125th Anniversary Review: Yeast Flocculation and Sedimentation in Brewing

Flocculation is prerequisite for bulk sedimentation of yeast during brewery fermentation. Although single yeast cells gradually sediment in green beer, this sedimentation rate is too slow without formation of large yeast flocs. The present review concerns the major determinants of yeast flocculation and sedimentation in brewery fermentations. Flocculation characteristics of yeast are strongly strain‐dependent and largely defined by which FLO genes are functional in each strain. In addition to the genetic background, several environmental factors affect flocculation. These can be, somewhat arbitrarily, classified as physiological factors, such as the calcium availability, pH, temperature and ethanol and oxygen concentrations in the medium or physical factors, such as cell surface hydrophobicity, cell surface charge and the presence of appropriate hydrodynamic conditions for the formation of large flocs. Once yeast flocs are formed, their size, shape and density and the properties of the surrounding medium affect the rate at which the flocs sediment. Higher gravity worts usually result in green beers with higher viscosity and density, which both retard sedimentation. Moreover, environmental factors during yeast handling before fermentation, e.g., propagation, storage and cropping, influence the flocculation potential of yeast in subsequent fermentation. Premature yeast flocculation (PYF) and the role of PYF factors are discussed. In conclusion, some potential options available to adjust yeast flocculation are described.     

EFFECT OF ENVIRONMENTAL CONDITIONS ON FLOCCULATION AND IMMOBILISATION OF BREWER'S YEAST DURING PRODUCTION OF ALCOHOL-FREE BEER

A method using immobilised yeasts has been developed and successfully applied for production of alcohol-free beer. The influence of environmental conditions present during alcohol-free beer production on the flocculation and immobilisation of the yeast Saccharomyces cerevisiae var. uvarum was investigated in the present study. In wort, the cells developed flocculation at the end of exponential growth, according to the NewFlo phenotype. In defined medium, the flocculation capacity appeared to be temporary and was lost rapidly during the stationary phase. No increase in cell wall hydrophobicity at the onset of flocculation was observed in either medium. Low growth temperatures increased flocculation capacity approximately four-fold, compared to growth at high temperatures. The optimum temperature for flocculation was at 25°C with cells grown at low or high temperature . A novel method using carboxyfluorescein-stained cells was developed to analyse the initial adhesion of cells to carrier. This method also allowed rapid analysis of the effects of immobilisation to DEAE-cellulose carrier during alcohol-free beer production process. It appeared that a high flocculation capacity stimulated adhesion to the DEAE-carrier .     

BIOCHEMICAL ASPECTS OF YEAST FLOCCULATION AND ITS MEASUREMENT: A REVIEW

Factors affecting flocculation of brewing yeast, our current understanding of the process and traditional methods of assessing flocculence are discussed in this review. In spite of extensive study during the last two decades, a number of uncertainties and controversies regarding yeast flocculation still remain. This confusion is due in part to the polyploid or aneuploid nature of most brewing strains studied. At present, uncertainty exists as to the number of genes involved in flocculation as well as the nature and mode of action of the resulting gene products. Another restriction to our investigation of flocculation is the lack of a fundamental and standard test method. Along with a general discussion of yeast flocculation, this report will note conflicting reports concerning the influence of temperature, the effect of metal ions such as magnesium and barium, and the importance of the carboxyl and phosphate groups in yeast cell—cell interactions. As well, past and current methods employed for the assay of brewing yeast flocculation will be discussed.     

Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

Industrial wort was fermented with a NewFlo phenotype ale yeast in lab‐scale cylindrical fermenters. The effects of various fermentation parameters and yeast cell wall properties on yeast flocculation were studied during 120 h fermentation. The evaluation of the cell volume during the fermentation revealed a non‐normal distribution (p < 0.05) at most fermentation times. Overall yeast cell size initially decreased during the first 24 h of fermentation then increased during the 24–60 h fermentation period. Cell size subsequently declined until the end of fermentation presumably due to floc settling. While yeast flocculation began after 24 h fermentation, most flocs remained in suspension until 60 h when the average turbulent shear rate caused by CO₂ evolution declined to below 8 s^?1. Both the Helm's flocculence and cell surface hydrophobicity values rapidly increased to high numbers from 24 h onward. Changes in the orthokinetic capture coefficient (α₀) value with fermentation time, measured in fermenting worts, indicated a significant increase (p < 0.001) after 24 h of fermentation. Presumably, this change was due to increases in ethanol and the decline in sugar concentration with time. Although a significant positive correlation (p < 0.05) was observed between zymolectin densities and cell surface areas, the total zymolectin level on yeast cell walls did not change significantly with fermentation time (p > 0.05). Interestingly, no significant difference existed in Helm's flocculation values of suspended and settled yeast cells (p > 0.05). The flocculation rate of LCC125 was readily inhibited by addition of glucose or maltose. Results suggest that fermentable sugar levels and shear force exert major influences on yeast flocculation during beer fermentations.     

THE RESPONSE OF BREWING YEASTS TO ACID WASHING

Brewing yeasts are inherently resistant to acid washing treatments but, under some conditions this resistance is diminished. Sixteen yeast strains, including both ale and lager strains, have been successfully washed using phosphoric acid (pH 2.1) and, in some cases, sulphuric acid (pH 2.0) or acidified 0.75% w/v ammonium persulphate (pH 2.8). Cell viability, fermentation performance and flocculation/fining behaviour were unaffected. However, changes could be observed in the yeast resulting from the washing treatment. These included alterations of the cell surface, as shown by scanning electron microscopy and leakage of adenosine triphosphate from the cells during the wash. Acid washing is successful provided that, i) food grade acid is used, ii) the acid is chilled before use, iii) the yeast and acid are well mixed, iv) the temperature of the yeast does not exceed 5°C during washing and, v) the yeast is pitched immediately after washing. ‘Unhealthy’ yeast (yeast which has been stored for long periods, heavily contaminated yeast, or yeast from slow fermentations) may respond poorly to acid washing and a shorter washing time, or higher wash pH value should be employed.     

Yeast flocculation: a dynamic equilibrium

The steady state in yeast flocculation is a dynamic equilibrium between flocculated and dispersed yeast cells. The free cell concentration is directly proportional to the total cell concentration and may be expressed as an equilibrium constant. Increased agitation decreases floc size and equilibrium constant whilst increasing floc-surface area and free-cell concentration. Values of equilibrium constant are influenced by agitation in a complex relationship probably involving the floc-surface area and floc momentum. Inhibition of flocculation by mannose and low pH is reversible and becomes greater with increased agitation. Both these inhibitions appear consistent with a weakening of flocculent bond strength by these inhibitors.     

Yeast flocculation: kinetics and collision theory

Flocculent yeast cells have an absolute requirement for mechanical energy input in order for flocculation to occur. Flocculation is arrested by cessation of energy input. The initial rate of flocculation increases as the square of the cell concentration. There is a minimum shaking speed to initiate flocculation and thereafter the initial rate of flocculation increases exponentially with the shaking speed. The minimum shaking speed for flocculation to occur increases with pH value. Activation energy for flocculation, derived from Arrhenius-like plots, varies with pH value. We propose that activation energy is required to overcome mutual repulsion between charged yeast cells and allow flocculent bonds to be formed.     

A Method to Detect Anti‐metabolic Factors in Fermentations

Premature yeast flocculation (PYF) has been described as the rapid settling of yeast cells during fermentation despite the presence of sufficient nutrients. PYF can cause negative impacts on beer quality and thus can be quite costly to brewers and maltsters. To investigate the causative agent of PYF, small‐scale fermentations were undertaken in both test tubes and cuvettes (15 and 3.5 mL respectively) using worts prepared from PYF‐positive and PYF‐negative malt samples. Fermentations were carried out using six malts, for up to seven days. Turbidity and extract values were monitored for all samples. The small scale (test tube) assay exhibited clear yeast cell flocculation differences between malts. In the cuvette assay the wort fermented, but the yeast cells settled out of suspension rapidly. While this property made the cuvette assay unsuitable for detecting PYF malt, it did allow for measurement of impaired sugar uptake by the yeast independent of yeast in suspension effects. All wort samples fermented in the cuvette assay showed a similar decline in apparent extract (p > 0.05), indicating that (at least in the samples studied) premature yeast flocculation was not caused by a decline in yeast activity. We believe the simple cuvette assay reported here could have application in the measurement of anti‐metabolic factors in fermenting media.     

