TWO-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF ENDOPOLYGALACTURONASES PRODUCED BY KLUYVEROMYCES MARXIANUS

The high resolution technique of two-dimensional electrophoresis is used to establish the endopolygalacturonase isoenzyme profile of Kluyveromyces marxianus, strain NCYC 587. We show that the use of this technique in nondenaturing conditions represents a powerful procedure for resolving the isoenzyme population and detecting their endopolygalacturonase activities. The enzyme staining method described is at least as sensitive as silver staining. The purified enzyme was shown to consist of nine isoenzymes. Their pIs ranged from 5.6 to 6.5 and they appeared to have similar molecular weights in the region of 43,000 daltons.     

THE EFFECT OF PITCHING YEAST AERATION ON THE PRODUCTION OF ACETIC ACID DURING FERMENTATIONS WITH BREWERS' YEAST: AN ENZYMATIC APPROACH

Aeration of pitching yeast signicantly increases the metabolism of acetate. This increase is particularly noticed at an enzymatic level, with special reference to the specific activity of the Fe⁺⁺⁺ linked pyruvate de-carboxylase which has been shown to be involved in the production of acetic acid. At the beginning of fermentations carried out with aerobic and anaerobic pitching yeasts, an increase in acetic acid production is observed, this is followed by reabsorption. Stabilisation in the concentration of acetic acid is observed until the end of fermentation. Initial and final concentrations of acetic acid obtained during fermentations were significantly higher in fermentations carried out with aerobic pitching yeasts than in fermentations carried out with anaerobic pitching yeasts.     

THE IDENTITY OF AN INHIBITOR IN WORT OF DIMETHYL SULPHOXIDE REDUCTASE FROM YEAST

Wort contains a substance which inhibits the reduction of dimethyl sulphoxide by yeast. The inhibitor has been purified by dialysis followed by chromatography on Sephadex G-15 and Dowex 50. It has been identified as methionine sulphoxide.     

CONSTRUCTION OF AN ENDOGLUCANOLYTIC BREWING YEAST STRAIN

An endo-β(1, 4)glucanase encoding gene from fungal origin has been expressed in a brewing yeast strain. The yeast transformation was carried out using a previously reported system based in the acquisition of cycloheximide resistance. The plasmid transferred to brewing yeast showed some changes in restriction pattern of the 2 μ portion after transformation while cycloheximide resistance marker and endoglucanase gene were not affected. The drug resistance phenotype showed by the recombinant yeast was highly stable in non-selective conditions. The endoglucanase enzyme was detected in cell-free culture medium and showed high activities in liquid and solid media.     

THE GENETICS OF YEAST FLOCCULATION

A short review of the development of yeast genetics in general, and with respect to flocculation in particular, is presented. At least three genes, two dominant and one recessive, confer flocculence, only one of these genes requiring to be present. The spontaneous gene mutation or mitotic segregation rates from flocculence to non-flocculence are high and are much higher than those rates in the reverse direction. Attempts were made to estimate the ploidy of some commercial strains of Saccharomyces cerevisiae by measurement of cell volume and DNA content.     

AN UPDATE OF ZINC ION AS AN EFFECTOR OF FLOCCULATION IN BREWER'S YEAST STRAINS

Zn⁺⁺ ion was observed to be a strong effector of the flocculation—deflocculation process in an in vitro system for strains of Saccharomyces cerevisiae at ion concentration ranges that are normal in conventional substrates, such as wort. All strains of Saccharomyces uvarum (carlsbergensis) examined in this study did not exhibit any flocculation response to Zn⁺⁺ ion. This test could be employed to distinguish between ale and lager flocculating yeast strains.     

CHEMICAL CHARACTERIZATION AND STEREOSPECIFIC ANALYSIS OF LIPIDS SYNTHESIZED BY CERTAIN YEAST STRAINS

Fatty acid analysis of lipids from yeast strains, Apiotrichum curvatum ATCC 10567, Cryptococcus albidus ATCC 56297, Lipomyces starkeyi ATCC 12659, and Rhodosporidium toruloides ATCC 10788, grown on whey permeate revealed palmitic, stearic, oleic and linoleic acids as the predominant fatty acids in the triacylglycerol fractions. The phospholipid fractions were dominated by oleic and linoleic acids. The stereospecific distribution of fatty acids in the triacylglycerol fractions of lipids from two yeast strains, Apiotrichum curvatum ATCC 10567 and Lipomyces starkeyi ATCC 12659, was determined and revealed the presence of almost entirely unsaturated fatty acids at the sn-2 position (91.9% and 82.8%, respectively). The dominance of unsaturated fatty acids (in the range of 61.5 to 72.3%) at the sn-1 position was also observed. Oleic acid was the predominant fatty acid at positions sn-1 and sn-2 for both yeast strains. Position sn-3 had the greatest concentrations of saturated fatty acids, with palmitic acid as the predominant fatty acid.     

CHARACTERIZATION OF BREWING YEAST STRAINS BY NUMERICAL ANALYSIS OF TOTAL SOLUBLE CELL PROTEIN PATTERNS

Total soluble cell proteins from 33 yeast strains from the brewing industry were extracted and subjected to polyacrylamide gel electrophoresis. Yeast strains were grouped by computerized numerical analysis of protein banding patterns. Three clusters were obtained at r>0.90. Cluster I contained 21 Saccharomyces cerevisiae lager beer strains. Cluster II comprised two strains isolated from beer with a phenolic off flavour and a third strain used for lager beer brewing. Cluster III consisted of two bottom ale yeasts. Protein patterns of yeast strains within each cluster corresponded closely or were identical. However, the intensity of certain bands often varied and the number of peaks recorded was not identical. These minor differences were reproducible and regarded as characteristic for the specific strains. Protein patterns can therefore be used to characterize or fingerprint individual yeast strains.     

