CLASSIFICATION OF BREWING YEASTS BY PRINCIPAL CO-ORDINATES ANALYSIS OF THEIR BREWING PROPERTIES

Numerical taxonomic techniques have been used to re-evaluate the findings of Walkey and Kirsop⁸ on practical brewing yeast classification. Principal co-ordinates analysis produced five groups each of which could be divided into two subgroups. The use of this technique for selecting yeast strains for the various types of fermentation systems is discussed.     

THE CLASSIFICATION AND NOMENCLATURE OF BREWING BACTERIA: A REVIEW

Improved taxonomic techniques have rationalized the classification of several bacterial taxa which can be isolated from the brewing environment and spoiled beer. This review outlines the changes that have been made and provides a nomenclature of brewery bacteria derived from the ‘Approved Lists of Bacterial Names’.²⁹     

CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

KAR1-MEDIATED TRANSFORMATION OF BREWING YEAST

Transfer of plasmid between nuclei has been observed in heterokaryons obtained from kar1 × KAR1 crosses³. On this basis, brewing yeast strain CC45 was forced to mate with K5-5A, a kar1 laboratory strain bearing plasmid YEpCR21. This multicopy plasmid carries the gene cyh2 and determines a dominant resistance to cycloheximide . Heteroplasmons having a CC45 nucleus and hosting YEpCR21 were obtained. Plasmid stability and brewing performance have been studied. The utility of kar1-mediated plasmid transference as an alternative method to transform brewing yeast is discussed. Advantages of YEpCR21 as a vector cloning for brewing yeast are considered as well .     

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.     

Beer metabolomics: molecular details of the brewing process and the differential effects of late and dry hopping on yeast purine metabolism

The flavour of beer is complex, based upon changes at the molecular level in the key raw materials, notably grain, hops and yeast, as well as during the process stages that comprise malting and brewing. As analytical techniques evolve in their sophistication and sensitivity, there are opportunities to delve ever more deeply into the fate of small molecules in brewing. To this end, ¹H nuclear magnetic resonance (NMR) metabolomics was used to follow the progression of 76 metabolites in four different late or dry hopped beers (brewed in triplicate) at five time points throughout the brewing process. The majority of the metabolites identified, including sugars, amino acids and nucleotides, significantly decreased in concentration from the start of the boil to post‐secondary fermentation, whereas energy‐related and fatty acid associated metabolites significantly increased in concentration as wort nutrients were consumed by the yeast. Adenine was significantly higher in the dry hopped brews than in the late hopped brews after both primary (p = 2.1 × 10^?6) and secondary (p = 2.7 × 10^?9) fermentation, while 2′‐deoxyadenosine (after primary, p = 1.1 × 10^?2, after secondary, p = 3.2 × 10^?5) and adenosine (after primary, p = 2.6 × 10^?8; after secondary, p = 3.1 × 10^?7)were significantly lower in the dry hopped beers at these time points. These results give molecular insight into the brewing process and the differential effects of hopping methods on yeast purine metabolism. Copyright © 2016 The Institute of Brewing & Distilling     

A RAPID,SIMPLE AND RELIABLE METHOD OF DIFFERENTIATING BREWING YEAST STRAINS BASED ON DNA RESTRICTION PATTERNS

DNA was isolated from polyploid brewing ale and lager yeast strains using a simple and rapid procedure which was a modification of a previously described method of Seehaus et al.¹⁴ The isolated DNA was cut with a number of restriction enzymes and subjected to agarose gel electrophoresis. Significant differences in banding patterns were observed between a Saccharomyces cerevisiae ale strain DNA and Saccharomyces uvarum (carlsbergensis) lager strain DNA, particularly with the enzyme Hpal. Differences were also observed between the banding patterns of digests from two ale strains, and from two lager strains. Use of this technique with appropriate restriction enzymes should prove useful for the rapid differentiation of brewing yeast strains.     

