GENETIC CONTROL OF THIAMINE EXCRETION AND OF ITS SUPPRESSION IN SACCHAROMYCES CEREVISIAE

Three non-linked loci were found to be involved in thiamine excretion in Saccharomyces cerevisiae, i.e. thi2 which is complementary with thi3 and thi4, the latter two being substitutable mutually but acting also together. Mutation thi3 was present already in strain Nα, which produced relatively high frequencies of excreters after UV treatment. Non-excreting ‘revertants’ of unstable excreters derived from another haploid strain contained recessive mutation thi2, so that mutation thi3 can be substituted also by the metabolic or physiological disorders ruling after UV treatment. Mutations thi2 were found also in all 43 analysed stable excreting mutants obtained by UV treatment of strain Nα which indicates that they are either inevitable for the excretion or are specifically selected by the selection method used. At least one suppressor locus of thiamine excretion was present in that strain of S. cerevisiae, which did not produce thiamine excreters after UV treatment in spite of its ability to synthetise thiamine.     

EFFECT OF ALKALINE-EARTH METAL SALTS ON FLOCCULENCE IN SACCHAROMYCES CEREVISIAE

Flocculation in three strains of Saccharoymces cerevisiae is inhibited by two classes of ionic substances: (1) the alkaline-earth metal ions Sr and Ba, and (2) Ca complexing substances. Sr appears to be competitive with Ca in the flocculence mechanism because the extent of inhibition depends mainly on the ratio of Sr and Ca concentrations. The observations imply that the Ca requirement for flocculation is by a specific, unique process, and not by the commonly presumed charge neutralization or Ca salt-bridging mechanism.     

SUGAR UPTAKE AND SUBSEQUENT ESTER AND HIGHER ALCOHOL PRODUCTION BY SACCHAROMYCES CEREVISIAE

The production of a number of esters and higher alcohols by brewing strains of Saccharomyces cerevisiae, in synthetic media containing only glucose, fructose or maltose as sole carbohydrate source, has been investigated. Results indicated that production of most volatiles was generally lower when maltose was the sugar being fermented, despite maltose-grown cells having higher viabilities and vitalities than glucose or fructose-grown cells. There was no significant difference in the levels of esters and higher alcohols produced during fermentation when glucose or fructose was metabolised, although strain variation was observed. Similar results were obtained when wort was supplemented with either glucose, fructose or maltose. Wort supplemented with maltose produced fewer volatiles, especially higher alcohols, than that which had a hexose sugar added. The activity of estersynthesising enzymes present in glucose or maltose PYN grown cells was also examined. Similar levels of ethyl acetate and isoamyl acetate were obtained when cells grown in either glucose or maltose PYN were disrupted and ester production monitored. The implications of these results for the fermentation of high-gravity worts are discussed.     

EXTRACELLULAR PROTEASE ACTIVITY IN A STRAIN OF SACCHAROMYCES CEREVISIAE

The isolation and analysis of a strain of laboratory yeast with extracellular protease activity is described. The proteolytic activity found in culture supernatants exceeded the parental strain by at least an order of magnitude and apparently was due neither to cell lysis nor to increased cell wall permeability. The extracellular proteases were heterogeneous in composition, consisting of possibly 3 aberrantly secreted intracellular proteinases. This extracellular protease activity was conferred by a single recessive mutation (epr1.1) in a gene displaying classical Mendelian inheritance. EPR1 was tentatively assigned to chromosome XV with loose linkage to the HIS3 gene. Strains carrying the mutant epr1.1) allele also possessed increased levels of secreted invertase activity and it is proposed that EPR1 is closely involved in the intracellular protein translocation pathway of yeast.     

INVOLVEMENT OF WALL COMPONENTS IN ADSORPTION OF SILICONE ANTIFOAM TO SACCHAROMYCES CEREVISIAE

The ability of Saccharomyces cerevisiae NCYC 366 to adsorb polydimethylsiloxane (PDS), dispersed in an aqueous emulsion containing stearate emulsifiers and cellulose-based thickeners, is diminished after treatment with trypsin, KOH and HF; treatment with β-glucanase initially caused an increase in binding which diminished on prolonged incubation. Trypsin and KOH removed most of the mannan, protein and phosphorus from isolated walls, while exposure to β-glucanase removed about half of the glucan, protein and phosphorus. HF treatment removed most of the phosphorus but had little effect on the contents of other wall components. Cells treated with trypsin or β-glucanase had pH-mobility curves similar to those of untreated cells. Those treated with KOH or HF had lower mobilities at all pH values. Treatment of cells with each of the reagents except β-glucanase lowered their capacity to bind fluorescein-labelled antibody. The ability of cells to bind fluorescein-labelled concanavalin A was decreased only after trypsin and KOH treatment. Treatment of PDS-containing cells with trypsin, β-glucanase or KOH caused release of bound silicone, the effect being smallest with β-glucanase treatment. It is concluded that binding of PDS-containing antifoam to yeast mainly involves ionogenic groups in the wall phosphomannan-protein.     

