R. Alex Speers

Flocculation of Saccharomyces cerevisiae

Abstract This paper reviews our current understanding of cell flocculation with particular emphasis of the process in brewing fermentations. While cell flocculation has been examined for over a century and has been the subject to a number of reviews in the early part of this decade, our view of the process is cloudy. Flocculation is affected by cell genetic behavior, cell age as well as the chemical and physical nature of the surrounding medium. Recently, a number of advances in our understanding of the genes governing the process have occurred. In conjunction with these genetic advances, new assay methods have also been developed. This review will discuss and update our current knowledge of cell flocculation and its use in brewing fermentations. ©

Miniaturizing the fermentation assay: Effects of fermentor size and fermentation kinetics on detection of premature yeast flocculation

This article reports on fermentation assays used to test the fermentability of malt, especially malt implicated in premature yeast flocculation (PYF). No standard small-scale method currently exists. The current study involved 12°C fermentations with three vessel configurations: a 4.4-cm high spectrophotometer cuvette, a 12.5-cm high test tube, and a 122-cm high “tall” tube fermentor. Worts tested were made from control and PYF malts. We included in our experiments the method of Jibiki and associates, using wort supplemented with 4% glucose and fermented at 21°C. The decline in Plato values (or real extract) was fit with a sigmoidal model. The model parameters and fermentor height were used to estimate CO2 evolution and average shear rates. It was confirmed that both fermentor height and fermentation speed are the major independent variables determining CO2 evolution and agitation within the fermenting wort. In examining the fermentation data, it is evident that the yeast did not stay equally suspended in all three fermentation vessels. When fermented at 12°C, yeast fell out of suspension rapidly in both the cuvette and test tube fermentors. In the tall tubes, the yeast remained in suspension as expected. The 21°C test tube fermentations supplemented with glucose had fermentation profiles similar to the tall tube fermentations but were complete in less than 72 hr. It is notable that these profiles could be used to distinguish between PYF and control malts. The minimum average shear rate required to keep yeast suspended was determined to be between 4 and 7.5 sec−1. Thus, when downscaling a fermentation assay by reducing the fermentor height, the rate of fermentation must be increased to maintain adequate shear rates. We confirmed that the PYF fermentation assay could be reduced to 15 mL, resulting in reduced labor, time, and material costs.

Modeling of orthokinetic flocculation of Saccharomyces cerevisiae

This study examined the flocculation behavior of two Saccharomyces cerevisiae strains expressing either Flo1 (LCC1209) genotype or NewFlo (LCC125) phenotype in a laminar flow field by measurement of the fundamental flocculation parameter, the orthokinetic capture coefficient. This orthokinetic capture coefficient was measured as a function of shear rate (5.95-223 s(-1)) and temperature (5-45 degrees C). The capture coefficients of these suspensions were directly proportional to the inverse of shear rate, and exhibited an increase as the temperature was increased to 45 degrees C. The capture coefficient of pronase-treated cells was also measured over similar shear rate and temperature range. A theory, which predicts capture coefficient values due to zymolectin interactions, was simplified from that developed by Long et al. [Biophys. J. 76: (1999) 1112]. This new modified theory uses estimates of: (1) cell wall densities of zymolectins and carbohydrate ligands; (2) cell wall collision contact area; and (3) the forward rate coefficient of binding to predict theoretical capture coefficients. A second model that involves both zymolectin interactions and DLVO forces was used to describe the phenomenon of yeast flocculation at intermediate shear ranges, to explain yeast flocculation in laminar flow.

TRFLP analysis reveals that fungi rather than bacteria are associated with premature yeast flocculation in brewing

Premature yeast flocculation (PYF) is a sporadic fermentation problem in the brewing industry that results in incomplete yeast utilization of fermentable sugars in wort. Culture-independent, PCR-based fingerprinting techniques were applied in this study to identify the associations between the occurrence of the PYF problem during brewery fermentation with barley malt-associated microbial communities (both bacteria and fungi). Striking differences in the microbial DNA fingerprint patterns for fungi between PYF positive (PYF +ve) and negative (PYF −ve) barley malts were observed using the terminal restriction fragment length polymorphism (TRFLP) technique. The presence of terminal restriction fragments (TRFs) of 360–460 bp size range, for fungal HaeIII restriction enzyme-derived TRFLP profiles appeared to vary substantially between PYF +ve and PYF −ve samples. The source of the barley malt did not influence the fungal taxa implicated in PYF. TRFLP analysis indicates bacterial taxa are unlikely to be important in causing PYF. Virtual digestion of fungal sequences tentatively linked HaeIII TRFs in the 360–460 bp size range to a diverse range of yeast/yeast-like species. Findings from this study suggest that direct monitoring of barley malt samples using molecular methods could potentially be an efficient and viable alternative for monitoring PYF during brewery fermentations.