Comparison of beer quality attributes between beers brewed with 100% barley malt and 100% barley raw material

BACKGROUND: Brewing with 100% barley using the Ondea^® Pro exogenous brewing enzyme product was compared to brewing with 100% barley. The use of barley, rather than malt, in the brewing process and the consequences for selected beer quality attributes (foam formation, colloidal stability and filterability, sensory differences, protein content and composition) was considered. RESULTS: The quality attributes of barley, malt, kettle‐full‐wort, cold wort, unfiltered beer and filtered beer were assessed. A particular focus was given to monitoring changes in the barley protein composition during the brewing process and how the exogenous OndeaPro^® enzymes influenced wort protein composition. All analyses were based on standard brewing methods described in ASBC, EBC or MEBAK. To monitor the protein changes two‐dimensional polyacrylamide gel electrophoresis was used. CONCLUSION: It was shown that by brewing beer with 100% barley and an appropriate addition of exogenous Ondea^® Pro enzymes it was possible to efficiently brew beer of a satisfactory quality. The production of beers brewed with 100% barley resulted in good process efficiency (lautering and filtration) and to a final product whose sensory quality was described as light, with little body and mouthfeel, very good foam stability and similar organoleptic qualities compared to conventional malt beer. In spite of the sensory evaluation differences could still be seen in protein content and composition. Copyright © 2011 Society of Chemical Industry     

CONTENTS OF CYTOCHROMES IN YEAST

The contents of cytochromes in yeast were determined quantitatively from the absorption spectra, using a solid cell paste of intact yeast. During the industrial production of baker's yeast, the contents of the cytochromes, particularly of cytochrome aa₃ at successive stages, increased gradually with increasing aeration. In semi-aerobically grown baker's yeast, the contents of cytochromes aa₃, b and c were 0·9, 2·9 and 2·9 × 10^?5 moles/litre of fresh yeast (total amount 6·7 × 10^?5 moles/litre), while in vigorously aerated commercial baker's yeast the respective values were 2·3, 4·8 and 5·2 × 10^?5 moles/litre (total amount 12·3 × 10^?5 moles/litre). In brewer's yeasts separated after the brewing process, the contents of cytochromes were markedly lower than in baker's yeast grown with limited aeration, whereas in top-fermenting yeast the total cytochrome content, aa₃ + b + c, was in some samples markedly higher, 7·1 × 10^?5 moles/litre, than in bottom-fermenting brewer's yeast, 2·4 × 10^?5 moles/litre. When brewer's bottom yeast was grown on a laboratory scale under increasing aeration, a maximum appeared in the cytochrome contents when aeration was moderate, and increased aeration inhibited the formation of cytochromes. The cytochrome contents in brewer's bottom yeast may exceed the amounts found in commercial baker's yeast. In addition to aeration, the type of metabolism influences the amounts of cytochromes in yeast.     

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.     

A Survey of Industrial Strains of Saccharomyces cerevisiae Reveals Numerous Altered Patterns of Maltose and Sucrose Utilisation

A model fermentation system was used to define the abilities of 25 Saccharomyces cerevisiae strains, representing the brewing, baking, winemaking and distilling industries, to utilise maltose and sucrose in the presence of glucose and fructose. Three categories of sucrose and maltose utilisers were observed; repressible, constitutive and non‐utilisers. In terms of fermentation kinetics, neither high rates of sucrose hydrolysis nor the early onset of maltose utilisation were correlated with reduced fermentation duration in the experimental system used. Instead better positive correlations were found between this parameter and biomass formation (R² = 0.62) and rates of maltose or monosaccharide removal (R² = 0.87 and 0.82, respectively). Additionally, invertase activity of brewing strains was seen to occur in two forms: cell‐associated and non‐cell‐associated. This survey exposed a number of novel phenotypes that could be harnessed as a means of producing strains with rapid and efficient utilisation of fermentable carbohydrates.     

PRODUCTION OF BEER USING IMMOBILIZED YEAST ENCODING α-ACETOLACTATE DECARBOXYLASE

Genetically modified brewer's yeast encoding α-acetolactate decarboxylase (α-ALDC) was tested in immobilized yeast bioreactors for main fermentation of beer. The α-ALDC enzyme produced by the transformant catalyzes the direct conversion of α-acetolactate to acetoin without formation of diacetyl. The long lagering period required for beer maturation in conventional brewing can thus be shortened or even omitted. Three different packed bed bioreactors were employed, with volumes of 1.6 dm³, 5 dm³ and 25 dm³. The 5 dm³ column had a slightly conical geometry in contrast to the others which had cylindrical shapes. Sintered glass beads were chosen as the carrier material on the basis of experiments with the parent strain. The brewing performance of the transformant compared well with that of the parent strain in the immobilized system. Fermentation, utilization of amino acids (including isoleucine, valine and leucine) and flavour formation were practically identical with both strains, the only difference being a marked decrease in the formation of diacetyl by the transformant. Small differences were, however, observed in the long-term biochemical stability. By using yeast encoding α-ALDC in the immobilized yeast system the total (primary and secondary) fermentation time could be reduced to approximately 2–6 days, compared with 3–6 weeks in a conventional batch process.     

