A RAPID SPECTROPHOTOMETRIC METHOD TO DETERMINE ESTERASE ACTIVITY OF YEAST CELLS IN AN AQUEOUS MEDIUM

A new rapid method to determine the total esterase enzymatic activity of yeast cells is proposed. In a sodium phosphate buffer a β-naphthol synthetic ester is hydrolized by cells, and the released β-naphthol is coupled with a diazonium salt (Fast Garnet GBC) in the presence of sodium dodecyl sulfate. The whole procedure is carried out in an aqueous buffer medium, and the resulting azo dye is directly evaluated by absorbance measurement at 524 nm. The analytical results from different assays were adjusted to a fixed cell concentration with a statistical procedure. The method shows good repeatability, reproducibility and detectability, and it requires simple equipment and instruments. It is therefore suitable both for routine analysis, as industrial yeast strain screening, and for yeast physiological studies, in order to improve the aromatic quality of fermented drinks.     

德国啤酒酵母和国内啤酒酵母发酵性能的研究

酵母菌种是啤酒酿造的关键,不同的菌种可用来酿造不同类型的啤酒。本试验对德国酵母和国内酵母的发酵性能进行了对比研究,总结出了二者之间的差异。     

THE POLYMORPHISM OF ESTERASES IN YEAST (SACCHAROMYCES CEREVISIAE)

In laboratory strains of yeast (Saccharomyces cerevisiae) two esterase loci, EST1 and EST2, are known. Two more, EST3 and EST4, were found in samples of wine yeast from 40 localities in Europe.     

STUDIES ON THE BINDING BETWEEN YEAST AND A MALT POLYSACCHARIDE THAT INDUCES HEAVY YEAST FLOCCULATION

A factor which causes heavy yeast flocculation of Saccharomyces cerevisiae 2036 was found to be associated with the malt husk by Axcell et al.¹ In the present work it was established that the factor is a polysaccharide and immunological techniques were used to show that the factor binds to the yeast cell surface during fermentation. It was shown that the factor is present at significantly higher concentrations in wort causing premature yeast flocculation than in normal wort. A particular malt husk extract, obtained using a mild aqueous extraction procedure, induced premature flocculation when added to fermentations in normal wort. In sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the malt husk extract, 4 protein bands (42,600; 17,500, 15,100 and 13,100 daltons) and a high MW (molecular weight) polysaccharide were identified. Antibodies were raised against electroeluted proteins as well as against the homogenized polyacrylamide gel containing the polysaccharide band. Enzyme-linked immunosorbent assays (ELISA) showed that the protein components were present in similar concentrations in premature flocculent and normal wort. In contrast, the high MW polysaccharide occurred at a significantly higher concentration in wort inducing premature flocculation than in normal wort. Immunogold electron microscopy showed that the high MW polysaccharide bound extensively to the surface of flocculent cells grown in premature flocculent wort. There was markedly less labelling on yeast cells grown in normal wort. Negligible labelling occurred when the yeast cells were incubated with antibodies against the different protein components of malt husk extract.     

RELATIONSHIP BETWEEN YEAST CELL PROLIFERATION AND INTRACELLULAR ESTERASE ACTIVITY DURING BREWING FERMENTATIONS

Flow cytometry was used for the determination of the intracellular esterase activity of unstressed and stressed Saccharomyces cerevisiae cells using fluorescein diacetate as substrate during brewing fermentations in EBC tubes. The determination of intracellular esterase activity by flow cytometry was compared with in vitro assays for determination of yeast esterase activity. The method was regarded valid with a high degree of reproducibility. Intracellular esterase activity during brewing fermentations was dependent on the yeast strain applied but independent of the wort compositions applied within this study. Further, the intracellular esterase activity during fermentation was correlated with cell proliferation determined by DNA staining and flow cytometry and by calculating the percentage of G1-phase cells. Yeast esterase activity in both unstressed and ethanol stressed cells followed a similar pattern during brewing fermentations. Furthermore, this pattern could be correlated with the percentage of G1-phase cells during fermentation indicating that the esterase activity was in some way related to cell cycle progression .     

