Disc Stack Centrifuge Operating Parameters

and Their Impact on Yeast Physiology

Paul H. Chlup1, Dominic Bernard1 and Graham G. Stewart1,2

ABSTRACT • reducing the yeast quantity from green beer before

the start of secondary fermentation
J. Inst. Brew. 114(1), 45–61, 2008
• beer recovery from cropped yeast
Hydrodynamic stresses imposed on brewing cells of Saccharo- • separation of the hot break after wort boiling
myces cerevisiae during beer processing can have a detrimental • removal of cold break and yeast at the end of
impact on beer quality. The use of centrifuges has become an
maturation.
efficient way to increase brewery throughput as they decrease
clarification times and improve fermenter and tank conditioning Yeast management and handling systems are influen-
efficiency. The effect of a disc stack centrifuge on yeast and beer tial in determining the physiological status of yeast, sub-
physical stability has been investigated. In this study, a com- sequent fermentation performance activity2 and clarifi-
mercial ale yeast strain has been subjected to different operating cation during lagering/maturation32. It has been docu-
conditions during centrifugation. Cell viability and intracellular mented that beer haze can result from the yeast cell wall
pH decreased due to processing conditions encountered during releasing mannan as it is processed during agitation in
yeast cropping with a centrifuge. A relationship has been estab- storage and centrifugation8,19,32,36. These reports suggest
lished that yeast cell wall mannan, an unfilterable haze con- that hydrodynamic stresses have the potential to inflict
stituent, as a function of G-force and centrifugation cycles, is re- damage to the yeast cell wall during beer production and
leased from the cell wall while concurrently, particle sizes be-
consequently increase beer haze. However, intermediate
tween 0.5-2.8 μm and beer haze increased. Furthermore, yeast
intracellular glycogen and trehalose levels were depleted as a haze formation due to agitation, centrifugation and their
result of centrifugation. relevant operating parameters still remain of considerable
interest to brewers. During the brewing process, yeast is
Key words: beer haze, centrifuge, flow cytometry, g-force,
subjected to numerous factors that individually or collec-
glycogen, intracellular pH, stress, trehalose, yeast.
tively impose stress upon yeast cells. Improperly de-
signed, transfer and environmental factors can cause ther-
INTRODUCTION mal, osmotic, ethanol, mechanical and shear stresses on
Economic sustainability procedures have been devel- yeast cells1,20,29,33,34,37. A recent investigation conducted by
Kanzleiter16 found that the implementation of newer mod-
oped and implemented by brewing companies due to in-
creasing environmental regulations, consumer pressure el centrifuges along with piping reconfiguration improved
and to demonstrate corporate responsibility. The incentive green beer quality compared to an older existing centri-
fuge system.
to optimize operating costs, while reducing processing
times, in order to gain a competitive advantage and max- Although the uses of a centrifuge during brewing are
imize revenue, is imperative to brewery survival. Brewers diverse, this study has concentrated on the effect it has on
yeast and particularly on yeast cell wall damage and the
continuously search for ways to exploit production effi-
ciency36. As a consequence, the disc stack centrifuge has resulting impact on beer quality and stability. The aim of
become a popular component of yeast management sys- this project was to better understand and quantify the ef-
fect of passing Saccharomyces cerevisiae brewing cells
tems in order to reduce fermentation, maturation and clar-
ification times and to control effluent treatment costs. through a disc stack centrifuge. In order to confirm that
Modern centrifuges produce forces in excess of 10,000 the deterioration of yeast was from the centrifugation
process, an excessive number of centrifugation cycles op-
times the earth’s gravitational constant, achieving solid
separation in seconds with reduced equipment volume. erating at two differing G-forces (high and low) was em-
Centrifuges have a number of applications in a brewery9 ployed.
The passage of yeast through the centrifuge exposes
and can be used for:
• cropping of non-flocculent yeast cultures at the end cells to mechanical and hydrodynamic shear stresses8,9,32.
of primary fermentation Previous reports have shown2,8,9,17,18,38 that these stresses
can cause a decrease in cell viability and flocculation, cell
wall damage, increased extracellular proteinase A (PrA)
1 InternationalCentre for Brewing and Distilling (ICBD), Heriot- levels, hazier beers and reduced beer foam stability. In
Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland. this study, biological indicators of yeast physiology such
2 Corresponding author. E-mail: [email protected] as viable and damaged cells, intracellular pH, glycogen
and trehalose levels as well as beer physical stability
Publication no. G-2008-0304-554 parameters including mannan residues, particle size, and
© 2008 The Institute of Brewing & Distilling beer haze have been employed to quantify the damage

