A FOAMING PROBLEM IN BREWER'S YEAST SLURRY DESTINED FOR DRYING FOR ANIMAL FEED

A foaming problem in unwashed brewer's yeast collected at the end of primary fermentation and sold by a Western Canadian brewery for drying to make animal feed is described. The factors responsible for foaming have been shown to be residual fermentable carbohydrates in the volume of beer accompanying the yeast into the special storage tank. Foaming did not ensue until temperatures were raised either in transport or in temporary storage at the drying plant. Some methods of eliminating the problem are outlined.     

SPHEROPLAST FUSION OF BREWER'S YEAST STRAINS

Spheroplasts of brewing polyploid yeast strains have been successfully fused with spheroplasts of haploid yeast strains. After regeneration of the cell wall, stable fusion recombinants were isolated. Genetic analysis of these recombinants revealed that they contained the genotype of both parents, sporulated well with each ascus containing four spores and were indeed diploid. Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.     

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.     

DISPOSAL OF EXCESS BREWER'S YEAST BY RECYCLING TO THE BREWHOUSE

Waste streams high in B.O.D. are produced by the brewing process itself. These wastes are wholesome but unwanted materials that were once part of or were in intimate contact with the brewing milieu. They are not inherently foreign to the process or the product, and disposal could be accomplished by recycling to the process. We have studied the effect on brewing processes and beer flavour of recycling waste yeast slurry to the mash mixer. Beer flavour is essentially unaffected by recycling, and of the brewhouse processes only lautering is slowed at higher recycle rates. Extract (up to 1% of the total) and soluble nitrogen can be recovered from the recycled yeast, but is strongly influenced by temperature during yeast storage and mashing. Worts made with recycled yeast tend to ferment more rapidly than normal worts and tend to have a lower end gravity.     

FLOCCULATION OF BREWER'S YEAST

Analyses of cell walls isolated from flocculent and non-flocculent strains of Saccharomyces cerevisiae revealed no appreciable differences in the levels of major components. Fractionation using ethylenediamine according to the method of Korn & Northcote furnished a Fraction A from flocculent walls which had higher levels of phosphorus than that from non-flocculent. 70% of the phosphorus of this fraction was incorporated as phosphodiester in a manno-protein. Walls from flocculent yeasts bound on average twice as much calcium as did walls from non-flocculent yeasts. Removal of the phosphorylated manno-protein decreased the capacity of the cell wall to bind calcium and rendered the wall non-flocculent, emphasizing the role of superficial phosphate groups in flocculation.     

LIPID COMPOSITION OF AEROBICALLY AND ANAEROBICALLY PROPAGATED BREWER'S BOTTOM YEAST

Aerobically grown pitching yeast is very rich in unsaturated fatty acids and sterol esters compared to traditional, anaerobic yeast. The principal fatty acids in aerobic yeast cells are unsaturated palmitoleic and oleic acids, whereas in anaerobic cells saturated palmitic acid predominates. The difference in fatty acid distribution between aerobic and anaerobic cells is most marked in the sterol esters. The fatty acids of phospho-lipids are more stable, although remarkable differences are observed. The sterols of aerobic cells are almost entirely in esterified form and zymosterol is the principal sterol. During the first hours of fermentation a rapid synthesis of palmitoleic acid is observed when anaerobic yeast is used for pitching and the wort is aerated. The synthesis of oleic acid requires more oxygen and time than is available under normal brewing conditions. When aerobic pitching yeast is used no more unsaturated fatty acids are synthesised and the lipid stores of pitching yeast are distributed among the daughter cells. The decrease in acetate ester production by aerobic pitching yeast is concluded to be due to a decrease in acetyl CoA synthesis, which may be caused by the high proportion of unsaturated fatty acids in membrane lipids.     

ANALYSIS OF SPORULATION IN BREWER'S YEAST: INDUCTION OF TETRAD FORMATION*

A programme for assessment of sporogenic ability has been applied to analysis of sporulation in a polyploid strain of Saccharomyces cerevisiae used in ale production. Final sporulation percentages in five single colony isolates were compared employing several agar media and the most sporogenic of these was selected for further study. Cultivation in liquid rather than agar media improved ascus production substantially. Analysis of effects on ascosporogenesis of temperature and of presporulation growth on various carbon sources led to identification of culture conditions for enhanced ascus formation. Sporulation at 21 instead of the usual 27°C gave significant increases in ascus yields. Substitution of glucose with acetate as the presporulation carbon source increased yields further; moreover, a marked induction of tetrads was noted. Data from comparison of effects on sporulation of fermentable versus non-fermentable carbon sources suggest increased tetrad production to depend closely on presporulation growth under conditions of complete carbon catabolite derepression. Although spore viabilities were typically low, as determined by tetrad analysis, the dramatic increases in sporulation obtained by manipulation of culture conditions permitted rapid isolation of an array of segregants. These included a- and α-maters for use in hybridisation and genetic characterisation .     