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.     

EFFECTS OF AN AUTOLYZED YEAST ON PHYSICAL AND SENSORY PROPERTIES OF FRANKFURTERS

Three experiments were conducted to determine effects of autolyzed yeast on frankfurter firmness, flavor, and yields. Smokehouse yields of laboratory prepared frankfurters (Experiment #1) were not affected (P < 0.05) by addition of autolyzed yeast (1%). Commercially produced frankfurters containing 0%, 1.0%, or 1.5% yeast (Experiment #2) or 0%, 0.75% or 1.0% yeast (Experiment #3) were subjected to sensory and yield evaluations. Frankfurters from Experiment #2, with 1% autolyzed yeast were more firm (P < .10) than control frankfurters. Frankfurters from Experiment #3 with 0.75% and 1.0% autolyzed yeast were more firm (P < .01, P < .10) than controls. Vacuum packaged frankfurters containing yeast (Experiments #2 and #3), held 2, 4, or 6 weeks at 2–5°C, had less purge than their respective controls. Autolyzed yeast appeaers to enhance frankfurter flavor and firmness while reducing purge in vacuum packaged product.     

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%.     

BIOTIN AND THE METABOLISM OF BAKER'S YEAST

Based on experimental results and the results presented in the literature a model for the metabolism of baker's yeast under biotin deficiency is presented. In these conditions the most sensitive point in the metabolism is the carboxylation of pyruvate to oxaloacetate catalyzed by the biotin-containing pyruvate carboxylase. Because the rate of glycolysis is not affected by biotin-deficiency pyruvate accumulates in the cells and is partially excreted into the medium. The high pyruvate pool in the cells means that the metabolism is mainly fermentative even in vigorously aerated cultures. The oxidation of pyruvate to acetyl-CoA proceeds almost unaffected, as can be seen by the production of elevated amounts of ethyl acetate by yeast grown under sub-optimal biotin concentrations. Acetyl-CoA carboxylase, which also has biotin as the prosthetic group, is not as sensitive to a deficiency of this growth factor as is pyruvate carboxylase. In yeast grown without biotin the amounts of fatty acids and lipids are the same or even higher than in cells grown under optimal conditions. The metabolism from oxaloacetate towards the TCA cycle and glutamate is not affected as strongly as is the metabolism to aspartate, which is present in cells in strictly limited amounts. This causes an over-production of metabolic intermediates, e.g. diazotizable arylamine and hypoxanthine as well as citrulline. Their conversion to normal cell constituents, purine nucleotides and arginine, is dependent on the aspartate available and is thus depressed by lack of biotin.     

THE PRODUCTION OF AROMA COMPOUNDS BY YEAST

The yeast plasma membrane regulates the movement of compounds into the yeast cell and of yeast metabolites from the cell into the medium. The rate of penetration of organic acids into the yeast cell depends on their lipophilic nature, and on their molecular size and degree of branching. During fermentation yeast synthesizes a vast number of aroma compounds. The numerically and quantitatively largest groups of aroma compounds include fusel alcohols, fatty acids and fatty acid esters. The yeast used and the fermentation conditions can influence the formation of aroma compounds. The yeast also has a profound effect on the formation of other aroma compounds, such as sulphur compounds and phenols. In addition to fermentation, the maturing of a beverage can also influence the aroma. During the maturing lactones, phenols and other compounds are extracted from the oak casks in which the beverage is aged. The presence of the so-called “whisky lactone”, β-methyl-γ-octalactone, is characteristic of a beverage that has been matured in oak.     

CONSTRUCTION OF FLOCCULENT BREWER'S YEAST BY CHROMOSOMAL INTEGRATION OF THE YEAST FLOCCULATION GENE FLO1

Yeast flocculation gene FLO1, located on chromosome I of Saccharomyces cerevisiae, has been cloned previously¹⁶. However, it has recently been found that the gene was an in-frame deletion derivative of the chromosomal intact FLO1 gene¹⁹. When introduced into non-flocculent industrial strains, including brewer's yeast, the latter gene, FLO1L, containing an open reading frame of 4,611 bp, conferred stronger flocculation than the former gene, FLO1S, containing an open reading frame of 2,586. By chromosomal integration of the ADH1-controlled FLO1L gene, “gene therapy” of the flocculation behaviour of the parent non-flocculent brewer's yeast was successfully achieved.     

啤酒废酵母在食品工业中的应用

啤酒废酵母富含蛋白质、核酸、维生素、矿物质等多种营养成分,在食品工业具有良好的开发应用前景,主要论述了啤酒废酵母在食品行业应用现状及前景.     

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.     

THE ATTACHMENT OF BREWING YEAST TO GLASS

The secondary conditioning of cask and bottled beer with live yeast is normally facilitated by ensuring the sedimentation of the yeast to the base of the package prior to dispense. Here we examine the resistance of yeast sediment to disruption in bottled conditioned beer by investigating the attachment of brewing yeast to glass. An attachment assay is described which, when applied to brewing yeast, demonstrated that these yeasts are not naturally adhesive. However, yeast may be encouraged to form an attached mat by the physiological manipulation of starvation. The significance of this observation is discussed in relation to the effect of beer composition on yeast attachment.     

啤酒废酵母综合破壁法提取酵母味素技术

本文研究了高压均质、酶法溶胞、冻融法三种细胞壁破碎法对啤酒废酵母抽提物（酵母味素）的得率、蛋白质的转化率及氨基氮溶出率的影响。实验结果,在适宜的自溶条件下,利用综合破壁法可使酵母抽提物中上述三项指标显著提高。