VOL. 114, NO. 1, 2008 45

which occurs to yeast as a function of cycles through a EXPERIMENTAL
centrifuge operating at high and low G-forces. Due to
centrifugal forces within the centrifuge numerous reac- Wort production in the ICBD pilot plant
tionary forces take place between liquid, solid, and discs All malt wort was produced in the ICBD 2 hL pilot
including shear stress. Along the bowl wall and support- brewery. The malt (Optic) was provided by Pure Malt
ing structures compression and tensile stress are pro- (Haddington, UK) and was stored at 11°C before use. The
duced. The forces acting on the disc stack and particle-to- all malt wort was brewed to a specific gravity of 10°Plato.
particle contact are proportional to the G-force. Thus, The malt was milled with a four hammer ‘Essex Major’
operating conditions such as revolutions per minute (rpm) mill (Christy-Hunt, UK) and mashed at a liquor/grist ratio
and flow rate influence forces within the centrifuge. of 2.5:1. The mashing temperature was maintained at
The flow cytometer is a powerful instrument capable 65°C for 1 h and the temperature was raised to 74°C for
of determining the physiological status of S. cerevisiae mashing off.
and aspects of beer stability in near real time11. Flow cy- A Meura 2001 42 kg capacity pilot mash filter (Meura,
tometry possesses technology that performs simultaneous Belgium) was used for mash separation. The wort was
multiparametic analyses of yeast physical and chemical collected in the kettle and boiled for 1 h achieving 8%
characteristics based on cell size, relative granularity and evaporation. Pride of Ringwood hop pellets were used to
fluorescence. The particles are transported in a fluid provide a beer of 16 IBU’s.
stream to the interrogation point. The extent to which the
particle scatters light is dependent on the surface topog- Yeast centrifugation
raphy and internal complexity. These characteristics are The yeast culture employed was an ale yeast strain do-
determined using a complex optical-to-electronic coupling nated by a local commercial brewery. A12°P wort fermen-
system that records the particle’s ability to scatter incident tation (2 hL), pitched with 1.2 × 107 cells/mL and a con-
laser light and the emission of fluorescence15,21,23. The centration of 12 ppm dissolved oxygen, was conducted in
cells that scatter light are defined by a gate, a numerical or order to have sufficient yeast biomass for passage through
graphical boundary, the peak mean (Pm) is the relative the centrifuge. Two separate regimes through the centri-
fluorescence intensity expressed as the x-mean channel. fuge were conducted, using an ale yeast strain, at differing
The differentiation of subpopulations, based on fluores- G-forces. Yeast slurry concentrations of 2.5 – 3.0 × 107
cence intensity, permits the classification of stressed and cells/mL (2 hL) were cycled through a Westfalia Separator
non-stressed11 populations of brewing yeasts. (Oelde, Germany) SC6-06-076 at the manufacturer’s
Flow cytometry methods were developed to measure suggested flow rate of 5-6 hL/h for this model. The disc
cell viability, damaged cells, intracellular pH (pHi), man- stack height is 150 mm and consists of 76 discs with a
nan residues and Pm of intracellular glycogen and tre- half-cone cone angle of 40°; each disc has an inner radius
halose11,12. Flow cytometry and fluorescent dyes provide a of 31 mm and an outer radius of 62 mm. The gap between
rapid and accurate means to quantify the viability and vi- the discs is 0.5 mm. The centrifuge bowl speed operated
tality of yeast during centrifugation cycles at differing G- at 8,000 and 12,000 rpm with an angular velocity of 1047
forces and throughout fermentation. The viability method and 1256 rad/s, which produces 8,900 (low) and 20,000G
developed distinguishes between the dead, alive and dam- (high), respectively, on the outer radius of the disc. The
aged cells. The probes propidium iodide (PI) and fluores- inlet pressure of the centrifuge was maintained at 0.1 MPa
cein diacetate (FDA) have been used as markers to deter- and the outlet pressure ranged between 0.3-0.4 MPa. Dur-
mine functioning cells. The vitality of the cells was deter- ing processing, the temperature of the beer was main-
mined by intracellular pH which employed the pH- tained via the centrifuge’s heat exchanger. The inlet
dependent fluorescent probe carboxy SNARF-4F temperature of the heat exchanger was 4°C and the outlet
(SNARF-4F). The ester form of this probe permeates the temperature was 5°C.
cell membrane and once inside the cell, it is cleaved by The yeast was collected by conducting partial dis-
esterases to release a pH-sensitive polyanionic probe charges of the centrifuge bowl at six minute intervals
which fluoresces44. SNARF-4F possesses two inversely during centrifuging and then maintained at 4°C in the col-
related emission signals at two different wavelengths, lection vessel. During preliminary trials it was determined
which makes it possible to calculate the pH from the ratio that discharging more frequently would dilute the centri-
between the Pm measured at the two wavelengths44. Yeast fuged yeast slurry significantly (process water is used to
macromolecular components such as intracellular glyco- flush the bowl). This would make it impractical to re-pitch
gen and trehalose, stained with Schiff reagent and the yeast into the 2 L fermentations. A manual discharge
concanavalin A (ConA) have been assayed by flow was completed after the centrifugation cycle was com-
cytometry. In addition, a flow cytometric method has pleted. At different operating G-force, yeast samples were
been developed to detect mannan, an unfilterable haze collected, passing through the centrifuge for a total of 9
constituent, released from the yeast cell wall due to cycles. Yeast slurry (1 L) was collected at the appropriate
hydrodynamic stresses during processing3. Flow cycle and the cell viability, damaged cells, intracellular
cytometry provides an innovative, rapid and viable pH, intracellular glycogen and trehalose, mannan, beer
option for brewers to evaluate yeast cells throughout the haze and particle size were determined. For fermentation
fermentation cycle and the influence that beer pro- trials, the yeast exposed to the high G-force was re-cycled
cessing equipment and operating conditions, in through the centrifuge, to a maximum of 15 cycles, and
particular the disc stack centrifuge, have on beer sta- subsequently pitched into the appropriate tank. Samples
bility and yeast quality. (700 mL) from the ale yeast were collected at 0 cycle