YEAST STRAIN AND THE FORMATION OF FLAVOUR COMPONENTS IN CIDER

Two apple juices were fermented at 8°C and 18°C using thirteen strains of Saccharomyces uvarum. Glucose, fructose, malic, lactic, isobutanol, 2–3 butanediol, isoamyl alcohol, ethyl acetate and titratable acidity were determined in the two resulting experimental ciders. This data was correlated by means of multiple regression equations derived from earlier work with the following organoleptic characteristics: sweetness, sourness, fruity, scented, sharp and irritating flavours and a “global hedonic score” for overall acceptability. A significant effect of yeast strain could be detected on all component concentrations except ethanol, ethyl acetate, amyl alcohols and titratable acidity. The amounts of malic acid and titratable acidity found in cider depends mainly on the source of the juice. The anticipated effect of the source of the juice on sourness and on sharpness and fruity flavours is therefore significant. Yeast strain is also expected to have a significant effect on fruity and sharp flavours as well as on overall acceptability.     

STUDIES ON YEAST DIFFERENTIATION USING ORGANIC ACID METABOLITES PART 2. STUDIES ON THE ORGANIC ACID METABOLITES PRODUCED BY YEASTS GROWN ON GLUCOSE

A survey using four strains of yeast was made to establish whether the organic acid metabolites produced by growth on synthetic medium using glucose substrate could provide a means of differentiation. While concentrations of the various acids produced were different, they were insufficiently unique to allow predictability of the strain from which they arose. High performance liquid chromatography on the resin Aminex HPX-87H provided an ideal monitoring base but a number of components could not be identified from retention times of commonly occurring organic acid metabolites. Chromatograms and charts showing changes occurring throughout the fermentation are included.     

酵母菌综合利用的研究进展

根据酵母菌的结构和组成，主要论述了酵母菌在食品、医疗保健、饲料、酿酒、生物工程等工业中的广泛应用。     

YEAST STRAIN AND KINETIC ASPECTS OF THE FORMATION OF FLAVOUR COMPONENTS IN CIDER

An apple juice was fermented at 8°C using twelve strains of Saccharomyces uvarum. Glucose, fructose, malic and L-lactic acids, isobutanol, 2,3 butanediol, isoamyl alcohol, ethyl acetate and titratable acidity were determined and monitored in the course of fermentation. It was observed that yeast strains differed from one another mainly in fermentation rates. When components were determined in ciders of the same remaining fructose concentration, rather than after the same fermentation time, the only significant effect of the yeast strain was on the amounts of glucose and ethanol in sweeter cider (fructose 34 g/litre), or on the amounts of glucose, acetic acid, isobutanol and amyl alcohols in dryer ciders (fructose 17 g/litre). A sensory analysis showed that standard deviations of concentrations of remaining or formed components in ciders fermented by different yeast strains, was sufficient to be detected by the taste panel. Added glucose in this range of concentration (1.5 g/litre) in cider reduced the perception of sourness while the addition of acetic acid (0.3 g/litre) reduced the perception of the scented flavour. Added Isobutnol (6 mg/litre) reduced the perception of sweetness. No significant effect of added isoamyl alcohol (30 mg/litre) could be detected. Chemical determinations were correlated by means of regression equations derived from earlier work with some organoleptic characteristics. No anticipated effect of the yeast strain could be detected on any studied flavour characteristic. It is concluded that in ciders of the same attenuation, the effect if it does exist, of the S.uvarum strain isolated from ciders, must be low.     

MALT FLAVOUR — TRANSFORMATION OF CARBONYL COMPOUNDS BY YEAST DURING FERMENTATION

Laboratory and pilot scale fermentations have been carried out using worts spiked with a range of carbonyl compounds, many of which are derived from malt. Aldehydes and vinyl ketones were chemically reduced during fermentation and could not be detected in the resulting beers; on the other hand certain saturated and non-conjugated unsaturated ketones were only partially reduced. The corresponding saturated alcohols and acetate esters were usually detected as transformation products.     

