Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

Yong-Quan Wan, 1) R.Alex Speers, 1)* Yu-Lai Jin, 2) and Robert J. Stewart3)
1) 2) 3)

Department of Food Science and Technology, Dalhousie University, Halifax, Canada. Ocean Nutrition Canada Ltd., Halifax, Canada.

Labatt-ITW Technology Department Americas, London

Abstract 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 in the first 24 h of fermentation then increased during 24-60 h. Cell size then declined until the end of fermentation. While yeast flocculation began after 24 h, 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 Helm's flocculence and cell surface hydrophobicity rapidly increased to high and stable values from 24 h onward. Although a significant positive correlation (p<0.05) was observed between zymolectin densities and cell surface area, the total zymolectin level on yeast cell walls did not change significantly with fermentation time (p>0.05). Changes in orthokinetic capture coefficient (0) value with fermentation time, measured in fermenting worts, indicated a significant increase (p<0.001) after 24 h of fermentation. Results suggest that fermentable sugar levels and shear force exert major influences on yeast flocculation in beer fermentation. Keywords: Brewing yeast; flocculation; zymolectin; orthokinetic capture coefficient.

Introduction The flocculation characteristics of a yeast strain are important to beer fermentation and clarification. Our understanding of how brewing yeast cells flocculate is still incomplete. While much of our understanding of the phenomenon has been gained by biochemical, molecular and genetic investigations, it is our belief that examination from a colloidal viewpoint is also required to more fully understand this industrially important process. In fact, the consideration of colloidal theory behind cell flocculation has been recognized in recent years.13,31,32 Yeast flocculation has been defined as, the reversible phenomenon wherein yeast cells adhere in clumps and either sediment rapidly from the medium in which they are suspended or 1

rise to the mediums surface.38 Flocculence, on the other hand, is the ability of the yeast cells to flocculate under optimal conditions, which is a cell wall property independent of environmental change. Thus, when studying flocculation one has to consider both the properties of the yeast cells as well as the effect of the environment. Although it is accepted that zymolectin binding and hydrophobicity are the main biochemical reasons causing cells to attach to one another,16,20 one also needs to be concerned with the rate at which cells collide to form flocs. Specifically, the rate of cell orthokinetic flocculation caused by shearing forces must be considered. Although yeast cells probably flocculate in turbulent shear fields during beer fermentation, the majority of studies on flocculation have employed simpler laminar flow fields.13,18,32 An expression describing flocculation rate of perfect spheres within a laminar shear fluid was modified by van de Van and Mason: 42
. Nt = e ( 4 0 0 / ) t N0

(1)

where Nt is the number concentration of particles (i.e., flocs) at time t, N0 is the initial number concentration of particles, 0 is the orthokinetic capture coefficient, & is the shear rate and o is the initial volume fraction of the particles. In this expression, the orthokinetic capture coefficient is the most important term because it represents the odds of two colliding cells or flocs to form a doublet floc or a larger floc. Speers and coworkers32 investigated the variation of the capture coefficient of four yeast strains in lager and ale media, respectively. It was found that orthokinetic capture coefficients were much higher than those predicted by Duszyk and Doroszewski12 based on the DLVO (Derjaguin, Landau, Verwey and Overbeek) theory. Thus, DLVO theory alone could not explain brewing yeast flocculation. It is believed that zymolectin binding and hydrophobic interactions are more important than DLVO forces in brewing yeast cell-cell interactions. Hsu et al.13 reported the effects of shear rates and temperature on the capture coefficient of two Saccharomyces cerevisiae strains (Flo1 LCC1209 and NewFlo LCC125) in a laminar flow field. A new theory modified from Long et al.18 was used to explain yeast flocculation in laminar flow. The capture coefficient of yeast cells in suspension was reported to be directly proportional to the reciprocal of shear rate, and exhibited an increase as the temperature was increased from 5oC to 45oC.13 However, all these studies on yeast flocculation were focused on the yeast cells propagated in standard yeast extract-peptone-dextone (YEPD) medium in vitro. To our knowledge, very few flocculation studies, if any, have been carried out in situ. Monitoring those factors (such as zymolectin density and shear force etc.) that affect flocculation during even lab-scale fermentations could provide us a new insight into the process of brewing yeast flocculation. In this study, lab-scale static fermentations were carried out with industrial brewers wort. During a 120-h fermentation, apparent extract (AE), cell size, floc size, flocculence, CO2 evolution rate (CER) and several yeast cell wall properties were examined. Cell wall properties including cell surface zymolectin density, cell surface hydrophobicity (CSH) and orthokinetic capture coefficient were investigated to examine their roles in yeast flocculation. Experimental Materials and Methods Yeast culture and growth - An ale brewing yeast strain LCC125 was obtained from the Labatt Culture Collection (London, ON). Strain LCC125 is a bottom cropping ale yeast exhibiting the NewFlo phenotype, which can be deflocculated by glucose and mannose.37 YEPD liquid medium was used as described by van Uden and Sousa.43 Isolated colonies were inoculated into 100 mL 2