SOME CONSIDERATIONS OF THE FLOCCULATION CHARACTERISTICS OF ALE AND LAGER YEAST STRAINS*

While some ale yeast strains are able to flocculate when cultured in a defined medium of glucose, ammonium salts, vitamins and ions, others require the presence of a nitrogen-containing inducer in the growth medium. On the other hand, all flocculent lager strains examined to date are able to flocculate after being cultured in a defined medium and do not appear to require the addition of inducer material to the growth medium. The inducer material present in wort has been identified as peptide. By the use of ion exchange chromatography the peptide fraction that induces flocculation has been found to contain a high level of acidic amino acid residues with a very similar structure to that reported for the α-factor involved in sexual agglutination of haploid α and a cells of Sacch. cerevisiae. Studies on the adsorption of Ca⁺⁺ ion by the cell wall failed to reveal any significant differences in total uptake between flocculent and non-flocculent cultures. It would appear that Ca⁺⁺ ions are bound less tightly by non-flocculent cells than by flocculent cells. The contribution of calcium to flocculation is not the absolute amount of this ion adsorbed by the yeast cell wall but rather the stereo-specific manner by which it is bound, i.e., its position relative to the three-dimensional structure of the yeast cell wall.     

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.     

Inverse Flocculation Patterns in Saccharomyces cerevisiae UOFS Y‐2330

An interesting yeast strain was uncovered which showed an inverse flocculation pattern when cultivated in chemically defined and complex media. When inoculated in a defined medium with glucose as a sole carbon source, this strain immediately flocculated strongly and lost this ability before stationary phase was reached. In a complex malt medium containing glucose, this yeast strongly flocculated throughout the exponential and stationary growth phases. This inverse pattern may be ascribed to a switch in sensitivity of the yeast to flocculate in the presence of glucose as well as pH level, which may, in turn, influence the availability of calcium ions. In both media, matured cells produced protuberances or “wrinkles” upon flocculation as observed by electron and immunofluorescence microscopy. These protuberances may be involved in cell adhesion during the flocculation process.     

EFFECT OF SOME MONOVALENT AND DIVALENT METAL IONS ON THE FLOCCULATION OF BREWERS YEAST STRAINS*

In agreement with previous reports, it has been found that both Mg⁺⁺ and Mn⁺⁺ ions can imitate Ca⁺⁺ as inducers of flocculation, though the intensity of the flocculation is considerably reduced. This reduction is not dependent upon the ionic concentration and a 10-fold increase in Mg⁺⁺ or Mn⁺⁺ from the normal concentration of 10 mg ion/litre fails to increase the flocculation intensity. Low concentrations (1–10 mg ion/litre) of Na⁺ or K⁺ induce flocculation in those strains displaying intense flocculation with Ca⁺⁺, but high concentrations of either Na⁺ or K⁺ (50–100 mg ion/litre) antagonize floc formation. It is suggested that divalent ions act by bridging cells through negative charges on the cell surface, whereas monovalent ions induce flocculation via a “counter ion” effect where the repellent forces of the negative charges on the cell surface are neutralized, thus allowing some floc formation due to hydrogen bonding or other types of non-ionic bonding between cells. The antagonism of high concentrations of monovalent ions towards flocculation may be due to the fact that all available cell surface charges are neutralized, resulting in insulation of the cells and thus preventing cell-to-cell hydrogen bonding.     

STUDIES ON THE EFFECT OF TEMPERATURE ON THE MOISTURE SORPTION CHARACTERISTICS OF POTATOES

An understanding of the moisture sorption characteristics of food systems is an important objective in food engineering, in particular with respect to the mass transport phenomena during drying. A standard gravimetric technique was employed to examine the influence of temperature on the adsorption and desorption behavior for potatoes. The type II sorption isotherms were analysed using the Guggenheim-Anderson-de Boer model, with the observed hysteresis phenomena displaying a temperature dependence. The moisture dependent net isosteric heats of sorption were determined using the Clausius-Clapeyron equation.     

