Pitching Yeast Research Articles

A New Approach for Detecting Spoilage Yeast in Pure Bottom-Fermenting and Pure Torulaspora Delbrueckii Pitching Yeast, Propagation Yeast, and Finished Beer

Beer quality is highly dependent upon yeast condition. Consequently, breweries should consider the quality of the pitching yeast. Spoilage yeasts such as wild types of Saccharomyces cerevisiae, S. cerevisiae var. diastaticus, and some non-Saccharomyces yeasts occur occasionally in pitching yeast and as secondary final-product contaminants. They negatively impact pitching yeast fermentation and beer flavor. In this study we used a novel device to measure the pressure in small-scale fermentation vessels and to detect five spoilage yeast strains of S. cerevisiae var. diastaticus in four pure pitching yeast strains of the brewing yeast Torulaspora delbrueckii. We investigated a method to detect the five spoilage yeasts in pure pitching yeast of four S. pastorianus strains. Pitching yeast was chosen for its high cell density, activity, adaptability, and therefore stress resistance, and because it could produce false positive results in the detection method. Cultivation in the vessel at 37°C in YM broth inhibited all the pitching yeasts but increased the spoilage yeast. Real-time polymerase chain reaction validated the method. Low spoilage yeast concentrations of 10 cells/mL and contaminated industrial samples were reliably detected. Minimal time was needed to prepare the sample and detect spoilage yeasts.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a monitoring tool for in-house brewer's yeast contamination: a proof of concept

Contamination of brewer's pitching yeast cultures with wild-type yeasts or bacteria is unwanted as it can corrupt the fermentation outcome and causes huge economic losses for the brewing industry. The applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a fast tool to monitor the purity of brewer's yeast cultures was investigated. This proof of concept was examined for a brewer's yeast strain contaminated with wild-type yeast and for bottled beer produced by fermentation with that particular contaminated brewer's yeast strain. The data demonstrated that MALDI-TOF MS is very suitable to discriminate between brewing and non-brewing yeast strains. Copyright © 2014 The Institute of Brewing & Distilling

High Expression Levels of Cell Wall Protein Cwp2p Decrease the Turbidity of Fresh Lager Beer by Reducing the Size of Haze Particles

Cwp2p is one of the major mannoproteins of the yeast cell wall. In this study, the CWP2 gene was placed under the constitutive TDH3 promoter and introduced into a lager production strain. When the obtained CWP2-enhanced strain was subjected to a fermentation trial, the resultant beer was less turbid and contained haze particles that were smaller than those in the control beer with the parental strain. Enhanced expression of CWP2 in the transformant strain resulted in a reduction in the size of haze particles, similar to a case in which the parental strain was propagated aerobically for the fermentation trial. The aerobically prepared parental pitching yeast cells exhibited a higher level of CWP2 mRNA expression during fermentation. These results suggest that the aerobic propagation of pitching yeast exerts a haze-reduction effect by changing the expression profiles of specific cell wall mannoproteins, including Cwp2p. Immunoblot analysis detected the green fluorescent protein-cell wall protein (GFP-Cwp2p) fusion protein in fresh beer when expressed in a lager brewing strain, indicating that a glycosylphosphatidylinositol (GPI)-anchored cell wall protein, such as Cwp2p, can be released into a solution under physiological conditions. The expression level of the aerobiosis-specific mannoproteins (e.g., Cwp2p) appeared to be a key factor in the determination of haze particle size and, accordingly, beer turbidity.

The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis

For beer wort fermentation the addition of unsaturated fatty acids has sometimes been suggested as an alternative to wort oxygenation. This can however negatively affect the synthesis of acetate esters and consequently beer flavour. This work investigates the effect of supplementing a cropped yeast with an unsaturated fatty acid on the fermentation performance of the pitching yeast. Cropped yeast is in a different physiological state to yeast pitched in unfermented wort. Using a synthetic medium for the fermentations, it was found that the incubation of cropped yeast with linoleic acid resulted in two important changes in the yeasts composition: (1) the ratio of unsaturated fatty acids to total fatty acids increased from 0.53 to 0.66 and (2) the ratio of trehalose to glycogen increased from 0.17 to 0.49. The performance of this yeast in subsequent fermentations was compared to unsupplemented yeast under three conditions: medium pre-aeration, de-aerated medium and de-aerated medium with newly added unsaturated fatty acid. It was found that the supplemented pitching yeast showed growth, attenuation and ethanol formation profiles similar to those obtained with unsupplemented yeast in pre-aerated medium, which simulated the normal brewing practice. Compared to fermentations with unsaturated fatty acids added to the medium, the supplemented cropped yeast did not induce a reduction in acetate ester synthesis. Results indicated that the supplementation of cropped yeast with unsaturated fatty acids could be an interesting alternative to wort oxygenation to restore the optimal membrane fluidity of the yeast.

Factors Affecting 5-Hydroxymethyl Furfural Formation and Stale Flavor Formation in Beer

5-Hydroxymethyl furfural (5-HMF) and furfural are index compounds for beer staling. A correlation exists between the organoleptically determined degree of staling flavor in beer and 5-HMF levels during storage. This suggests that controlling 5-HMF formation during brewing might provide useful clues for improving the flavor stability of beer. Various experiments performed to control the levels of 5-HMF show that active oxygen and low pH in a finished beer enhance the formation of 5-HMF during storage. Using antioxidative mashing and selecting yeast strains with higher cellular volume are effective, practical methods for regulating these levels. Cell sizes of yeast strains (calculated from photographic images of about 100 cells) were found to correlate with the beer pH levels produced by those strains. Using pitching yeast with a larger cell size resulted in a higher pH level of beer and a lower level of 5-HMF.