CALCIUM IN FLOCCULENCE OF SACCHAROMYCES CEREVISIAE

A quantitative method was adopted for measuring flocculation intensity of yeast photometrically. In three strongly flocculent strains of Saccharomyces cerevisiae examined with this method, flocculation intensity depended on ionic strength of the medium as well as on Ca concentration, and was maximum at about 0·01 ionic strength. At this optimum ionic strength, when free Ca concentration was varied in stabilized complexing systems, a transition occurred at about 10^?8 m Ca between flocculent and nonflocculent states. At higher Ca concentrations, flocculation intensity was nearly constant. The observed transition is at much lower Ca levels than other effects noted in the literature.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

ADSORPTION OF SILICONE ANTIFOAM BY SACCHAROMYCES CEREVISIAE

Polydimethylsiloxane (PDS) dispersed in an aqueous emulsion containing stearate emulsifiers and cellulose-based thickeners is adsorbed to the walls of Saccharomyces cerevisiae NCYC 366 during growth in a defined medium. Including PDS at concentrations up to 30 μg per ml of medium has no effect on the duration of the lag phase or the rate of exponential growth. Cells suspended in buffer containing PDS take at least 2–4 h to become saturated with PDS. Cells saturated with PDS do not differ from PDS-free organisms in respiratory activity, but have a slightly lower electrophoretic mobility at pH values between 4.0 and 7.0. About half of the PDS on saturated cells appears in the supernatant when they are converted into sphaeroplasts, but proportionately less PDS is released into the supernatant from cells containing less PDS. The equilibrium constant for binding of PDS to isolated walls is about five times as great as for binding to cells. PDS is taken up by isolated walls faster than by intact organisms. Incubation of walls saturated with PDS in buffered EDTA leads to release of PDS from the walls, but incubation in the presence of 8-m urea does not.     

ON THE DIFFERING RATES OF FRUCTOSE AND GLUCOSE UTILISATION IN SACCHAROMYCES CEREVISIAE

Fructose is utilised slower than glucose when the two sugars are fermented separately. This phenomenon occurs in a growth promoting medium as well as in brewers wort when using the brewing yeast Saccharomyces cerevisiae. The use of a fructose adjunct in wort at concentrations of 2% w/v and above, may result in residual concentrations of fructose to remain at the end of fermentation and consequently taint the beer with a sweet off-flavour. Glucose and fructose have no effect on maltose utilisation. Thus they do not exert catabolite repression on the maltose membrane transport system in the particular brewing strain of S. cerevisiae under investigation, when fermented in brewers wort.     

EFFECT OF ENVIRONMENTAL CONDITIONS ON FLOCCULATION AND IMMOBILISATION OF BREWER'S YEAST DURING PRODUCTION OF ALCOHOL-FREE BEER

A method using immobilised yeasts has been developed and successfully applied for production of alcohol-free beer. The influence of environmental conditions present during alcohol-free beer production on the flocculation and immobilisation of the yeast Saccharomyces cerevisiae var. uvarum was investigated in the present study. In wort, the cells developed flocculation at the end of exponential growth, according to the NewFlo phenotype. In defined medium, the flocculation capacity appeared to be temporary and was lost rapidly during the stationary phase. No increase in cell wall hydrophobicity at the onset of flocculation was observed in either medium. Low growth temperatures increased flocculation capacity approximately four-fold, compared to growth at high temperatures. The optimum temperature for flocculation was at 25°C with cells grown at low or high temperature . A novel method using carboxyfluorescein-stained cells was developed to analyse the initial adhesion of cells to carrier. This method also allowed rapid analysis of the effects of immobilisation to DEAE-cellulose carrier during alcohol-free beer production process. It appeared that a high flocculation capacity stimulated adhesion to the DEAE-carrier .     

DETERMINATION OF FERMENTABLE SUGARS AND NITROGENOUS COMPOUNDS IN WORT BY NEAR- AND MID-INFRARED SPECTROSCOPY

Near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were evaluated for the analysis of brewery worts. Good calibrations were obtained for maltose (SEP_NIR 2.6 g/l, SEP_MIR 2.0 g/l), glucose SEP_NIR 0.6 g/l, SEP_MIR 0.4 g/l) and the sum of fermentable sugars (SEP_NIR 2.8 g/l, SEP_MIR 2.6 g/l) with both methods. Satisfactory calibration was obtained for maltotriose (SEP_NIR 1.4 g/l, SEP_MIR 1.5 g/l), nitrogen (SEP_NIR 122 mg/l, SEP_MIR 92 mg/l) and free alpha-amino nitrogen (SEP_NIR 29 mg/l, SEP_MIR 23 mg/l). MIR spectroscopy gave slightly better results, but sample handling was more difficult than in the NIR region. The NIR measurement is simple and rapid and could be used for on-line process control during mashing. No sample preparation other than filtration is needed.     

FLOCCULATION IN SACCHAROMYCES CEREVISIAE AS INFLUENCED BY WORT COMPOSITION AND BY ACTIDIONE

The stage during the fermentation of wort at which cells flocculate is shown to vary with the extent of exponential growth, so that the use of worts with a high content of assimilable nitrogen leads to delayed flocculation. Additionally, the presence of a high concentration of lysine, which inhibits post-exponential growth, retards flocculation still further and may prevent its occurrence. Addition of actidlone to a growing culture inhibits further increase in cell mass and prevents flocculation occurring, although the use of sugar continues; cell wall synthesis is restricted and mannan production is particularly repressed by actidione treatment.     