46 JOURNAL OF THE INSTITUTE OF BREWING

(control), 7 and 15 cycles for fermentation trials. These yeast population, acquired in log 3 mode, enabled on for-
yeast slurries were stored overnight at 4°C prior to re- ward scatter and thresholds set so that cell debris was ex-
pitching into 10°P wort. cluded from data acquisition. The fluorescence measured
on FL2 (590 nm) and FL3 (680 nm) parameters were ac-
Wort fermentation quired in log 4.
Fermentations were carried out in triplicate in 2 L Alcohol fixation
graduated cylinders. The pitching rate was adjusted to
give a viable cell count of approximately 1.0 × 107 The alcoholic fixation enables the permeation of large
cells/mL. Dissolved oxygen concentrations of 10-12 ppm dye molecules through the cell membrane. The mem-
were introduced into the wort in each 2 L graduated cylin- branes of living cells are able to selectively mediate the
der. The fermentation temperature was maintained con- passage of molecules. Dead cells lack this regulation
stant at 21°C. For glycogen and trehalose determinations, function and easily allow substances like large fluores-
2 mL of fermenting wort was taken by pipetting from the cence molecules to enter the cell. The surface structure of
mid point of the graduated cylinder (800 mL mark). Intra- the cells is deformed by means of water removal. The de-
cellular glycogen and trehalose were measured throughout naturation does not alter the composition of protein com-
the course of fermentation. pounds and many intracellular macromolecules such as
DNA and glycogen are preserved during the alcoholic fix-
Viability measurements using the ation. Samples of cell suspensions were collected from
Partec CyFlow SL flow cytometer the fermentation vessels and were immediately immersed
Yeast cell viability was measured by the method de- in 10 mL cold 70% (v/v) ethanol and incubated for at least
scribed by Chlup et al.2 Flow cytometric analysis was 24 h at 4°C. Cells treated in this manner can be stored at
4°C for up to one month prior to analysis.
performed on the CyFlow SL flow cytometer (manufac-
tured by Partec GmbH, Münster). Data analysis was Intracelluar glycogen measurements using
performed afterwards using the FloMax software version the Partec CyFlow SL flow cytometer
2.4e provided by Partec.
Glycogen staining was carried out using acriflavine ac-
Intracellular pH measurements using the cording to the procedures of Gharton et al.6 and Hutter11
Partec CyFlow SL flow cytometer with minor modifications. The Schiff reagent was pre-
A slightly modified version of the method for assessing pared as follows, diluted HCl solution (1:10) was pre-
intracellular pH in S. cerevisiae cells developed by Valli et pared with distilled water and 15 mL of this dilute acid
al.44 was used. This method measures the spectral proper- was used to dissolve 0.5 g of acriflavine (Sigma, UK).
ties of the probe SNARF-4F (Invitrogen, UK). A 5 mM K2S2O5 (0.5 g) was dissolved in 85 mL distilled H2O. This
stock SNARF-4F solution was prepared from anhydrous K2S2O5 solution was then added to the acriflavine solu-
DMSO. The loading buffer (20µM) was made by quanti- tion. After 24 h, 0.3 g charcoal was added and filtered.
tatively transferring the stock solution with pH 3.0 Sample aliquots (2 mL) of alcohol fixed (70% v/v
McIlvaine buffer. Since SNARF-4F is very susceptible to EtOH) yeast suspensions were added to 4 mL PBS buffer.
hydrolysis, the stock solution was used immediately for The yeast suspensions were centrifuged for 10 min at
sample preparation. For every measurement, the appropri- 4,000G (10°C). The supernatants were discarded and the
ately diluted yeast slurry (< 1000 events/s) sample of 1 washing step repeated. The resulting pellets were re-sus-
mL was collected and centrifuged at 8,000G for 1 min and pended in 1 mL periodic acid solution (5 mg/mL of H2O),
re-suspended in 250 µL of loading buffer. After incubation vortexed for 10 sec and incubated for 10 min at 20°C.
at 28°C for 11 min on a shaker, the cells were collected by Samples were then centrifuged at 4,000G for 10 min. The
centrifuging at 8,000G for 1 min and re-suspended in pellets were re-suspended in 4 mL PBS buffer, centri-
1000 µL of pH 3.0 McIlvaine buffer. The samples were fuged at 4,000G for 10 min and re-suspended in a reaction
placed on ice and analysed by flow cytometry. Each chamber with 55 μg/mL Schiff reagent. The suspensions
aforementioned sample was measured 3 times. In situ cal- were incubated for 1 h at 20°C in the dark. After the incu-
ibration must be completed for each experiment as pHi bation period, the samples were centrifuged at 4,000G for
varies depending on the growth phase of the cells44. Cell 10 min (10°C) and the resulting pellets were re-suspended
samples were collected and loaded as previously de- in 4 mL PBS buffer. This washing step was repeated and
scribed. After reaction with the loading buffer, each sam- the pellets re-suspended in a final volume of 2 mL in PBS.
ple pellet was re-suspended in 500 µL of McIlvaine buffer The probe was excited using the solid state laser at 488
of varying pH. After addition of 1.5 µL of 9.7 mM am- nm. All data were acquired in log 4 mode and threshold
photericin B, to final concentration of 30 µM SNARF, the settings were enabled on forward scatter so that cell
cells were incubated at 37°C for 1 h on a shaker and then debris was excluded from data acquisition. A total of
analysed by flow cytometry. The sample volumes were 30,000 (centrifugation cycles) and 45,000 (fermentations)
adjusted to a final volume of 1000 μL prior to analysis events were recorded for analysis. The forward scatter
using the appropriate buffer. All solutions are light sensi- (FSC) verse side scatter (SSC) dot plot was used to identi-
tive therefore care must be taken to minimise light expo- fy and gate the yeast population. The fluorescence was
sure. All samples and solutions were kept in the dark and measured on FL1 (520 nm) parameter. Each aforemen-
on ice while awaiting analysis. The forward scatter and tioned sample was measured 3 times. Data analysis was
side scatter dot plot was used to identify and gate the performed afterwards using the FloMax software.

VOL. 114, NO. 1, 2008 47

Intracellular trehalose measurements Visualization of non-centrifuged and
by the Partec CyFlow SL flow cytometer centrifuged yeast by Environment Scanning
The intracellular trehalose content was stained accord- Electron Microscope (ESEM)
ing to the method of Hutter et al.12 that utilises the lectin- Fifty mL aliquots of the appropriate sample were cen-
fluorochrome-conjugate ConA (Invitrogen, USA). Sample trifuged at 4°C (730G) for 10 min. The resulting pellet
aliquots (2 mL) of alcohol fixed (70% v/v EtOH) yeast was frozen at -40°C and then dried for 48 h. The freeze
suspensions were added to 4 mL PBS. The yeast suspen- dried yeast was coated with gold-palladium and subse-
sions were centrifuged for 10 min at 4,000G (20°C). The quently reconstituted with water before analysis. Obser-
supernatants were discarded and the washing step repeat- vations were conducted by a Phillips FEI XL30 FEG-
ed. The resulting pellets were re-suspended in 10 μg/mL ESEM. ESEM provides high magnification observation of
ConA (1 mg/mL of PBS). The suspensions were incubat- yeast cells without damaging the constituents. The ESEM
ed for 20 min at 20°C prior to flow cytometric analysis. column was equipped with a multistage differential pres-
All analyses were carried out as previously described for sure pumping unit, a pressure of 1-20 torr was maintained
glycogen staining. A total of 30,000 (centrifugation cy- in the observation chamber.
cles) and 100,000 (fermentations) events were acquired
for each determination. Each sample was measured 3 RESULTS AND DISCUSSION
times.
Cell viability and damage
Mannan residues determined by the Partec
during centrifugation
CyFlow SL flow cytometer
The data obtained from this investigation validated pre-
Mannan residues were measured by the method de- vious reports2,8,9 that yeast cells that have undergone cen-
scribed by Chlup et al.3 trifugation exhibit lower cell viabilities when compared
Mannan and glucose measurements by HPLC with those which had not been centrifuged. This experi-
ment further examined the effects of G-force upon cells as
Mannan and glucose was measured by the method de- a function of centrifugation cycle. In order to establish a
scribed by Chlup et al.3 relationship between centrifugal G-force and effects on
Beer haze the physiological status of yeast, it is necessary for yeast
cells to possess an analogous yeast history and physio-
Samples were centrifuged at 730G and degassed. A logical state. The yeast strain used in the current study
Hach 2100N Turbidimeter (Hach Co., Loveland, CO) was experienced similar fermentation conditions. The yeast
employed for analysis. Each aforesaid sample was meas- cell culture exposed to a high G-force underwent a 72 h
ured 3 times. conditioning at 10°C and the low G-force yeast was
Particle size conditioned for an additional 48 h. Therefore, all cells in
this investigation were under nutrient-limiting conditions
The particle size and its distribution for all samples and were in stationary phase.
were measured with the Malvern Mastersizer 2000 The high and low G-force yeast cultures were cycled
(Worcestershire, UK) utilizing laser diffraction. Samples through the disc stack centrifuge for a total of 9 cycles for
were prepared as described in the mannan residue meth- each G-force. The yeast cell viability was measured be-
od3. Approximately 10 mL of sample was used for each fore centrifugation and after passage through the centri-
measurement. The feed rate was set at 2500 and particle fuge for 3 cycles, 6 cycles, and 9 cycles. The study found,
size distribution was recorded. Each abovementioned as would be expected, that yeast cultures exhibited greater
sample was measured 3 times. decreases in viability at higher G-force compared to cells
at lower G-force. It has been reported20 that yeast cells in
stationary phase develop thicker cell walls than when in
exponential phase and this increase in cell wall thickness
promotes cell membrane protection and could explain the
increased resilience of the yeast subjected to the lower G-
force. The additional 48 h conditioning period of the low
G-force yeast may have partially contributed to it being
more resilient than the high G-force yeast, but most likely
the hydrodynamic forces within the centrifuge at high G-
force resulted in cell damage and thus a decrease in cell
viability (Fig. 1). Cell wall composition and topography
will vary among different yeast strains37. Therefore, it is
possible that this particular ale strain was naturally more
susceptible to hydrodynamic stresses than other yeast
strains.
It was not possible to confirm from these experiments,
where yeast cell damage occurred within the centrifuge.
Fig. 1. Viability and damaged cells as a function of G-force and Three potential areas where yeast cells experience hydro-
centrifugation cycle. dynamic stresses that decrease viability and vitality are:

48 JOURNAL OF THE INSTITUTE OF BREWING

(1) centrifuge inlet, (2) in between disc stack gaps, and (3) Beer haze, mannan and particle size
yeast discharge. Cell death results from passive conse- as a function of G-force
quences and degenerative or active processes46. The for-
mer type of cell death is termed necrosis and the latter The impact on the physical damage to yeast cells and
apoptosis46. Necrosis is a consequence of passive cell consequent physical beer stability is dependent on centri-
injury. Alternatively, apoptosis is driven by a complex fuge operating parameters. Hydrodynamic forces and
process known as programmed cell death. Morphological yeast cell interaction within the gap of the disc stack cre-
and physiological consequences resulting from necrosis ates collisions among the yeast cells producing kinetic en-
differ from apoptosis. The differences mainly concern cell ergy causing cellular damage. Increasing G-force will ex-
shape and cell structural features. pose yeast cells to detrimental effects of hydrodynamic
Flow cytometry viability analysis discriminates be- forces. Release of mannan during mechanical agitation of
tween necrotic and apoptotic cells allowing for quantifica- yeast slurries in conjunction with an increase in beer haze
tion and identification of populations. The viability assay has been previously documented3,19,40. It has been reported
differentiated between dead, living, and damaged cell that beer haze can result from the yeast cell wall releasing
populations stained with FDA and PI, quantifying yeast mannan2,8,35,36 as it is processed during centrifugation. Ad-
sub populations. Cell esterases, capable of hydrolyzing ditionally, several reports have confirmed mannan, which
FDA to fluorescein, indicated living cells, and PI, a nu- is unfilterable from the medium, to be a constituent of
cleic acid stain that permeates compromised membranes, haze material19,35,38. Haze was derived from pieces of
distinguished dead cells from live ones. Damaged cells material released from the yeast wall exterior as cells
stain with both FDA and PI. It was determined that passed through the centrifuge. Moreover, McCourtie and
necrotic and apoptotic cells represent damaged and dead Douglas22 determined that Candida albicans cell wall
cells, respectively. composition and adherence are a function of environmen-
At the end of 9 cycles, with the culture exposed to high tal conditions, which implies that S. cerevisiae is similarly
G-force during centrifugation, viability decreased by prone to damage inflicted by stressful treatment4. ESEM
92.4% and following exposure to low G-force by 79.6% analysis (Figs. 2A and B) provided visual evidence of
(Fig. 1). Additionally, a larger percentage of yeast cells yeast cell damage and the release of cellular wall compo-
subjected to low G-force (37.5% compared with 2.5% for nents as a result of disc stack centrifugation at high G-
high G-force) were associated with damaged cells. The force.
higher proportion of damaged cells of low G-force yeast
culture compared to high G-force implies that these cells
are still able to function and are more resilient to centrifu-
gal forces whereas the low proportion of damaged cells at
high G-force remains constant indicating a permanent loss
of functioning cells. These results suggest that yeast cells
as a function of G-force and centrifuge cycle, experience
a damage cell threshold (Fig. 1). Damage cell threshold,
the transition from the necrotic to the apoptotic cell state,
is encountered when the centrifugation hydrodynamic
stress is sufficient to cause death to the cell. A damaged
cell’s capacity to function or proliferate, after exposure to
hydrodynamic stresses, is not fully understood and re-
quires further investigation.
Both yeast cell cultures demonstrated the same general
trend of a decrease in viability as a function of centrifuga-
tion cycle. At high G-force, the viability decreased by
83% (3 cycle), 47% (6 cycle), 12% (9 cycle) whereas the
low G-force decreased by 41% (3 cycle), 25% (6 cycle),
and 52% (9 cycle). The percentage of damaged cells for
low G-force culture increased by 34% between the control
and cycle 3, then decreased 7% between cycle 3 and 6 and
a further reduction of 7% between cycle 6 and 9 was ob-
served. The percentage of damaged cells for the high G-
force strain was fairly consistent (1 – 2.5%), throughout
centrifugation cycles. These results provide evidence that
the amount of G-force applied to yeast cells during centri-
fugation contributes to a decrease in viability and influ-
ences the damaged cell threshold. However, because only
one yeast strain was studied, it is necessary to conduct
further testing that considers additional yeast strains, their
history and physiological status in order to develop a ho- Fig. 2. (A) Yeast cells before passage through a disc centri-
listic theory of the effects of yeast centrifugation on beer fugation or exposure to high G-forces. (B) Yeast cells following
quality. passage through a disc centrifugation at a high G-force.