of YEPD broth in 250 mL Erlenmeyer flasks. Incubation was carried out at 30oC while shaken at 100 rpm for 24 h. Fermentation - Industrial wort (17.5oP) obtained from Oland Brewery Ltd. (Halifax, NS) was adjusted to 12oP with distilled water and then immediately autoclaved at 121oC for 15 min. Before pitching yeast, wort was aerated with pure oxygen through a 0.45 m membrane to give an oxygen level of approximate 10-12 ppm. Yeast starter for beer fermentation was prepared in two steps. One preculture step was carried out in YEPD medium as described above. Yeast cells collected were then inoculated to next preculture in 12oP wort (100 mL in a 250 mL flask) at 1.0106 cells/mL. The final yeast count of this second preculture was 4.5107 cells/mL. All the precultures were grown at 30oC for 24 h and shaken at 100 rpm. Yeast cells were then harvested by centrifuge (500g for 2 min). Cell number per gram wet yeast recovered were determined by suspending 1 g yeast in a sterile standard buffer (pH 4.5, 0.1 M sodium acetate containing 5% v/v ethanol) with 10 mM EDTA and counted with a hemacytometer counting chamber according to the American Society of Brewing Chemist (ASBC) method.2 The yeast cells collected were then pitched into wort at 1.3107 cells/mL. Aliquots (250 mL) of worts were evenly dispersed into glass cylindrical fermenters (1.22 m in height and 18.5 mm in inner diameter). A total of four independent static fermentations were undertaken at 17oC. Yeast harvest and wash - The basic yeast wash procedure was modified from the method of DHautcourt and Smart.11 After fermenting for a certain time, yeast cells were harvested, washed and centrifuged (500g for 2 min) in distilled water for three times. The yeast cells were then suspended in the standard buffer with 10 mM EDTA. Measurement of yeast cell size - Yeast samples were examined using a Nikon Alphaphot-2 microscope (Nikon Corp., Tokyo, JPN) at a 400 magnification. The system was calibrated using a stage reticle. The major and minor axes of 60 cells were measured randomly by an eyepiece micrometer. The volume of a single yeast cell (V) was calculated based on the assumption that the yeast cell generally conforms to the shape of a prolate ellipsoid according to the following equation: 4 (2) V = ab 2 3 where a is half of the major axis and b is half of the minor axis of yeast cells. Yeast flocculation assay - Yeast flocculation was measured by Helms method modified by Jin and Speers15 from the ASBC standard method.3 CO2 evolution rate measurement - A U-shaped glass tube (5 mm in diameter and 2 mL in volume) was connected to the fermentor for measurement of CO2 evolution rate (CER). A 0.2 mL aliquot of 1% sodium dodecyl sulfate was added to the test tube to produce bubbles during CO2 evolution. After 3 min of equilibrium, the time that a meniscus of bubble to flow through 0.5 mL in the tube was recorded. This measurement was carried out for five consecutive times at the fermentation temperature of 17oC. Determination of orthokinetic flocculation - The orthokinetic capture coefficient (0) determination procedure was adapted from the method used by Speers et al.32 with the following modifications. A total of 1.0107 cells/mL of washed yeast cells were suspended in a degassed, fermenting wort taken from the fermenter at set fermentation time. As well, 1.0107 cells/mL of washed yeast were suspended in the standard buffer containing 1.0 mM CaCl2. The deflocculated 3