YEAST ESTERASES AND AROMA ESTERS IN ALCOHOLIC BEVERAGES

It has been shown that the proportion of fatty acid ethyl esters retained by the yeast cell increases with increasing acyl chain length as the ester becomes more lipid soluble. The distribution of esters depends on the yeast strain and on the fermentation temperature; larger amounts of esters were found to transfer from the cells into the medium at higher temperature. It was shown that esterase activity is located both inside and outside the yeast cell plasma membrane. Intact yeast was capable of hydrolysing the ethyl esters of caproic, caprylic and capric acid. Acetate esters, were hydrolysed only very slowly or not at all. The hydrolytic activity of baker's yeast was studied with ethyl caprylate as substrate. The hydrolysis was very fast at the beginning. The equilibrium attained depended not only on the concentration of ester and alcohol but also on the pH, a higher amount of ester remaining in solutions of lower pH. It was also shown that the esterases possess appreciable ester synthesizing ability and an equilibrium was attained by incubating yeast with caprylic acid and ethanol. The experiments described show that the ester level in an alcoholic beverage, such as beer, is not dependent solely on the ester concentration formed during fermentation: in the presence of yeast the level can be shifted in either direction by changing temperature, pH or alcohol concentration — or the amount and type of yeast.     

CAMBRIDGE PRIZE LECTURE: STUDIES ON YEAST PHYSIOLOGY-IMPACT ON FERMENTATION PERFORMANCE AND PRODUCT QUALITY*

The physiology of brewing yeast is of pivotal importance in securing and maintaining beer quality. Fundamental studies on aspects of yeast physiology are critically evaluated, areas considered include alternative approaches to yeast propagation and methods for strain differentiation. The proposed role of glycogen in fuelling lipid synthesis and other early events in fermentation is discussed, as are methods for measurement of this reserve polysaccharide. As an extension of this work, a new and novel approach to the improved control of fermentation is described. The interaction between yeast physiology and beer flavour is considered. Evidence is presented that the synthesis of higher alcohols and volatile esters contribute to the regulation of intermediary metabolism in yeast during fermentation.     

STUDIES ON YEAST DIFFERENTIATION USING ORGANIC ACID METABOLITES PART 1. DEVELOPMENT OF METHODOLOGY USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

The separation of organic acids on the column Aminex HPX-87H from Bio-Rad is described. The relationship between column temperature and retention time was studied for both sulphuric and phosphoric acid eluants and an analytical scheme using 0.2% (v/v) phosphoric acid was developed. While the procedure is designed for the commonly occurring non-volatile acid metabolites it was demonstrated that conditions could be established for the analysis of fatty acids to C₁₀. Difficulties in the qualitative and quantitative analysis of organic acids in beer are discussed.     

YEAST BLOTTERS: SURVIVAL OF YEAST AND BACTERIA

The survival of yeast on yeast blotters with and without contaminating bacteria was monitored to determine whether or not microscopic analysis of yeast after shipment on blotters might be influenced by growth of such bacteria. To examine blotter yeast, they were resuspended and roughly balanced to known opacity by colorimeter—then precisely by hemacytometer. Suspensions were then analysed for survival by plating. It was determined that bacteria and yeast both die quickly on blotters but demonstrate varying levels of survival. It was verified that viable counts from blotters are not practical for enumeration purposes although qualitative knowledge can be gained from viable cell assessment.     

DIFFERENTIATION OF BREWING YEAST

Current methods for the differentiation of brewing yeasts are described and their performance critically discussed. Although these methods are widely used they are non-specific and require confirmation by other tests. Emergent approaches such as pyrolysis gas chromatography or the use of gene cloning techniques are considered and their application reviewed. It is concluded that development of such methods could offer a unique ‘fingerprint’ for each brewing yeast based on a single test.     