VOL. 114, NO. 1, 2008 49

 The impact that G-force has on beer haze and mannan The particle size analysis gave further insight into the
residues has been investigated (Figs. 3 and 4). Beer haze extent of yeast cell damage produced by centrifugation
increased following both high and low G-force, compared (Figs. 5A and B). Particles sizes ranging between 0.02 and
to the control and haze increased 32.9 EBC or 264% (cycle 5.02 μm were measured after the sample had been
3), 25.2 EBC or 202% (cycle 6) and 27.9 EBC or 224% concentrated by ultraspeed centrifugation. Figs. 5A and B
with high G-force. The increase in beer haze at the low G- illustrate the distribution of particle sizes expressed in
force was not as extreme compared to the control, as in- percentage. Particle analysis for high G-force clearly indi-
creases of 5.7 EBC or 16% (cycle 3), 4.6 EBC or 17% and cates size increases between 1.0-2.8 μm as yeast was
4.0 EBC or 15% (cycle 9) were observed. Mannan residues passed through the centrifuge. Particle sizes between
increased as a function of high G-force compared to the 0.56-1.4 μm increased, compared to the control, increases
control, where increases of 8.5 × 105 or 106% (cycle 3), 1.5 of 0.8 or 60% for cycle 3, 0.5 or 39% for cycle 6 and 2.7
× 105 or 18% (cycle 6), and 4.1 × 105 or 51% (cycle 9) were or 202% for cycle 9 were observed. Additionally, there
observed. Initial increase in mannan residues was antici- was a rise in particle size between 1.4-2.8 μm, increases
pated but the subsequent decline during the later cycles was of 2.2 or 49% (cycle 3), 1.5 or 32% (cycle 6) and 4.9 or
not expected. Williams and Wiseman43 showed that a man- 110% (cycle 9) was observed. Particle sizes between 2.8-
nan-enzyme complex from the cells was released by os- 5.0 μm exhibited a dissimilar pattern, reductions of 4.4 or
motic shock. Moreover, osmotic shock and shear stress 37% particle volume (cycle 3), 3.2 or 27% (cycle 6) and
caused the loss of cell surface fimbriae and flocculation4. 1.6 or 14% (cycle 9) were detected.
Thus, the high G-force promoted a large portion of the At low G-force, there was a minimal particle size fluc-
mannan to be broken off initially and the continuous cy- tuation between 0.56-2.8 μm, except particle sizes be-
cling through the centrifuge facilitated efficient separation tween 1.4-2.8 μm for cycle 9 increased 3.8 or 38%. Parti-
consequently reducing both beer haze and mannan resi- cle sizes between 2.8-5.0 μm decreased as a function of
dues. However, reduction below the original mannan resi- centrifugation cycle number compared to the control, de-
due concentration was not achieved. In contrast, at low G- creases of 0.6 or 3% (cycle 3), 3.5 or 20% (cycle 6) and
force, mannan residues decreased compared to the control, in 8.0 or 44% (cycle 9) have been observed. Yeast wall
which a reduction of 9.6 × 105 or 35% (cycle 3), 8.5 × 105 or breakage at high G-force was proportionally larger as
43% (cycle 6), 8.5 × 105 or 50% (cycle 9) was discernible. demonstrated by increasing particle size between 0.56-2.8
These data suggest, as would be expected, that at low G- μm which ultimately contributes to beer haze. Contrary to
force, yeast cells are resistant to hydrodynamic stress. high G-force, the data shows evidence that yeast cells sub-
jected to low G-force did not result in yeast cell breakage
as particle size did not increase. Particle size for low G-
force (Fig. 5B) were > 2%, whereas high G-force (Fig.
5A) particle size were < 2%. This implies that separation
is more efficient at high G-force because smaller particle
size percentages were observed.
Quantification of mannose/glucose ratio
by HPLC
Malcorps et al.21 reported that intracellular glycogen
particles are released during primary fermentation due to
stressful conditions, which can cause an unfilterable haze.
Thus, the sugar composition of the supernatant before and
Fig. 3. Haze and mannan residues as a function of high G-force after centrifugation was analysed, following acid hydroly-
and centrifugation cycle. sis, by HPLC to determine the mannose and glucose con-
centrations2. The HPLC results obtained from this study
determined that glucose levels in the supernatant did not
increase after the yeast slurry had been centrifuged at ei-
ther high or low G-force (Fig. 6). This indicates that the
hydrodynamic forces caused by centrifugation in these ex-
periments did not cause release of glycogen particles. Ad-
ditionally, the mannose/glucose ratio increased as the
number of centrifugation cycles increased (Fig. 7), con-
firming the flow cytometer results that hydrodynamic
forces elicited the release of mannan residues from the
yeast cell wall and not glycogen particles from the yeast
cytoplasm.
Intracelluar pH measurement
by flow cytometry
The intracellular pH (pHi) measurements were based
Fig. 4. Haze and mannan residues as a function of low G-force on the assay described by Valli et al.44 The method obtains
and centrifugation cycle. the pHi distribution within a cell population based on the

50 JOURNAL OF THE INSTITUTE OF BREWING

 Fig. 5. (A) Particle size (%) as a function of high G-force and centrifugation
cycle. (B) Particle size (%) as a function of low G-force and centrifugation cycle.

Fig. 6. Glucose concentrations of yeast slurry supernatants cen-

trifuged at high and low G-forces determined by HPLC analysis
Fig. 7. Mannose/glucose ratio of centrifuge yeast slurry super-
pH dependent emission spectra of the probe SNARF-4F. natant determined by HPLC analysis.
A ratiometric calculation based on the inversely related
emission signals, the Pm expressed as x-mean channel, at Intracelluar glycogen measurement
590 nm and 680 nm determines pHi. The pHi dependent
by flow cytometry
shift is illustrated with unstained and cells stained with
SNARF-4F (Figs. 8A and B). The yeast cell population Various trials were conducted using unstained cells and
was gated in the FSC versus SSC dot plot. A threshold cells stained with Schiff reagent. The data generated were
was defined to eliminate debris in the sample. used to determine where the viable/depleted cell popula-

VOL. 114, NO. 1, 2008 51

 Fig. 8. (A) The peaks represent the autofluorescence and debris of an unstained Intracellular pH
sample. Alternatively, stained cells with SNARF exhibits a significant pH-dependent emission shift
from yellow-orange to red fluorescence under acidic and basic conditions, thus, allowing the ratio
of the fluorescence intensities at two emission wavelengths to be used for quantitative determina-
tions of intracellular pH. (B) SNARF-4F stained cells with pH dependent shift observed. SNARF-
4F dye is loaded into the sample as cell-permeant AM ester acetate. The ester form of this probe
permeates the cell membrane, once inside the cell it is cleaved by esterases to release a pH sensitive
polyanionic probe which fluoresces. SNARF-4F undergoes a shift to longer wavelengths upon de-
protonation of its phenolic substituent. The pH-dependent spectral shifts exhibited by SNARF-4F
allows for in vitro calibration of the pH response in terms of the ratio of fluorescence intensities
measured at two different wavelengths, typically 590 nm and 680 nm, and excitation at 488 nm.

tions and debris appeared on the histograms and dot plots the FSC versus SSC dot plot. The gating on the FSC and
generated by the flow cytometer. Figs. 9A, B and C dem- SSC determined the viable and depleted cells and the Pm
onstrate the fluorescence produced by the Schiff reagent characterised. Yeast cells stained with Schiff reagent fluo-
and how these patterns can be used to separate viable and resced at 520 nm (FL1). The threshold was adjusted such
depleted cell populations. Gating was used to define the that the debris and autofluorescence of the unstained cells
yeast cell population. Data from within this population were not included in the measurement.
can be plotted on dot plots and histograms for further
analysis. A single parameter histogram views one param- Intracelluar trehalose measurement
eter against the number of events, whereas dot plots with by flow cytometry
range markers display the fluorescence data from events The method for the assessment of intracellular treha-
in the gated region. The yeast cell population was gated in lose content was based on the method described by Hutter