yeast suspensions were loaded onto the flat plate of a Bohlin VOR rheometer (Bohlin Instruments, Cranbury, NJ) equipped with a 5o, 3.0 cm diameter cone. All samples were sheared at 20C and a range of 9.27 to 23.3 s-1 to simulate the shear rate estimated by CER values at each fermentation time. At set periods (0, 5, 10, 15, 20, 25 and 30 min), a small portion of the samples was carefully transferred to a hemacytometer and the floc density (Nt) was counted. The 0 was calculated according to equation 1. The cell volume fraction (o) of yeast suspensions was calculated as the product of the cell concentration (cells per mL) times the average volume of the yeast cells (mL per cell) at each fermentation time. Determination of zymolectin binding sites - The zymolectin binding site assay was modified from Hsu et al.13 Stock solutions of 0 to 200 g/mL of an FITC-avidin fluorescent probe (Molecular Probes, Inc., Eugene, OR) were prepared in the standard buffer. A 100 L aliquot of the stock solutions was added into 4-sided clear cuvettes (Fisher Scientific, Whitby, ON) with 1500 L yeast suspension (2.0106 cells/ml) in the standard buffer. Then 1500 L of the standard buffer with 2.1 mM CaCl2 was added. The suspension was vortexed for 10s and centrifuged (Centra-MP4R, IEC, Needham Heights, MA) at 1240g (3000 rpm) for 3 min. Fluorescence intensity measurements of the supernatant was performed at an excitation wavelength of 495 nm and an emission wavelength of 520 nm with 10 nm slit widths with a spectrophotofluorimeter (LS 50, Perkin-Elmer Ltd., Beaconsfield, GBR). A standard curve was measured at the same day with the same procedure. The non-specific binding of probe (background) was determined with 10mM EDTA in the standard buffer. Langmuir analysis of the binding data was used to derive the density of zymolectin binding sites. Cell surface hydrophobicity assay - The determination of cell surface hydrophobicity (CSH) was based on the method of Jin et al.17 modified from Akiyama-Jibiki et al.1 A hydrophobic resin (phenyl-Separose CL-4B, Sigma Chemicals Co., St. Louis, MO) was used to determine CSH.

Results Fermentation attenuation - As can be observed in Figure 1, the change in apparent extract (AE) (as measured in degrees Plato) with fermentation time was described by a sigmoid-shaped logistic function (smooth line) as recently reported by Speers et al.: 34 PD Pt = + P ( B ( t M )) 1+ e (3) where PD represents the change in the Plato value during the fermentation (i.e., P0 - P). Pt, P0 and P are the Plato values at time t, the initial Plato value and the Plato value at equilibrium time, respectively. B is the specific consumption rate at time M, the midpoint or inflection of the curve. The data from four independent fermentations was pooled and equation 3 was fit to the data using the non-linear regression model (Systat 10.1, SPSS Inc., Chicago, IL). The parameters estimated by this model are shown in Table I. Among the major advantages of using this nonlinear modeling technique, one is that it can be used to monitor the fermentation attenuation before taking actions once the fermentation shows evidence of a serious process deviation.34 Corrected R2

Parameter

Estimated

Asymptotic Standard Error

Value PD B M P 9.87 -0.09 27.06 3.21

(ASE) 0.32 0.00 0.98 0.08 1.00 1.00 1.00 1.00

Table I. Variation of logistic model parameters. N.B. The total number of points of each fit represents a minimum of 30. All regressions were significant (P< 0.001). All four fermentation trials showed similar fermentation rates, and the resulting beers all fermented to an end AE that was rather low and very similar (Figure 1, scattered points). This result confirms that adequate and consistent lab-scale fermentations can be achieved.

14

12

Apparent extract (oP)

10

2 0 20 40 60 80 100 120 140

Fermentation time (h)

Figure 1. Change in apparent extract with fermentation time. Symbols ( independent fermentations.

) represent four

Evaluation of yeast cell size The change in yeast cell volume during fermentation is presented in Figure 2. This plot depicted is known as a Box plot as reported by Tukey.44 The horizontal 5

bars indicated by solid and dash-dot lines in this plot represent the median and mean cell volumes, respectively. The upper and lower ends of the box (or hinges, UH and LH, respectively) represent the medians of the upper and the lower subgroups of the data as split by the median value. A high fence (HF) and low fence (LF) beyond which possible outliers lie are defined as: HF = UH + 1.5 (UH LH) (4a) LF= LH 1.5 (UH LH) (4b) Data points that lie outside of the fences (outliers) are denoted by a solid dot. The vertical line in the box represents the range of data points, which lie within the inner fence.

