significant inhibition on growth of these brewing yeasts were 50 μg/ml ZEA for both yeast strains, and 10 μg/ml FB₁ for the lager strain and 50 μg/ml for the ale strain.     

THE SPORULATION AND MATING OF BREWING YEASTS

A study has been made of the sporulating behaviour of twenty selected brewing strains of yeast, and the mating activity of the products of sporulation. ‘Lager’ yeasts (strains of Saccharomyces carlsbergensis) in general sporulated to a lesser degree and more slowly than ‘ale’ yeasts (strains of Saccharomyces cerevisiae) and produced 1-or 2- spored asci compared with 2-or 3- spored asci for the latter yeasts. Most of the parent strains of S. cerevisiae were shown to be heterozygous for mating type, and they were all probably either triploid or aneuploid. Two of the strains of S. carlsbergensis were apparently homozygous for mating type and also triploid or aneuploid. The compatibility system favours outbreeding of yeasts, ‘ale’ yeasts being more compatible with ‘lager’ yeasts than with other ‘ale’ 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.     

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.