52 JOURNAL OF THE INSTITUTE OF BREWING

et al.12 Analysis was conducted using unstained cells and ATPase, which acts as a proton (H+) pump14. This proton
cells stained with ConA. These examinations were used to pump plays a significant role in cell proliferation activity
establish where the viable/depleted cell populations and and fermentation regulation10. The transmembrane H+ gra-
debris are discernible on the histograms and dot plots gen- dient, which is generated, is the driving force for the
erated by the flow cytometer. The fluorescence produced transport of nutrients (for example, maltose and amino ac-
by ConA is illustrated and how these patterns can distin- ids) across the cell membrane44. The pHi homeostasis is
guish viable and depleted cell populations (Figs. 10A, B very important, as key enzymes in glycolysis and gluco-
and C). Gating and instrument settings were similar to neogenesis are regulated by cascade reactions which are
those described for intracellular glycogen determination. pH dependent (c-AMP dependent protein kinases; phos-
phorylase, glycogen synthase, trehalase, fructose-1,6-bis-
Intracelluar pH during centrifugation phosphatase, 6-phosphofructo-2-kinase)14. Imai et al.14
Research on yeast physiology has indicated that the found that under acidic conditions, cells possessing a high
cell’s ability to maintain and regulate its pHi is crucial to H+ extrusion activity were more vital than cells having a
ensure proper functioning of cellular processes44. The pHi low extrusion activity.
in yeast cells is regulated by the plasma membrane The findings from these experiments established that

Fig. 9. (A) Intracellular glycogen of control yeast cells (prior to centrifugation) stained by Schiff reagent. A bimodal cell population
was observed. The viable cells (R1) fluoresce more intensely than depleted cells (R2). The viable cells are larger in size and have a
different internal granularity compared to depleted cells. A bimodal population implies yeast with differing physiological conditions
were present in the cell population. (B) Glycogen peak mean (Pm) of viable cells. These cells possess higher proliferation capacity than
depleted cells. A fraction of this population is mother and daughter cells, which potentially have a larger glycogen content. A Pm
increase was observed during anaerobic and nutrient rich conditions. (C) Depleted cells glycogen peak mean (Pm) represents the
population with lower glycogen levels, proliferation capabilities and exhausted cells. Lower glycogen levels imply decreased
fermentation activity of the cell population. (D) A confirmation of the bimodal distribution of glycogen content in the control cell
population. 3D plot generated by WinMDI software (http://facs.scripps.edu/software.html).

VOL. 114, NO. 1, 2008 53

the pHi of yeast cells and viability was interrelated during 0.54 pHi or 9.9% higher than the high G-force. For cycle
centrifugation cycles. Additionally, the pHi was dependent 6, the pHi difference between the G-forces was less
on the amount of G-force applied during centrifugation dramatic; the low G-force slurry experienced a 0.36 pHi or
(Fig. 11A). Initially, the pHi of the high G-force yeast 6.5% increase compared to the high G-force. A difference
slurry had a higher pHi compared to the low G-force yeast of 0.67 pHi or 12.5% was observed at cycle 9 between
slurry, a difference of 0.25 or 4.5% is observed, which high and low G-force. In agreement with Imai and Ohno13,
may have resulted from the extra 48 h conditioning period. these results provide a correlation between decreasing pHi
The data revealed that upon centrifugation, the high G- and increasing numbers of dead cells. Consequently, the
force yeast slurry pHi decreased, whereas, the low G-force pHi provides insight into yeast physiology and indicates
yeast slurry increased. This trend continued throughout yeast cell functionality loss due to high G-force operating
the duration of centrifugation. During the first 3 centrifu- conditions encountered when passed through a disc stack
gation cycles the pHi of the low G-force yeast slurry was centrifuge.

Fig. 10. (A) Intracellular trehalose of control yeast cells stained by ConA. Similar to intracellular glycogen, a bimodal cell population
was observed. The viable cells (R1) fluoresced more intensely than depleted cells (R2). The two distinctive fluorescent properties
represent subpopulations within the pitching yeast. (B) Trehalose peak mean (Pm) of viable cells. Trehalose is a cell wall constituent
comprised of α-glucopyranose and is an osmoprotectant. The viable cell population Pm has a low trehalose level at the beginning of
fermentation. Trehalose Pm level increases at the beginning phase of proliferation signifying the yeast’s adaptation to a new environ-
ment. (C) Trehalose peak mean (Pm) of depleted cells. Environmental factors such as the depletion of nutrients, high ethanol and CO2
concentrations coincide with an increase in trehalose content. High trehalose concentrations indicate the cells’ ability to withstand
stressful conditions during fermentation. (D) Bimodal distribution of trehalose in the control cell population. 3D plot generated by
WinMDI software. There is a higher proportion of depleted cells with higher fluorescence intensity content at 0 h compared to the via-
ble cells. This may indicate that these cells are quiescent.

54 JOURNAL OF THE INSTITUTE OF BREWING

Intracellular glycogen at two carbohydrate and promotes the synthesis of lipids during
different operating G-forces aerobic conditions, stimulating cell membrane structural
integrity25. Quain26 established three distinct phases of
The use of yeast cell glycogen content as a sensitive metabolic activity of yeast intracellular glycogen through-
parameter to evaluate physiological status and fermenta- out fermentation, consisting of an initial rapid decrease in
tion performance has been well documented by many aerobic conditions, accumulation during the lag and early
brewing scientists1,11,24,25,30,37. Glycogen serves as a reserve exponential phase of fermentations when wort nutrients

Fig. 11. (A) Intracellular pH as a function of G-force and centrifugation cycle. (B)
Intracellular glycogen as a function of G-force and centrifugation cycle. (C) Intra-
cellular trehalose as a function of G-force and centrifugation cycle.

VOL. 114, NO. 1, 2008 55

are available, and glycogen reduction during the station- cuit with CO2. Agitation of the yeast culture during mix-
ary phase. Stewart and Russell37 confirmed Quain and ing and transfer coupled with an aerobic atmosphere pro-
Tubb’s27 report while demonstrating a relationship between vided the opportunity for dissolved oxygen assimilation.
intracellular glycogen reduction and nutrient exhaustion The results obtained from this experiment confirm pre-
during fermentation. An elevated level of glycogen util- vious reports28,37 that yeast cells in stationary phase exhib-
ization during stationary phase reduces the opportunity of ited low intracellular glycogen concentrations compared
cell proliferation in subsequent fermentations37. Further- to exponential phase cells (Figs. 11B and 12). The peak
more, the intracellular glycogen level in the stationary mean (Pm) of the high and low G-force control possessed
phase cells is a function of yeast strain, fermentation similar values of 6.0 and 9.2. It was observed that the low
conditions, and wort gravity37. G-force centrifuged yeast increased to 55.2 Pm or 497%
The use of flow cytometry has shown11 that fermenting after cycle 3, for cycle 6 increased 66.0 Pm or 19.2% and
yeast populations consist of different glycogen levels and cycle 9 increased to 68.1 Pm or 3.1%. An alternative trend
physiological states. Intracellular glycogen was measured was observed for the high G-force yeast which had a mar-
over the course of the centrifugation cycles at two differ- ginal increase of 9.4 Pm or 54% after cycle 3, followed by
ent G-forces (Fig. 11B). The results show that centrifuged a cycle 6 increase of 50.1 Pm or 436% and a cycle 9 in-
yeast exhibited higher levels of intracellular glycogen crease of 1.9 Pm or 3% was observed.
compared to non-centrifuged yeast. It is plausible to at- Aforementioned reasons may have contributed to the
tribute this unexpected result to the use of compressed air increase in intracellular glycogen; unfortunately, dis-
to transfer the yeast slurry from the holding vessel to the solved oxygen was not measured during the course of the
disc stack centrifuge. Additionally, care was not taken centrifugation cycles. Consequently, it is difficult to posi-
throughout the experiment to purge the centrifugation cir- tively state that the increase in glycogen content was a re-