500

Yeast cell volume (m3)

400

300

200

100

12

24

36

48

60

72

84

96

108

Fermentation time (h)

Figure 2. Range of yeast cell sizes in suspension at different fermentation times. A statistical test for non-normality of the data the Kolmgorov-Smirnov test was performed on each cell size dataset. In most cases (except 36 h and 72 h), the test indicated that the data were non-normally distributed (p<0.05). This finding is not surprising given the occurrence of both large numbers of mother cells and daughter cells. Examination of the change in cell volume with fermentation indicated a complex pattern. Initially the individual cell volume decreased at 12 h presumably due to cell budding. As fermentation proceeded, the individual cell volume increased up to a maximum at 60 h. This second drop in individual cell volume may be due to the onset of flocculation. This observation agrees with Cahill et al.7 The size of brewing yeast previously has been related to cell age whereby young cells are small and get progressively larger as they age.4 Also in senescence studies,5,14 yeast cells are subject to morphological, metabolic and genetic modifications. Such modifications include an increase in size6 and alterations to the shape and surface appearance of the cell.22 While it is not suggested that individual cell size alone controls the cells flocculation ability, one would expect a relationship between cell volume fraction and flocculation behavior.15 The mean cell volume at 6

different fermentation times is also required to calculate the orthokinetic capture coefficient (as discussed later). Evaluation of yeast flocculence during fermentation - It has been reported that flocculent brewing yeast cells, mostly NewFlo strains, lose their ability to flocculate in the early stage of growth and regain it at the late exponential or stationary phases.10,28 The flocculation ability of NewFlo strain LCC125 was examined over the 120-h fermentation. A rapid increase in flocculence as measured by the modified Helms method was observed in the beginning of fermentation (p<0.01) (Figure 3).

100 98

Helm's flocculence (%)

96 94 92 90 88 86 84 82
0 6 12 24 36 48 60 72 96 108 120

Fermentation time (h)

Figure 3. Comparison of flocculation ability of LCC125 at different fermentation times.

Thereafter all samples showed highly flocculent behavior as measured by the Helms test (>85%). This result agrees with the previous report by Jin and Speers.15 Although the diminished flocculence at an early stage of fermentation can be partly explained by the low cell concentration in suspension,15 the effect of decreased cell size due to yeast budding and division (Figure 2) cannot be excluded. It has been generally accepted that aged cells have an increased tendency to flocculate, whereas new cells have lower flocculation ability than older budded cells.5,26 As can be observed in Figure 4A, the floc size in fermentation suspension increased from 12 h to a maximum at 48 h. As fermentation attenuation reached 90% at 60 h (data not shown), the suspended yeast cells sedimented at a very rapid rate, leaving few yeast cells in suspension at the end of fermentation (Figure 4B).

Cells in suspension (x107/mL)

12 10 8 6 4 2 0
(A)

7 Floc size (cells/floc) 6 (B) 5 4 3 2 1 0 0 12 24 36 48 60 72 84 96 Fermentation time (h)

Figure 4. Change in (A) cells and (B) average floc size in suspension during fermentation. Changes of yeast cell surface hyhrophobicity during fermentation - Cell surface hydrophobicity (CSH) has been reported as one of the contributors to flocculation of yeast.1,17,25,27,39-41 In order to examine CSH, the suspended yeast cells at different fermentation times were harvested, washed, counted, and then tested for their CSH in the standard buffer. CSH was expressed as a percentage of cells adhering to the phenyl-Sepharose CL-4B column. As can be observed in Figure 5, the CSH of yeast (LCC125) increased rapidly in the exponential phase of fermentation but readily reached high and stable levels in the stationary phase.