EFFECT OF STORAGE ON THE NUCLEIC ACID COMPOSITION OF BAKER'S YEAST*

General changes are described in the nucleic acid content of yeast stored for 20 days under extreme conditions (35 °C). The total amount of nucleic acid increased in the first phase of 6–8 days storage. In the second phase, during which the yeast started to autolyse, the nucleic acid content diminished, first slowly and then more quickly. The quantity of acid-soluble nucleotides increased with storage. The amount of dead yeast cells is low during the first period, but starts to increase quickly during the second period. Changes in the rRNA, 5S RNA, tRNA, mRNA and DNA, fractionated on MAK columns, were investigated over a 20-day storage period. The contents of the RNA fractions increased initially during storage. The amount of rRNA attained its maximum value after 5 days, followed by DNA (7 days), then tRNA (10–12 days) and lastly 5S RNA; thereafter they all decreased. The mRNA fraction diminished rapidly: after only two days' storage the incorporation of labelled uracil into mRNA had declined by about 90%, showing that the ability to synthesize mRNA was soon lost. The short half-life of mRNA ensures its absence in yeast stored for longer periods.     

COMPARATIVE STUDIES OF FIVE POROUS SUPPORTS FOR YEAST IMMOBILISATION BY ADSORPTION/ATTACHMENT

Five different porous carriers for brewing yeast immobilisation were compared and evaluated for suitability in a fluidised bed reactor system. Carrier morphology as revealed by scanning electron microscopy, yeast cell load per mass of carrier, minimum fluidisation velocity, and practical aspects of carrier usage were investigated. Electron microscopy revealed both surface attachment and absorption of yeast within carrier pores, depending on the carrier. Carrier pore structure was found to be important: carriers with deep subsurface cavities and narrow pore entrances gave highest levels of yeast immobilisation. The carrier of choice was the smallest diameter porous glass carrier, SIKUG41. SIKUG41 exhibited no practical physical limitations, gave second highest yeast cell loading, and the second lowest minimum fluidisation velocity.     

THE ESTERASES OF BAKER'S YEAST. I. ACTIVITY AND LOCALIZATION IN THE YEAST CELL

The activity of esterase in baker's yeast cells and in cell fractions was estimated using 2-oxoglutaric acid diethyl ester, p-nitrophenyl acetate and α- and β-naphthyl acetate as substrates. The esterase hydrolysing 2-oxoglutaric acid ester was shown to be located inside the cell, both directly by an enzymic method and by indirect evidence. The activities of the esterases hydrolysing aryl esters were found to vary greatly with the different substrates and estimation methods used. The presence of esterase activity towards phenyl and naphthyl esters in both cell wall digests and sphaeroplast lysates confirms the localization of at least one esterase in both sides of the plasma membrane barrier. Depending on the method of evaluation, values between 80 and 40% of the total were obtained for the proportion of esterase activity located outside the plasma membrane, the most reliable values being about 50–65%.     

FLOCCULATION—SOME OBSERVATIONS ON THE SURFACE CHARGES OF YEAST CELLS

Surface charges on flocculent and non-flocculent yeast cells have been measured by micro-electrophoresis. Yeasts were grown both in calcium deficient and in complete medium and particular attention was paid to changes as cells passed from a logarithimic to a stationary phase of growth. Many of the ionogenic groups contributing to surface charge are situated well within the cell wall; charges on cells from the calcium-deficient medium were higher than on cells from the complete medium. A pH-dependent rearrangement of an underlying surface protein layer is postulated for the flocculent yeast Saccharomyces cerevisiae Strain NCYC 1109. No evidence of a similar rearrangement was found with other flocculent strains examined. The results are discussed in relation to ‘calcium bridge’ formation.     

ON THE MEASUREMENT OF THE FLOCCULATION CHARACTERISTICS OF BREWERS' YEAST

The capacity of certain yeast strains to flocculate is important to the brewing industry. So is the determination of the flocculation characteristics of a yeast strain. In this study we subdivided the flocculation characteristics into three phenomena. A proposal for the most suitable method to quantify each phenomenon is given. For this, four parameters (bond strength, floc size, settling rate and number of single cells) that serve as a measure to these phenomena have been studied. Next to this, attention is payed to the influence of environmental conditions (temperature, calcium concentration, pH and the hydrodynamic conditions during the test) on the result of the test. During this part of the study the flocculence of the yeast cells was constant, so the effect of the yeast on the results of the test is excluded. It turned out that the temperature of the medium and the hydrodynamic conditions during the test most strongly influence floc formation. Next to this, medium viscosity is important if the flocculation characteristics are quantified via settling experiments.