Fig. 12. (A) Intracellular glycogen peak mean (Pm) of viable cells as a function G-
force and fermentation time. (B) Intracellular glycogen peak mean (Pm) of depleted
cells as a function of G-force and fermentation time.

56 JOURNAL OF THE INSTITUTE OF BREWING

sult of oxygen assimilation. Additionally, investigations centrifuged yeast could be a result of newborn and senes-
by Slaughter and Nomura33 found that decreased viability cent cells being damaged during the centrifugation pro-
did not necessarily correlate with a decline in glycogen cess. Therefore, these yeast cells were not quantified in
content, which was confirmed in this study. the viable cell population. Furthermore, these cells could
have been predisposed to necrosis in the centrifuge result-
Intracellular glycogen levels ing in loss of functionality.
during fermentation Figs. 12A and B provide results that establish that gly-
Fermentation trials were conducted with yeast that had cogen levels oscillate during the first 48 h of fermentation
been passed through the centrifuge for 15 cycles at high which confirms the Hutter11 findings. It has been report-
G-force. A bimodal distribution of glycogen relative fluo- ed26,28,37 that as glycogen is metabolised during the early
rescence intensity was observed indicating that within the stages of fermentation, for the synthesis of lipids one
yeast population there were two populations with quantifi- would anticipate an initial reduction followed by an in-
able physiological states (Figs. 9A and D). R2 represents crease as the yeast undergoes regeneration of its glycogen
the depleted cell population (Fig. 9C). Additionally, the levels. The anticipated decrease in glycogen levels at the
viable cells in the second peak possess higher prolifera- start of fermentation was not observed. The discrepancy
tion capacity (Fig. 9B). observed between the results obtained by flow cytometry
The bimodal distribution was observed throughout the and those reported by conventional glycogen determina-
fermentations. Although, at the beginning of fermentation, tions requires further investigation. In a comparison of the
centrifuged yeast cells possessed larger depleted cell pop- viable cell population of control to cycle 7, differences of
ulations. The fact that centrifuged yeast did not show the 27 Pm or 16% (0 h), 23 Pm or 8% (8 h), 89 Pm or 34% (28
same bimodal distribution (Figs. 13A, B and C) as non- h) and 86 Pm or 36% (48 h) were observed. Similarly,

Fig. 13. (A) Intracellular glycogen of yeast cells after 7 cycles through the centrifuge. These cells
represent a decrease in functionality of the cell population. Due to stress resulting from high G-
force the depleted cell glycogen content increases. (B) A decrease in glycogen peak mean (Pm) of
viable cells due to passage through a centrifuge results in the cell population’s ability to proliferate
decreasing. (C) Glycogen peak mean (Pm) of depleted cells has increased proportionally to viable
cells after centrifugation. These cells are newborn and senescent cells being destroyed during the
centrifugation process and thus eliminated from the viable cell population.

VOL. 114, NO. 1, 2008 57

comparing viable cell populations of control to cycle 15, and Bailey5 reported that immobilized yeast had a higher
differences of 6 Pm or 3% (0 h), 41 Pm or 13% (8 h), 96 Pm macromolecule composition than free cell systems. The
or 34% (28 h) and 167 Pm or 51% (48 h) were shown. data from the fermentation trials displayed a clear rela-
After 48 h fermentation, the glycogen declined steadily tionship between depleted glycogen Pm levels as a func-
throughout the course of fermentation. There were only tion of centrifugation cycle (Figs. 12A and B). Therefore,
marginal differences of glycogen Pm between cycle 7 and centrifuged yeast cells may have insufficient glycogen to
cycle 15. “fuel” lipid metabolism thus restricting cell growth and
The glycogen Pm displayed a similar trend for the de- fermentation performance.
pleted cells, when comparing the control to cycle 7, differ-
ences of 9.5 Pm or 10% (0 h), 66 Pm or 44% (8 h), 60 Pm or Intracelluar trehalose at two
36% (28) and 43 Pm or 27% (48 h) were found. Evaluation different operating G-forces
of the control and cycle 15 glycogen Pm, differences of 6.3 Stewart and Russell37, Slaughter and Nomura33 and
Pm or 6% (0 h), 74 Pm or 47% (8 h), 67 Pm or 40% (28 h) Majara et al.20 have presented evidence that intracellular
and 89 Pm or 58% (48 h) were revealed. Correspondingly, trehalose levels increased as a response to stress. The role
only minor fluctuations of glycogen Pm existed between of intracellular trehalose as a yeast cell membrane stabi-
cycle 7 and cycle 15. In the study by Shen et al.31, a cor- lizer has been reported by Thevelein39. Majara et al.20
relation between diminished glycogen content in immobi- found that cellular trehalose levels accumulated propor-
lized cell systems compared to a free cell system speculat- tionally to wort density indicating that it serves as an os-
ed that lower glycogen levels were attributed to counter- moprotectant during high gravity wort fermentations. Ad-
acting stresses. The same investigation concluded that ditionally, yeast cells with elevated levels of intracellular
immobilized yeast cells were not able to accumulate as trehalose possess increased osmotolerance, thermotoler-
much glycogen as free cells. This data provides verifica- ance, and ethanol tolerance37,45. Therefore, high levels of
tion that hydrodynamic stresses have potential to unfa- intracellular trehalose are associated with yeast cells that
vourably affect yeast cell physiology. Alternatively, Doran are resistant to stressful conditions.

Fig. 14. (A) Intracellular trehalose peak mean (Pm) of viable cells as a function G-
force and fermentation time. (B) Intracellular trehalose peak mean of depleted cells as
a function of G-force and fermentation time.