100

Hydrophobicity (%)

95

90

85

80

75 0 6 12 24 48 72 96 120

Ferm entation tim e(h)

Figure 5. Comparison of cell surface hydrophobicity at different fermentation times. The reduced CSH of yeast cells in the exponential phase can be explained by the occurrence of large numbers of daughter cells or virgin cells that are reported to be significantly less hydrophobic than their older counterparts.24 During fermentation, most of CSH values for the NewFlo strain LCC125 were greater than 90%. The Helms flocculation ability (%) of the yeast strain was found to positively correlate with CSH (r=0.52, p<0.05). The variation of CSH partially explains the change in flocculation ability of yeast cells during fermentation. This result supports the previous reports on relationships of CSH and flocculent strains.1,17

Change of mean shear rate during fermentation - Investigations have shown that the shear force is required for flocculation process.36 Flocculation has been observed in many situations, noticeably breweries, where considerable agitation is generated by CO2 evolution. It has become apparent to brewing researchers in recent years that one should consider this agitation caused by CO2 evolution during fermentation when considering the flocculation process. Unfortunately, until recently, little information has been available in the brewing literature on this topic. However, it is possible to evaluate the average shear rate in turbulent flow assuming the energy generating the turbulence is merely due to CO2.9,30,33 The values of agitation power, mean shear rate, and Kolmogoroff scale dimension were thus calculated and presented in Table II. These shear rate calculations were valid since the Kolmogoroff scale dimension is much larger than the cell and floc diameter.

Time (h) 12 24 36 48 60 10

Fermenter Volume (10-4m3) 2.50 2.50 2.50 2.50 2.50

Liquid Height (Hf, m) 0.88 0.88 0.88 0.88 0.88

Agitation Power Mean Shear Kolmogoroff -4 Rate (s-1) Scale (10 W) Dimension (m) 1.00 1.60 1.02 0.63 0.22 16.24 20.89 16.82 13.34 7.96 299.49 260.67 289.12 323.41 417.99

Table II. Mean shear rates due to CO2 evolution. As can be observed in Figure 6, there was a rapid increase in the fermenter shear rate from 12 h to 24 h (p<0.001). The highest value of 21 s-1 was reached after 24 h of fermentation, indicating a maximum rate of fermentation. After 24 h, the shear rate in fermenter declined sharply to below 8 s-1 at 60 h, when most of the yeast cells/flocs sedimented (Figure 4). A shear rate of 80 s-1 has been reported as the critical shear rate to breakup doublet yeast cells.32 Thus, the shear rates presented here indicate that agitation within the fermenter may not be sufficient to maintain dispersed single cells.

Figure 6. Change in shear rates calculated by CO2 evolution during fermentation.

25

Shear rate by CO2 evolution (s-1)

20

15

10

0 0 12 24 36 48 60 72

Fermentation time (h)

10

A linear regression showed that the suspended yeast flocs in suspension were positively correlated with the mean shear rate (Figure 7, r =0.96, p<0.001). Thus, the agitation or shear force caused by CO2 evolution during fermentation is a very important factor in both facilitating the formation of yeast flocs and holding the suspended flocs.
Flocs in suspension ( 10 7flocs/mL)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 5 10 15 20 25 Mean shear rate (s-1)

Figure 7. Yeast flocs in suspension as a function of mean fermenter shear rate. Zymolectin densities of yeast cells - Since being first reported by Miki et al. in the early 1980s,20,21 the lectin-carbohydrate bonding theory has been accepted as an important mechanism causing yeast flocculation. It was later summarized that specific surface proteins known as zymolectins present on flocculent yeast cells bind to mannose residues of mannan molecules on neighboring cell surfaces.35 Calcium ions are required to maintain a correct conformation of the zymolectin binding sites.16 As the mannose residues (ligands) are always present in the cell walls of both nonflocculent and flocculent cells, a critical factor for flocculation is clearly the presence or absence of the zymolectins. Quantitative determination of zymolectins has been attempted to explain yeast flocculation.13,17,19,23 As can be observed in Figure 8, the zymolectin densities on yeast cells were not significantly affected by fermentation time (p>0.05).

11

Zymolectin density (106 sites/cell)

3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 12 24 36 48 60 72 84 96

Fermenation time (h)

Figure 8. Change in zymolectin density with fermentation time.