Improving uncertainty in Widmark equation calculations: alcohol volume, strength and density

The Widmark equation is probably the most commonly used calculation for medicolegal purposes. Recently the National Research Council (USA) and the Forensic Science Regulator (UK) have called for the uncertainty of all results to be given with all forensic measurements and calculations. To improve the uncertainty of measurement of results from Widmark calculations we have concentrated on the uncertainties of measurement involved in the calculation of amount of alcohol, that of the volume of alcohol, the concentration of alcohol and the density of alcohol as previous studies have investigated some of the other factors involved. Using experimental studies, the scientific literature and legal statutes, we have determined revised and improved uncertainties of the concentration of ethanol for Widmark calculations for both the USA and UK. Based on the calculations that we have performed we recommend the use of Monte Carlo Simulation for the determination of uncertainty of measurement for Widmark Calculations.

Colloidal examination of worts associated with premature yeast flocculation

This paper reports on a study of the colloidal properties of yeast fermented in control and premature yeast flocculation (PYF) worts. A lager yeast strain was fermented in both control and PYF worts and analyzed for cell wall properties. Yeast from the PYF wort exhibited more flocculation than the control. The PYF fermentation yielded yeast with less negative zeta potential than the control (P < 0.001). When both yeasts were resuspended in beer (filtered through a 10-kDa filter), there was no difference in the surface charge of the control and PYF yeasts (P > 0.05). This suggests that wort colloids or trub larger than 10 kDa caused a reduction in surface charge. Cell separation forces estimated by floc breakup through a capillary indicated PYF yeast flocs exhibited higher apparent separation force. The orthokinetic capture coefficients of control and PYF yeasts showed significant differences in capture coefficient values between both of the yeast suspensions, with the PYF capture coefficient values being higher from 72 hr of fermentation onward (P < 0.05). A physical mechanism of PYF has been proposed that involves electrostatic interaction between wort particles (positively charged toward the end of fermentation) and yeast (negatively charged through fermentation).

Compositional, ultrastructural and nanotechnological characterization of the SMA strain of Saccharomyces pastorianus: Towards a more complete fermentation yeast cell analysis

Nano scanning Auger microscopy (NanoSAM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been used in materials science research for some time, but NanoSAM, in particular, has only recently been applied to biological specimens. Here, the first concurrent utilization of NanoSAM, TOF-SIMS and microscopic techniques for the examination of a standard beverage fermentation strain of Saccharomyces pastorianus uncovered the presence of intracellular networks of CO2 in fermenting cells. Respiring cells produced few bubbles and instead had large internal vacuolar structures. Transmission electron microscopy analysis also showed osmiophilic layers at the cell exterior of fermenting cells that became more prevalent with fermentation duration, while osmiophilic layers were largely absent in respiring cells. TOF-SIMS analysis showed a compositional difference at the exterior and interior of SMA cells and between fermenting and respiring cells. Fermenting cells also appeared to have different 3-OH oxylipin profiles compared to respiring cells based upon examination with immunofluorescence microscopy. The results of this work and further study using these materials science techniques will substantially enhance our understanding of the chemical, ultrastructural and metabolic changes that occur in fermentation yeasts.

Storage Temperature Impacts on Anthocyanins Degradation, Color Changes and Haze Development in Juice of “Merlot” and “Ruby” Grapes (Vitis vinifera)

This study evaluated the degradation kinetics of selected anthocyanins and the change in polymeric color, browning index, and haze development of grape juices from “Merlot” and “Ruby” grape cultivars stored at 5, 25, and 35°C for up to 360 days. Five major anthocyanins namely malvidin-3-O-glucoside (M3G), delphinidin-3-O-glucoside (D3G), petunidin-3-O-glucoside (Pt3G), peonidin-3-O-glucoside (Pn3G), and cyanidin-3-O-glucoside (C3G) were identified. Juice from “Merlot” had significantly higher (p < 0.05) content of all individual anthocyanins as compared to “Ruby.” During the long-term storage, total, and individual anthocyanins from both cultivars degraded following first-order reaction kinetics at the rate strongly dependent on temperature. At the end of the storage, noticeably higher loss of anthocyanins (95–99.9%) was observed at 25 and 35°C as compared to storage at 5°C [50–60% (“Merlot”); 74–81% (“Ruby”)]. Considerably lower rate of decay was observed at 5°C (k = 0.01–0.04) as compared to 25 (k = 0.04–0.14) and 35°C (k = 0.05–0.14) storage temperatures. The most temperature sensitive anthocyanin compounds were C3G (Ea = 66.5 kJ/mol) and D3G (Ea = 63.3 kJ/mol). At higher storage temperatures, significant (p < 0.05) and strong negative correlations were observed between anthocyanin concentrations and the levels of haze, polymeric and brown color development during storage. Storing grape juice, at lower temperature conditions could reduce the continuous loss of biologically active anthocyanins as well as the development of haze and brown color.