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.     

ABSENCE OF NUTRITIONAL REQUIREMENT FOR UNSATURATED FATTY ACIDS BY BREWER'S YEAST IN MALT WORT

Addition to de-aerated malt wort of ergosterol dispersed in the non-lipid detergent Tergitol allows satisfactory growth of oxygen-requiring cells of brewing yeast. Any nutritional requirement for unsaturated fatty acid for viability and growth of such cells (as distinct from effect on metabolite balance, e.g. of esters) can therefore be supplied by the normal constituents of a typical malt wort.     

THE FATE OF LIVE BREWERS' YEAST SLURRY IN BOVINE RUMEN FLUID

Live brewers' yeast slurry was incubated under carbon dioxide at 27°C and 39°C in 0·1% peptone solution and in bovine rumen fluid which had been clarified by removal of the population of bacteria and protozoa normally present. Numbers of viable yeast in both media remained constant for 12 h at 27°C; at 39°C loss in viability was 81 % in peptone and 94% in rumen fluid during the same period. When glucose was added to clarified or unclarified rumen fluid containing yeast slurry and incubated for 6 h at 39°C, ethanol was produced. Ethanol production was prevented if the slurry was treated with heat or chemical preservatives before addition to the rumen fluid. Unclarified rumen fluid from a steer fed a brome-alfalfa hay-grain ration contained 10²–10³ yeasts and moulds per ml. The results suggested that the feeding of live brewers' yeast slurry to ruminants could result in ethanol toxicity if fermentable carbohydrate were also present, though many of the yeast cells would succumb to heat inactivation at normal rumen temperatures. This risk could be eliminated by prior treatment of the slurry with heat or chemical preservatives.     

METABOLIC QUOTIENTS OF RESPIRATION-DEFICIENT MUTANTS OF BREWER'S YEAST AND THE APPEARANCE OF A NEGATIVE PASTEUR EFFECT

Four industrial strains of bottom brewer's yeast and a group of their spontaneous respiration deficient (RD) mutants were tested for rates of metabolism of glucose and maltose under aerobic and anaerobic conditions. Q_o2 of all the RD mutants tested (26 isolates) ranged from 1·9 to 6·8 μl/mg yeast dry weight on glucose and was lowered to about one-half on maltose although the original strains had the same Q_o2 values on both sugars. No isolate showed any increase in glucose fermentation in aerobic conditions as compared with the original strains and the decrease of Pasteur effect found in certain isolates was always accompanied by a strong decrease of glycolysis in both aerobic and anaerobic conditions. Two mutants showed a strongly negative Pasteur effect, for their fermentation rates were higher in aerobic conditions than in anaerobic ones. Two other mutants showed a strong negative Pasteur effect only on maltose. The ratio of Q^N_2CO2 on glucose in most mutants was significantly lower than in their parental strains.     

HEAT PROCESSING OF SPENT BREWER'S YEAST

Moist heat treatments of brewer's yeast at cell populations of approximately 106/ml resulted in biphasic but otherwise typical survival curves. Brewer's yeast was shown to suffer either thermal injury or death over the tested temperature range of 47–53°C. Recovery from thermal injury was possible in liquid media at 20–30°C. Death kinetics were predictable and reproducible. At cell concentrations found in industrial yeast slurries ～5 × 10⁵/ml), pasteurization of spent yeast could not be planned by assuming normal first order kinetics, as dying cells released materials which altered the thermo-resistance of survivors. Under simulated yeast slurry conditions however, with protective cell supernatant (menstruum) obtained after vigorous heating of slurried yeast, a phantom thermal death time curve was obtained which could be used to accurately predict yeast kill (decimal reduction time) at slurry concentrations and at any temperature.     