It is interesting to note that, while the zymolectin level of the cell surface did not change substantially, the Helm flocculation value increased to near 100% in the first 24 h (Figure 3). Presumably the initial flocculence increase (a cell wall property) is due to a noticeable increase in hydrophobicity as observed in Figure 5. However, changes in these wall properties cannot solely be used to explain yeast flocculation behavior in a fermenter since environmental factors such as shear force and nutrients may play important role in this intriguing phenomenon. Interestingly, a significant but weak positive correlation was observed between yeast cell surface area and zymolectin densities (r=0.31, p<0.05, Figure 9). This result indicates old cells, due to their bigger size and higher levels of zymolectins, should be more flocculent as has been reported previously.24,25

12

4.0 3.5
Zymolectin density (106 sites/cell)

3.0 2.5 2.0 1.5 1.0 0.5 0.0 10 20 30 40

2

50

60

Cell surface area (m )

Figure 9. Correlation of cell surface area and zymolectin density. Variation of the orthokinetic capture coefficient - The orthokinetic flocculation due to fluid flow is accepted to be the major mechanism for yeast cell-cell interaction.13,16,32,36 The 0 in equation 1 is the most important term as it reflects the tendency of yeast cells to bind after they collide in a given environment. Determining the 0 value can help us understand the flocculation behavior of yeast cells in shearing flow field. As it can be observed in Figure 10, the change in 0 values with fermentation time, measured in degassed, fermenting worts, indicated a significant increase (p<0.001) after 24 h of fermentation. It is suggested that, with the depletion of nutrients, the flocculation tendency readily increased.

13

0.6 Capture coefficient 0.5 0.4 0.3 0.2 0.1 0.0

(A)

25

Shear rate tested (s )

20 15 10 5 0 0 6 12 24 48 72 96

(B)

-1

120

Fermentation time (h)

Figure 10. Changes in capture coefficients with fermentation time (A) as a function of fermenter shear rate (B): () fermenting wort; () 0.1 M sodium acetate buffer containing 1 mM Ca2+. This result supports the observation in Figure 4 that maximum floc size was formed in fermenter after 48 h of fermentation. No significant difference (p>0.05) of 0 values (measured in fermenting worts) between 0 and 48 h was found. Although flocs in fermenting worts were observed in laminar shear field before 48 h, the bond strength of flocs was found weak and readily dispersed. However, when the same samples were sheared in standard buffer (containing 1.0 mM CaCl2), the 0 values were significantly higher (p<0.001). Flocs were formed tightly and not readily dispersed in laminar shear field. As the flocculation ability and zymoletcin densities of yeast cells did not change substantially throughout the fermentation, it is suggested that the diminished floc bond strength, mainly formed by zymolectin bonding, were probably affected by environmental factors, such as higher sugar concentration, amino nitrogen level, pH value, ethanol concentration etcetera. Fermentable sugars (glucose, maltose and galactose) were reported to induce progressive loss of flocculation of a NewFlo type strain while the other nutrients (nitrogen and sulphur sources or other minor nutrients) were unable to do this.29 The incubation of yeast cells in 4% (v/v) ethanol did not induce a flocculation loss.29 Generally, pH value is considered to be a minor factor under brewing conditions. Yeast cells may flocculate within the wide range of pH 2 to pH 8.8 However, it has been shown that yeast flocculation is optimal in slightly acid conditions, with pH values ranging from 4.5 to 5.8.15 Further investigation on the effect of pH indicated no significant (p>0.05) difference of 0 values existed 14