FLOCCULATION MECHANISMS OF TOP AND BOTTOM FERMENTING BREWING YEAST

The flocculation behaviour of a large set of top and bottom fermenting brewing yeasts was investigated. Bottom and top fermenting strains flocculated according to different mechanisms. Bottom strains flocculated in the stationary growth phase in the presence of sufficiently high Ca²⁺ and sufficiently low sugar concentrations; these strains possessed a lectin-mediated flocculation mechanism. Top strains flocculated in the stationary growth phase without addition of Ca²⁺, only in the presence of sufficiently high concentrations of ethanol. Some of the top strains were inhibited with mannose, but not with sucrose or galactose, while others were not inhibited by any of these sugars. The different sensitivity of flocculation of top and bottom strains with respect to ethanol may be related to the hydrophobicity of the cell surface. Flocculation models for bottom and top fermenting yeasts were proposed. It is suggested that, besides the sugar inhibition pattern, the sensitivity of flocculation with respect to ethanol should be included as an additional parameter for classification of brewing yeasts .     

THE INFLUENCE OF PANTOTHENATE CONCENTRATION AND INOCULUM SIZE ON THE FERMENTATION OF A DEFINED MEDIUM BY SACCHAROMYCES CEREVISIAE

Laboratory fermentations were carried out using defined media containing from 0.02 to 0.40 mg pantothenate 1^?1 and a brewing strain of Saccharomyces cerevisiae at inocula of 1 × 10⁶ to 1 × 10⁷ cells ml^?1, Cell size, increase in cell number, increase in yeast weight, production of ethanol. n-propanol, isobutanol, isoamyl alcohols, acetaldehyde and ethyl acetate were measured. All the properties were influenced to some extent by pantothenate concentration and/or inoculum size but no general correlations were found. At high inoculum levels variation in the pantothenate concentration above 0.1 mg 1^?1 did not have a major effect on the total quantity of volatile compounds produced but did alter the relative proportions of the individual compounds.     

THE INFLUENCE OF A COLIFORM BACTERIUM ON FERMENTATION BY YEAST

Hopped wort (20°C) was inoculated with brewing yeast alone (10⁷ cells/ml), or with Citrobacter freundii (10⁵ cells/ml) or with both organisms at the concentrations stated. The mixture grew somewhat more quickly than the pure yeast culture, the oxidation-reduction potential and S.G. fell more quickly but the pH less rapidly. The mixed culture produced more ethanol and various organic acids but less volatile carbon. Dimethyl sulphide was produced in the mixed culture but not in the pure yeast culture. It is concluded that C. freundii can have a profound influence on yeast fermentation, consistent with its pure culture characteristics.     

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre^?1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre^?1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.     

CLONING OF A YEAST GENE WHICH CAUSES PHENOLIC OFF-FLAVOURS IN BEER

A gene (POF1) has been cloned, which confers upon yeast (Saccharomyces cerevisiae) the ability to decarboxylate phenolic acids such as ferulic and trans-cinnamic acid. This property was previously shown to be a cause of phenolic off-flavour production in wort fermentations. The identity of the cloned gene was confirmed as POF1 by gene disruption techniques. Southern blotting of total genomic DNA revealed that sequences homologous to POF1 are conserved in Pof^? brewing strains of Sacch. cerevisiae. The transformation of a Pof^? lager strain with the cloned POF1 gene led to the production of an aroma characteristic of a phenolic off-flavour, when the transformed strain was used in wort fermentations. This latter observation suggests that the Pof^? phenotype of brewers' yeast is specifically due to the absence of a functional POF1 gene.     

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.     

CHARACTERISATION OF AMYLOLYTIC BREWING YEAST

Amylolytic brewing yeast can be used for the production of low carbohydrate beer and for maximizing fermentation efficiency. In this paper we describe the characterisation of amylolytic brewing yeast in which the STA2 (DEXI) gene, which codes for an extracellular glucoamylase, was cloned under two different promoters; PGK (phosphoglycerate kinase) and GPD1 (sn-glycerol-3-phosphate dehydrogenase) present on episomal plasmids. Both amylolytic strains were shown to ferment and degrade wort as efficiently as the control strain supplemented with an exogenous commercial glucoamylase, despite reduced intracellular glycogen levels (30% of wild-type). However, the nature of the promoter on the expression plasmid was shown to influence both the growth rate of the amylolytic strains and the stability of the plasmids during non-selective growth. One of the strains containing plasmid pDVX4 (GPD promoter) was found to show high levels of stability when tested in ten successive pilot scale (8Hlitre) fermentations.