RELEASE OF PHOSPHATE BY FERMENTING BREWER'S YEAST

When brewer's yeast (Saccharomyces carisbergensis) was pitched into a solution of fermentable sugar, phosphate, potassium and magnesium ions were rapidly released into the medium. On further incubation the released phosphate was re-absorbed. In the presence of fermentable sugar, n-butanol markedly increased release of phosphate and calcium ions inhibited the leakage, but these reagents had little effect when yeast was suspended in water. Yeast cells grown on a medium deficient in inositol or biotin and inositol, when subsequently exposed to fermentable sugar, released more phosphate than did cells grown on a sufficient medium. Leakage was dependent upon sugar uptake and fermentation; leakage was not inhibited by sodium azide or by 2,4, dinitrophenol, but was inhibited by fluoride, iodo-acetate or uranyl ions. From these results it was concluded that the leakage of inorganic ions was dependent on an increase in the permeability of the cytoplasmic membrane during sugar utilization.     

STRAIN SPECIFIC RESPONSE OF BREWER'S YEAST STRAINS TO ZINC CONCENTRATIONS IN CONVENTIONAL AND HIGH GRAVITY WORTS*

The effect of zinc on a variety of yeast strains is extensively documented in the literature. However, due to the varied experimental protocols employed in each study there is little opportunity to directly compare the strain specificity of this ion. In the present study, the response of six yeast strains (three Saccharomyces cerevisiae (ale type) and three S.cerevisiae (lager type)) to altered zinc concentrations, in both high (1080 OG) and conventional (1048 OG) gravity worts, was investigated. Varying the initial wort zinc concentration in both gravities had an effect on the ethanol production, rate of fermentation, cell number and sugar uptake of all six strains studied. The extent of the response was found to be dependent upon the zinc concentration, the strain employed and wort gravity employed.     

PITCHING RATE AND YEAST ACTIVITY DURING FERMENTATION OF BREWER'S WORT

Departure from a normal pitching rate during batch fermentation of brewer's wort by strains of Sacchoromyces cerevisiae results in disproportionate changes in the time needed for fermentation. Variations in pitching rate alter the ability of the yeast to utilize maltose and this mainly determines the rate of fermentation of the wort. These changes in yeast activity modify the effect of altered yeast concentration due to pitching rate and are responsible for the disproportionate changes in fermentation time.     

FERMENTATION OF SYNTHETIC MEDIA CONTAINING GLUCOSE AND MALTOSE BY BREWER'S YEAST

Brewer's yeast previously cultured on either glucose or maltose as the sole source of carbohydrate has a period of exclusive utilization of glucose when grown on synthetic media containing glucose and maltose. Only when glucose has fallen to a low level does fermentation of maltose begin. This behaviour is thought to be due to an effect of glucose on the maltose permease.     

FURTHER EVIDENCE FOR THE CROSS-BRIDGING HYPOTHESIS FOR FLOCCULATION OF BREWER'S YEAST

Mannan-protein has been extracted from the walls of various yeast strains. The range of side-chains of the mannan varies from strain to strain but differences do not correlate with flocculence. There is no correlation between flocculence and the levels of acidic amino acids present in the walls. The capacity of cells to flocculate by cross-bridging is proportional to the phosphate groupings present in the outer layer of the mannan-protein. The level of phosphate in the outer layer is sufficient only in the case of flocculent strains to enable binding of bivalent cations between adjacent cells. Scanning electron microscopy failed to reveal surface features of the walls relevant to flocculation.     

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.     

PRODUCTION OF SHOCHU ON A COMMERCIAL SCALE FROM POST-DISTILLATION SLURRY BY A NEWLY DEVELOPED RECYCLING PROCESS

During commercial scale fermentation for the production of rice shochu using post-distillation slurry in place of the first-stage mash, it was difficult to stir the mash from the first to the third day because of the high viscosity of the mash. Shochu was therefore fermented with a two-step addition of cooked rice, adding rice on the first and third days, to reduce the viscosity of mash during the first few days. Even though the ethanol concentration of the mash after 9 d was 17.2 v/v% and was a little lower than that obtained in laboratory scale tests, the yield of pure ethanol was 473 ml kg^?1 of rice and this value was higher than the average value (450 ml kg^?1) when shochu is made in the standard manner on a commercial scale. The shochu produced on a commercial scale by the newly developed recycling process was very mild and smooth, and received high scores in sensory tests.     

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.