between pH 5.2 and 3.7 (data not shown). Although the molecular mechanism that leads to the flocculation loss, induced by specific sugars, such as mannose and glucose etc., is still not known, the loss of flocculation of LCC125 caused by fermentable sugars can be easily explained by the zymolectin hypothesis. It is proposed that fermentable sugars may block the zymolectin binding sites and thus inhibit flocculation. It is proposed that fermentable sugar level (especially glucose and maltose) in wort should be considered as one major factor affecting brewing yeast flocculation. Discussion Previously, researchers have assumed that flocculation was primarily caused by zymolectins and, that since flocculation begins in the stationary phase then zymolectins must be somehow activated at this phase of growth. However, no verification of this theory has been ever demonstrated. In this work, the level of zymolectins on the cell wall appear to be more or less constant during the fermentation. This finding is supported by work of Carlton and United Breweries, (Melbourne, AUS, i,e,. Fosters) who noted no change in FLO1 expression during fermentation (Personal communication, P. Rogers, 2005). Thus, yeast cells appear to reach maximum flocculation potential after the increase in cell wall hydrophobicity occurs at 24 h (Figure 5). From this point on, yeast cells are able to flocculate and are prevented from doing so by the presence of sugars and turbulent flow in the (still fermenting) wort. Acknowledgement A discovery grant awarded by the Natural Sciences and Engineering Research Council of Canada to RAS is acknowledged. References 1. Akiyama-Jibiki M., Ishibiki, T., Yamashita, H. and Eto, M., A rapid and simple assay to measure flocculation in brewers yeast. MBAA Tech. Quar., 1997, 34(1), 278-281. 2. American Society of Brewing Chemists., Yeast-4. Microscopic yeast cell counting. In: Methods of Analysis, 8th ed., American Society of Brewing Chemists: St. Paul, MN. 1992. 3. American Society of Brewing Chemists., Yeast flocculation by absorbance method. J. Am. Soc. Brew. Chem., 1996, 54(4), 245-248. 4. Autriaco, N.R., Review, To bud until death: The genetics of aging in the yeast Saccharomyces. Yeast, 1996, 12(7), 623-630. 5. Barker, M.G. and Smart, K.A., Morphological changes associated with the cellular aging of a brewing yeast strain. J. Am. Soc. Brew. Chem., 1996, 54(2), 121-126. 6. Bartholomew, J.W. and Mittwer, T., Demonstration of yeast bud scars with the electron microscope. J. Bacteriol., 1953, 65(3), 272-275. 7. Cahill, G., Walsh, P.K. and Donnelly, D., Improved control of brewery yeast pitching using image analysis. J. Am. Soc. Brew. Chem., 1999, 57(2), 72-78. 8. Calleja, G.B., Cell aggregation. In: The Yeast, 2nd ed., A.H. Rose and J.S. Harrison, Eds., Vol. 2, Academic Press: London, GBR, 1987, pp. 165-238. 15

9. Delente, J., Akin, C., Krabbe, E. and Ladenberg, K., Carbon dioxide in fermenting beer. Part II: Physical mechanisms of yeast suspension-mathematical evaluation of natural agitation and fermenter design. MBAA Tech. Quar., 1968, 5, 228-234. 10. Dengis, P.B., Nelissen, L.R. and Rouxhet, P.G., Mechanisms of yeast flocculation: Comparison of top- and bottom-fermenting strains. Appl. Environ. Microbiol. 1995, 61(2), 718-728. 11. D'Hautcourt, O. and Smart K.A., Measurement of brewing yeast flocculation. J. Am. Soc. Brew. Chem., 1999, 57(4), 123-128. 12. Duszyk, M. and Doroszewski J., Poiseuille flow methods for measuring cell-to-cell adhesion. Cell Biophys., 1986, 8(2), 119-131. 13. Hsu, J.W.C., Speers, R.A. and Paulson, A.T., Modeling of orthokinetic flocculation of Saccharomyces cerevisiae. Biophys. Chem., 2001, 94(1/2), 47-58. 14. Jazwinski, S.M., An experimental system for the molecular analysis of the aging process: The budding yeast Saccharomyces cerevisiae. J. Gerontol., 1990, 45(3), B68-B74. 15. Jin, Y-L. and Speers, R.A., Effect of environmental conditions on the flocculation of Saccharomyces cerevisiae. J. Am. Soc. Chem., 2000, 58(3), 108-116. 16. Jin, Y-L. and Speers, R.A., Flocculation of Saccharomyces cerevisiae. Food Res. Int., 1998, 31(6/7), 421-440. 17. Jin, Y-L., Ritcey, L.L., Speers, R.A. and Dolphin, P.J., Effect of cell surface hydrophobicity, charge and zymolectin density on the flocculation of Saccharomyces Cerevisiae. J. Am. Soc. Chem., 2001, 59(1), 1-9. 18. Long, M., Goldsmith, H.L., Tees, D.F.J. and Zhu C., Probabilistic modeling of shearinduced formation and breakage of doublets cross-linked by receptorligand bonds, J. Biophys., 1999, 76(2), 1112-1128. 19. Masy, C.L., Henquinet, A. and Mestdagh, M.M., Fluorescence study of lectin-like receptors involved in the flocculation of the yeast Saccharomyces cerevisiae. Can. J. Microbiol., 1992, 38(5), 405-409. 20. Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L., Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae. J. Bacteriol., 1982, 150(2), 878-889. 21. Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L., Repression and induction of flocculation interactions in Saccharomyces cerevisiae. J. Bacteriol., 1982, 150(2), 890899. 22. Mortimer, R.K. and Johnston, J.R., Life span of individual yeast cells. Nature, 1959, 183(4677), 1751-1752. 23. Patelakis, S.J.J., Ritcey, L.L. and Speers, R.A., Density of lectin-like receptors in the FLO1 phenotype of Saccharomyces cerevisiae. Appl. Microbiol., 1998, 26(4), 279-282.