Acid Washing and Serial Repitching a Brewing Ale Strain ofSaccharomyces cerevisiaein High Gravity Wort and the Role of Wort Oxygenation Conditions†

Acid washing pitching yeast is an effective method for removing bacterial contamination, but if the yeast is washed incorrectly decreased fermentation performance and beer quality problems may result. Various factors can affect the acid resistance of yeast strains during brewery fermentations. Yeast from shaking flask experiments was more resistant to the combination of high gravity and acid washing conditions than yeast cropped from static fermentations. Yeast harvested from static high gravity wort (20° Plato; 1.083 OG) fermentations was more adversely affected by acid washing than yeast from standard gravity (12° Plato; 1.048 OG) wort. Wort oxygenation resulted in enhanced yeast fermentation performance and healthier yeast crops when yeast was serially repitched into 20° Plato wort. Yeast cropped from fermentations with air saturated high gravity wort responded poorly when acid washed. These results suggest that the structure of the plasma membrane particularly the sterol and fatty acid composition, may have an important role in tolerating high gravity wort and acid washing conditions.

Effects of High-Gravity Brewing and Acid Washing on Brewers' Yeast

Washing pitching yeast with bactericidal substances such as phosphoric acid to eliminate bacterial contamination is common practice in many breweries. Washing pitching yeast incorrectly will adversely effect the yeast and result in decreased fermentation performance. When yeast was acid washed and repitched into 12°P wort, no decrease in fermentation performance was observed but, after serially repitching in 20°P wort, a small decrease in viability was observed over the first 24 hr of the fermentation. The fermentation profile of yeast serially repitched in 20°P wort matched that of the control yeast. On examination of the wort sugar profile, a decreased rate at which glucose was removed from the wort was observed, which resulted in a lag phase before maltose could be utilized from 20°P wort. No adverse effect on glucose and maltose utilization from 12°P wort was noted.

Detection of Pediococcus spp. in brewing yeast by a rapid immunoassay.

A membrane immunofluorescent-antibody test was developed to detect diacetyl-producing Pediococcus contaminants in brewery pitching yeast (yeast [Saccharomyces cerevisiae] slurry collected for reinoculation). Centrifugations at 11 and 5,100 x g separate yeast cells from bacteria and concentrate the bacteria, respectively. Pelleted bacteria resuspended and trapped on a black membrane filter are reacted with monoclonal antibodies specific for cell surface antigens and then with fluorescein-conjugated indicator antibodies. Whether pitching yeast is contaminated with pediococci at 0.001% is determined in less than 4 h. The sensitivity of the assay is 2 orders of magnitude below the Pediococcus detection limit of direct microscopy.

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.

CLEANSING CONTAMINATED PITCHING YEAST WITH NISIN

The ability of solutions of nisin to cleanse pitching yeast of contaminating gram-positive lactic bacteria (at levels of about 1% by cell numbers) was compared to that of three different acid-washing procedures: ammonium persulphate, acidified ammonium persulphate or phosphoricacid. 1000 international units ml−1 of nisin killed all the cells of more sensitive bacterial strains in less than 30 minutes, equivalent to the killing ability of the acid-washing procedures, and more than 99·9% of cells of a more resistant strain in 2 hours. Washing contaminated pitching yeast with nisin has several advantages over acid-washing methods. It has no effect on yeast viability and vitality, and, in subsequent fermentations, the yeast shows unchanged flocculation characteristics and fermentative performance.

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.

Distribution of Bacterial Contaminants in a South African Lager Brewery

Bacteria isolated from contaminated pitching yeast, fermenting wort and beer samples from a South African lager brewery over a one‐year period were tentatively identified by an improved, rapid diagnostic procedure as pediococci (41%), homofermentative lactobacilli (5%), heterofermentative lactobacilli (9%), Acetobacter spp. (7%), Gluconobacter spp. (13%) and Hafnia protea (25%). Pediococci and lactobacilli dominated samples taken from fermentation, storage and ‘bright’ beer tanks but were absent from pitched wort samples, from collection vessels and the single pitching yeast sample investigated. Acetic acid bacteria and H. protea were widely distributed in collection vessel, fermentation and storage tank samples, and H. protea was isolated from recycled pitching yeast.

WILD YEASTS IN PITCHING YEAST

Examination of pitching yeasts from various breweries by the method involving plating on an agar medium containing lysine, revealed the presence, in most samples, of “wild yeasts” in the proportion of about one cell per million cells of pitching yeast. Whereas many of the “wild yeasts” belonged to species previously recorded from this source and from spoiled beer, others do not appear to have been noted before in brewer's yeast. Pichia membranaefaciens, Candida myeoderma, Toruloptis colllculosa and Torulopsh Inconspieua were of common occurrence, and a total of thirteen other species was observed on different occasions.

METHOD FOR THE MEASUREMENT OF WILD YEAST INFECTION IN PITCHING YEAST

A medium incorporating lysine as described by Walters & Thiselton favours the growth of those wild yeasts which may form contaminants in brewery pitching yeast; such a medium, modified by the addition of agar, provides a convenient means for quantitative estimation of the degree of infection.

Nutrition of a Strain of Brewer's Yeast Requiring p-Aminobenzoic Acid

ONE of us recently reported1 strains of brewer's yeast requiring p-aminobenzoic acid for growth in a synthetic glucose – mineral salt medium. Two additional strains similarly requiring p-aminobenzoic acid have now been isolated from a brewery pitching yeast.