58 JOURNAL OF THE INSTITUTE OF BREWING

 The degree of stress resistance and the amount of tre- lar findings of Rambeck and Simon41, Souza and Panek34
halose synthesized are dependent on the physiological and Shen et al.31 At 8 h when compared to the control, vi-
state of the yeast cultures. Non-growing cells were shown able cells trehalose Pm level had a difference of 114 Pm or
to be more stress resistant, whereas growing cells pro- 36% (cycle 7) and 98 Pm or 31% (cycle 15) with the de-
duced more trehalose in response to the applied stress. pleted cells 114 Pm or 36% and 111 Pm or 57%, respec-
Guldfeldt and Arneborg7 indicated that increased levels of tively. This indicates a rise in trehalose Pm for the centri-
trehalose in pitching yeast sustained high cell viability fuged cells, although not to the degree of the control.
during the initial stages of fermentation and resulted in Pratt-Marshall24 reported that ale yeast fermenting in 20o
higher carbohydrate utilisation rates and increased pro- Plato wort exhibited higher trehalose levels than the same
duction of isoamyl alcohol and iso-butanol. Alternatively, yeast fermenting in 12o Plato wort and that the yeast fer-
procedures that affect membrane integrity such as autoly- menting in the high gravity wort showed a trehalose peak
sis, heating, drying and treatment with organic solvents, after 24 h of fermentation. Although depleted cells did not
caused rapid trehalose degradation34,41. Neutral trehalases, depict this peak, the viable cells population of centrifuged
which mediate the degradation of trehalose42, are activated yeast (cycle 7 and 15) had a comparable peak after ap-
and result from a rapid increase in cAMP levels in yeast39. proximately 16 h of fermentation. These findings are in
The abovementioned research indicates that trehalose agreement with the results of Pratt-Marshall24, where
levels may be useful in evaluating yeast vitality. Boulton1 stressed cells maintained higher trehalose levels towards
found that the degradation of trehalose correlates with a the end of fermentation.
rapid loss of viability1,33. The data obtained from these ex-
periments (Fig11C) confirm these findings. The trehalose CONCLUSIONS
Pm of the high and low G-force control possess Pm values
of 67 and 95, respectively. The higher level of trehalose The objective of these experiments was to determine
Pm might be partially attributed to the additional 48 h whether the passage of a brewers’ yeast strain through a
yeast conditioning period. Trehalose Pm decreased as a disc stack centrifuge at differing operating G-force condi-
function of centrifugation cycle, compared to the control. tions had a negative effect on cell viability, vitality and
High centrifuge G-force yeast, resulted in trehalose reduc- beer physical stability. Yeast cropping by centrifugation
tions compared to the control of 53.4 or 21% (cycle 3), exposes yeast to extreme shear stress. The viability of the
22.1 or 67% (cycle 6) and 31.8 or 58% (cycle 9) were yeast culture tested decreased as it was re-cycled through
determined. A similar diminishing trehalose Pm trend was the centrifuge. There were proportionally higher percent-
observed for the low G-force yeast, compared to the con- ages of damaged yeast cells that were exposed to the low
trol, decreases of 26.5 or 72% (cycle 3), 30.9 or 67% G-force implying that these cells were more resistant to
(cycle 6), 33.1 or 66% (cycle 9) were observed. Low tre- hydrodynamic forces during centrifugation. The results in
halose content reflects low stress tolerance of centrifuged Fig. 1 demonstrate the damage cell threshold, the transi-
yeast cells. Shen et al.31 found a similar reduction of intra- tion from the necrotic to the apoptotic cell state, due to
cellular trehalose in immobilized yeast systems and exposure to hydrodynamic stresses within the centrifuge.
Slaughter and Nomura33 described a similar pattern during The results also provided evidence that the beer haze in-
stress induced fermentations. creased, mannan residues were produced, and particle size
of < 2.8 µm increased as exposure to high G-force in the
centrifuge was applied. The physiological state of the
Intracellular trehalose levels
yeast was modified due to the processing conditions en-
during fermentation countered during yeast cropping. Glycogen and trehalose
As previously mentioned, the intracellular concentra- levels were depleted. These changes in cellular properties
tion of trehalose has been reported20 to play an important influence metabolic activity and reduce stress resistance.
part in the cells’ ability to withstand adverse environmen- The flow cytometer allows for a graphic representation of
tal conditions. Fermentation trials were conducted with the different subsets within a yeast population, and it can
yeast that had been passed through the centrifuge for 15 be used to perform various analyses such as cell viability,
cycles at high G-force (Figs. 14A and B). Throughout the damaged cells, intracellular pH, mannan residues and the
fermentation cycle, a bimodal distribution of trehalose Pm determination viable and depleted intracellular macromol-
was observed indicating that within the yeast population ecules. During these experiments, the centrifuge was op-
there were analogous populations with different physio- erated under conditions similar to those encountered dur-
logical characteristics (Figs.10A and D). R2 represents the ing commercial production. However, shear stresses may
depleted cell population (Fig. 10C) which has a lower occur with or without the use of a centrifuge, leading to
trehalose Pm. Moreover, the viable cells in the second decreased beer quality and stability. It is important for
peak contained a higher trehalose Pm (Fig. 10B). Alterna- yeast management systems that utilize centrifuges be de-
tively, centrifuged yeast cells displayed higher levels of signed and operated properly to minimize negative conse-
depleted trehalose Pm (Figs. 15A, B and C). quences on beer quality. Recent investigations found that
The initial elevated trehalose Pm levels would seem to implementation of newer model centrifuges and rede-
substantiate the findings of Majara et al.20 and Pratt- signed pipe work improved green beer quality. From this
Marshall22 that trehalose concentrations were higher at the experiment, it can be concluded that the passage of yeast
beginning of fermentation. Consequently, compared to the cells through a disc stack centrifuge, at high G-force,
control, at 0 h, viable yeast cells during cycle 7 and 15 de- strongly adversely influences yeast physiology and beer
creased by the same amount of 241 Pm or confirmed simi- physical stability.

VOL. 114, NO. 1, 2008 59

 Fig. 15. (A) Intracellular trehalose of yeast cells following 7 cycles through the centrifuge. De-
pleted cells increased proportionally compared to viable cells. (B) Trehalose peak mean of
viable cells. A large decrease in viable cells is observed after centrifugation. These cells do not
possess stress resistant attributes which may have an affect on fermentation performance and
subsequent repitching. (C) Trehalose peak mean of depleted cells. These cells represent the pop-
ulation which has been passed through the centrifuge. There is a larger degree of depleted cells
with higher trehalose content, which implies these cells were stressed as a function of passage
through the centrifuge.

ACKNOWLEDGMENTS tion of Saccharomyces cerevisiae attached to gelatin. Bio-

technol. Bioeng., 1986, 28, 73-87.
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We also thank Graham McKernan for assistance in the pilot glycogen content in single neutrophil leucocytes using micro-
brewery, Jim MacKinlay for assistance with the particle size model of leucocytes glycogen. J. Histochem. Cytochem., 1975,
analysis and Dr. Jim Buckman for support with the Environmen- 23, 59-64.
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content at pitching on performance during brewing fermen-
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