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24. Powell, C.D., Quain, D.E. and Smart, K.A., The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation. FEMS Yeast Res., 2003, 3(2), 149-157. 25. Rhymes, M.R. and Smart, K.A., The relationship between flocculation and cell surface physical properties in a FLO1 ale yeast. In: Brewing Yeast Fermentation Performance, K.A. Smart, Ed., Blackwell Science: Oxford, GBR, 2000, pp. 152-159. 26. Smart, K.A., Aging in brewing yeast. Brew. Guard., 1999, 128(2), 19-24. 27. Smit, G., Straver, M.H., Lugtenberg, B.J.J. and Kijne, J.W., Flocculence of Saccharomyces cerevisiae cells is induced by nutrient limitation, with cell surface hydrophobicity as a major determinant. Appl. Environ. Microbiol., 1992, 58(11), 37093714. 28. Soares, E.V. and Mota, M., Flocculation onset, growth phase, and genealogical age in Saccharomyces cerevisiae. Can. J. Microbiol., 1996, 42(6), 539-547. 29. Soares, E.V. and Vroman, A., Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae. J. Appl. Micro., 2003, 95(5), 325-330. 30. Speers, R.A. and Ritcey, L.L., Towards an ideal flocculation assay. J. Am. Soc. Brew. Chem., 1995, 53(4), 174-177. 31. Speers, R.A., Durance, T.D., Odense, P., Owen, S. and Tung, M.A., Physical properties of commercial brewing yeast suspensions. J. Inst. Brew., 1993, 99(2), 159-164. 32. Speers, R.A., Durance, T.D., Tung, M.A. and Tou, J. Colloidal properties of flocculent and nonflocculent brewing yeast suspensions. Biotechnol. Prog., 1993, 9(3), 267-272. 33. Speers, R.A., Patelakis, S.J.J., Paulson, A.T. and Oonsivilai, R., Shear rates in brewing operations. MBAA Tech. Quar., 2004, 14(3), 241-247. 34. Speers, R.A., Rogers, P. and Smith B., Non-linear modeling of brewing fermentations. J. Inst. Brew., 2003, 109(3), 229-235. 35. Speers, R.A., Smart, K. and Stewart, R., Zymolectins in Saccharomyces cerevisiae. J. Inst. Brew., 1998, 104, 298. 36. Speers, R.A., Tung, M.A., Durance, T.D. and Stewart, G.G., Colloidal aspects of yeast flocculation: a review. J. Inst. Brew., 1992, 98(6), 525-531. 37. Stewart R. J., Russell I. and Stewart G.G., Characterization of affinity-purified cell wallbinding proteins of Saccharomyces cerevisiae: Possible role in flocculation. J. Am. Soc. Brew. Chem., 1995, 53(3), 111-116. 38. Stewart, G.G. and Russell, I., Yeast flocculation. In: Brewing Science, Pollock, J. R.A., Ed., Academic Press: Toronto, ON, CAN, 1981, pp. 61-92. 39. Straver, M.H. and Kijne, J.W., A rapid and selective assay for measuring cell surface hydrophobicity of brewers yeast cells. Yeast, 1996, 12(3), 207-213. 40. Straver, M.H., Kijne, J.W. and Smit, G., Cause and control of flocculation in yeast. Trends in Biotechnol. 1993, 11(6), 228-232. 17

41. Straver, M.H., Traas, V.M., Smit, G. and Kijne, J.W., Isolation and partial purification of mannose-specific agglutinin from brewers yeast involved in flocculation. Yeast, 1994, 10(9), 1183-1193. 42. van de Van, T.G.M. and Mason, S.G., The microrheology of colloidal dispersions. VII. Orthokinetic doublet formation of spheres. Colloid Polymer Sci., 1977, 255, 468-479. 43. van Uden, N. and Sousa, L.D.C., Yeasts from the bovine caecum. J. Gen. Microbiol., 1957, 16(2), 385-395. 44. Zar, J.H., Biostatistical Analysis. Prentice-Hall Inc.: Englewood Cliffs, NJ. 1974.

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