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.     

STUDY OF THE PHENOMENON OF AGGLOMERATION IN THE YEAST SACCHAROMYCES CEREVISIAE

Agglomeration or “grittiness” is detrimental to bakers' yeast quality. Gritty yeast only partially resuspends when mixed in water, most of it remaining as macroscopic cell aggregates. A macroscopic sedimentation test was developed for measuring agglomeration intensity. Expression of the gritty phenotype was investigated in two strains (N176 and GB1) of Saccharomyces cerevisiae grown on a 14-liter scale by varying fermentation conditions of agitation and aeration. Results show that yeast agglomeration is different from yeast flocculation, and is determined by both strain genetic background and environmental factors. The gritty phenotype was expressed in the strain prone to agglomeration (N176) when dissolved oxygen was limiting in the fermenter. Gritty cells had a lower phosphorus and lipid concentration and a higher protein concentration at the surface of the cell, and a higher amount of whole cell and cell wall proteins and calcium than non-gritty cells. Some proteins were also extracted from gritty cells with sodium hydroxide or mercaptoethanol, that were not present in non-gritty cells. Agglomeration did not result in major differences in the structure or composition of the structural cell wall mannoprotein (CWMP). A model for agglomeration is proposed: proteins (cognors) activated by Ca²⁺ (cofactors) to increase their binding capacity bind the mannans (cognons) of adjoining cells; binding is facilitated by the lower phosphorus and lipid concentration at the surface of gritty cells.     

STUDY OF THE FLOCCULATION OF SACCHAROMYCES DIASTATICUS NCYC 625

Yeast flocculation presents a great interest for the industry of fermentation but its mechanism is still not fully understood. In order to enlighten this mechanism, the flocculation of Saccharomyces diastaticus NCYC 625 was studied. As with other Saccharomyces strains, the effects of different factors affecting flocculation and deflocculation (pH, temperature, medium, EDTA, cations…) suggest a lectin-like binding between adjoining cells. The genetic determinism of flocculation is nuclear and not mitochondrial. Although Saccharomyces diastaticus NCYC 625 could be classified in the FLO1 phenotype according to Stratford and Assinder⁴², allelism tests show that the gene involved in the flocculation control is not allelic with FLO1 or FLO5 and possibly different from FLO8 .     

GROWTH OF NORMAL AND ARGININE-REQUIRING STRAINS OF SACCHAROMYCES CEREVISIAE ON SELECTED NITROGEN COMPOUNDS

Two arginine-requiring mutant yeast strains grew on arginosuccinic acid or argininic acid in presence of ammonium sulphate but failed to do so on ornithine, citrulline or glycocyamine. The metabolic lesion in these mutants appears to lie therefore in the enzyme system involved in completing the guanidino group of arginine. Arginine could not be replaced by various peptides containing it. Histidine depressed growth on arginine of both the deficient yeast and its parent but growth was somewhat stimulated by certain other amino acids. To some extent the need for all amino acids was met by the use of hexamethylenediamine.     

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:      

SELECTION AND BIOCHEMICAL CHARACTERISATION OF SACCHAROMYCES CEREVISIAE AND KLOECKERA APICULATA STRAINS ISOLATED FROM SPANISH CIDER

The behaviour of different strains of Saccharomyces cerevisiae and Kloeckera apiculata in apple juice fermentation was studied. Ethanol production was higher for Saccharomyces strains while residual sugars and ethyl acetate content was higher for Kloeckera strains. Kl. apiculata fermented products showed the lowest amount of higher alcohols and the lowest content in organic acids with the exception of acetic acid, so this yeast produced an increment in volatile acidity. On the basis of ethyl acetate, hydrogen sulfide, and acetic acid production, fermentative ability, potassium metabisulfite resistance and sporulation percentage, one strain from Sacch. cerevisiae could be employed as starter for making cider.     

ONE HUNDRED YEARS OF YEAST RESEARCH AND DEVELOPMENT IN THE BREWING INDUSTRY

The effect that yeast research and development on yeasts has had on the brewing industry is traced from the time of Pasteur to the present time.     

THE EFFECT OF OSMOTIC PRESSURE ON THE INTRACELLULAR ACCUMULATION OF ETHANOL IN SACCHAROMYCES CEREVISIAE DURING FERMENTATION IN WORT

An intracellular accumulation of ethanol was observed in Saccharomyces cerevisiae during the early stages of fermentation (3 h) in wort. However, after 12 h fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure in wort by adjusting the carbohydrate concentration, from 10° to 20° plato, was observed to cause an increase in intracellular ethanol concentration and glycerol production. Although intracellular ethanol concentration increased, no adverse effect was observed on cell growth and fermentation rates. Thus, increasing the carbohydrate concentration in wort to at least 20° plato did not affect cell growth and fermentation rates, but did result in the production of more ethanol.