STUDY OF THE FLOCCULATION OF SACCHAROMYCES DIASTATICUS NCYC 625

Yeast flocculation presents a great interest for the industry of fermentation but its mechanism is still not fully understood. In order to enlighten this mechanism, the flocculation of Saccharomyces diastaticus NCYC 625 was studied. As with other Saccharomyces strains, the effects of different factors affecting flocculation and deflocculation (pH, temperature, medium, EDTA, cations…) suggest a lectin-like binding between adjoining cells. The genetic determinism of flocculation is nuclear and not mitochondrial. Although Saccharomyces diastaticus NCYC 625 could be classified in the FLO1 phenotype according to Stratford and Assinder⁴², allelism tests show that the gene involved in the flocculation control is not allelic with FLO1 or FLO5 and possibly different from FLO8 .     

QUANTIFICATION OF YEAST FLOCCULATION

A sedimentation method based in the Helm's flocculation test was improved. In this method, all steps were standardised: initial cell concentration, agitation, sampling and determination of settled cells. The results obtained with the sedimentation test do not differ significantly (P = 0.05) from those obtained with the Stratford test; besides, the precision of the two methods do not differ significantly (P = 0.05). The results presented clearly demonstrated that the method improved in this work is rigorous for quantification of yeast flocculation and allows the differentiation of flocculation ability of the strains. The respective micro-flocculation tests were also compared; Stratford micro-flocculation test was selected as producing the most meaningful results.     

ROMATIC COMPOUNDS AND SUGARS IN FLOCCULATION OF SACCHAROMYCES CEREVISIAE

Phenol, aniline, benzyl alcohol and pyridine in the range 0.1 m to 0.3 m appreciably lowered the floc dissociation temperature, T_f , of two strains of Saccharomyces cerevisiae, without affecting flocculation intensity, F. Cell surface inactivation and Sr salt addition lowered F but not T_f . Mannose lowered F specifically, over fructose, glucose and maltose. Results are interpreted as a specific complementary site binding in yeast flocculence.     

Mitochondrial Associated Yeast Flocculation ‐The Effect of Acetylsalicylic Acid

Mitochondrial function is generally accepted as important for expression of yeast flocculation. In this study, a correlation between mitochondrial activity and flocculation is demonstrated using the XTT reduction assay. The mitochondrial activity of strongly flocculent cells was higher than those of weakly flocculent cells and cells cultivated in the presence of acetylsalicylic acid. Furthermore, we show the first oxylipin‐containing flocculation binding sites on yeast cell surfaces using scanning electron microscopy. We propose that in addition to zymolectin‐mediated flocculation, oxylipin interactions may also play a role in yeast flocculation.     

啤酒酵母絮凝机理研究进展

啤酒酵母絮凝机理的假说有絮凝共生假说、类外源凝集素假说、类外源絮凝素假说等。啤酒酵母絮凝表型可分为FLO1型和NewFLO型;对啤酒酵母絮凝基因的研究有结构基因——见O基因家族、调节基因及其他相关絮凝基因。对啤酒酵母絮凝性状的改良研究可应用于调控啤酒酵母絮凝性状,利于啤酒生产;利用酵母絮凝性状构建絮凝选择栽体;利用絮凝素蛋白构建酵母细胞表层展示体系;利用酵母絮凝蛋白对细菌的吸附,应用于医疗行业。     

COLLOIDAL ASPECTS OF YEAST FLOCCULATION: A REVIEW

In this review, areas of colloid science including the DLVO theory, flocculation kinetics and suspension rheology are outlined and their applicability to the study of yeast flocculation discussed. Specifically, fundamental methods of predicting cell-cell interaction energies, orthokinetic flocculation rates and rheological flow properties of flocculent suspensions are detailed. While the application of these theories to brewing systems is somewhat difficult, they may aid our understanding of brewing yeast flocculation. The limited information available on the colloidal nature and properties of brewing yeast cells is also summarized.