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The response of brewing yeast to acid washing

Article in Journal of the Institute of Brewing · September 1989

DOI: 10.1002/j.2050-0416.1989.tb04642.x

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J. Inst. Brew., September-October, 1989, Vol. 95,pp. 347-354 347

THE RESPONSE OF BREWING YEASTS TO ACID WASHING

By W. J. Simpson and J. R. M. Hammond

(Brewing Research Foundation, LyttelHall, Nutfield, Redhill, Surrey, Great Britain, RH14HY)

Received 6 December 1988

Brewing yeasts are inherently resistant to acid washing treatments but, under some conditions this resistance is
diminished. Sixteen yeast strains, including both ale and lager strains, have been successfully washed using
phosphoric acid (pH 2.1) and, in some cases, sulphuric acid (pH 2.0) or acidified 0.76% w/v ammonium persulphate
(pH 2.8). Cell viability, fermentation performance and flocculation/fining behaviour were unaffected. However,
changes could be observed in the yeast resulting from the washing treatment. These included alterations of the cell
surface, as shown by scanning electron microscopy and leakage of adenosine triphosphate from the cells during the
wash.
Acid washing is successful provided that, i) food grade acid is used, ii) the acid is chilled before use, iii) the yeast
and acid are well mixed, iv) the temperature of the yeast does not exceed 5°C during washing and, v) the yeast is
pitched immediately after washing. 'Unhealthy' yeast (yeast which has been stored for long periods, heavily
contaminated yeast, or yeast from slow fermentations) may respond poorly to acid washing and a shorter washing
time, or higher wash pH value should be employed.

Key Words: Yeast, phosphoric acid, sulphuric acid, ammo
nium persulphate, fermentation.
Introduction
Acid washing has been used by some brewers for many years
as a means of eliminating contaminant bacteria from pitching
yeast. The recent finding14 that bacteria, associated with
pitching yeast are responsible for the production of apparent
total N-nitroso compounds (ATNC), during the fermentation
process, has rekindled interest in the technique. Many reports
have appeared over the last eighty years which suggest that
acid washing can affect yeast performance. Examples of the
effects include reduced yeast viability11-23'24, reduced yeast
'vitality'6-19125 reduced rate or degree of
fermentation1'3'4'18'20"25'30 and changes in yeast-quality para
meters such as flocculation, fining, size of yeast crop and
excretion of cell components2-3'8-™'11-17'21'25'*6.
Acid washing can efficiently eliminate the bacteria re
sponsible for production of ATNC from yeast slurries5.
However, because concern had been expressed regarding
side-effects associated with acid washing, work was initiated
at the Foundation with the following aims: i) to identify the
effects of acid washing procedures on yeast performance with
particular regard to fermentation rate, flocculation and fining
performance and ii) to select, or devise, an acid washing
method which had no deleterious effect on the yeast and
which was effective at eliminating those bacteria involved in
the production of ATNC. This paper describes the results of
work concerned with the first of these aims.
Experimental
the yeast was propagated in all-malt wort16 before washing
whereas in other cases the yeasts were washed as received.
The latter were washed either as slurries in beer (50% solids
as packed cell volume) or, where appropriate, pressed yeast
was reslurried in AnalaR water (BDH Ltd., Poole, Dorset,
England, Product No. 36050) before washing (one part yeast,
by weight, was mixed with one part water by volume). The
amount of acid required to acidify the yeasts was assessed by
titrating a separate sample with acid as previously
described27. All manipulations and treatments were carried
out at 4CC except in one set of experiments in which the
temperature was varied between 4°C and 25°C.
Assessment of Yeast Viability and Activity
Yeast viability was assessed using the methylene blue
staining method. A minimum of 400 cells were counted for
each determination. Viability was assessed before washing,
and at intervals during the wash. A 20-50 fold dilution step
ensured that acid or persulphate carried over with the yeast
did not affect the accuracy or precision of the staining
procedure. The methylene blue staining technique has been
used extensively in this work and although under some
conditions it may not give an absolute estimate of cell
viability9 it does give an indication of changes in viability.
The specific oxygen uptake rate value was determined,
both before and after washing, for Sacch. cerevisiae NCYC
1681 and ale strain L at 25°C using the method of Kara et a/.15
Control experiments showed that the carry-over of acid into
the test did not affect the q02 value of the yeast. Acidification
Power (AP) values for each yeast were assessed prior to
washing using the method described previously.16

Yeast Effects of Ethanol

The following strains were used, i) Ale — Saccharomyces
cerevisiae NCYC 1236, NCYC 1681 and eight strains from
commercial breweries (A, B, C, D, E, F, G, L), ii) Lager—
Saccharomyces cerevisiae (syn. carlsbergensis, syn. uvarum)
NCYC 1324, NCYC 1342 and four strains from commercial
breweries (H, I, J, K). The test strains included both
flocculent and non-flocculent strains, head forming and
non-head forming strains and strains with both low and high
attenuation limits. Four of the srains were provided by
breweries in the belief that they responded poorly to acid
washing. At least three of the other strains had probably not
been previously acid washed.
Washing Techniques
All yeasts were washed with phosphoric acid at a pH value
of 2.1; in addition some of the yeasts were washed with
acidified 0.75% w/v ammonium persulphate at a pH value of
2.8 (strains NCYC 1681, A, F, K) and/or sulphuric acid at a
pH value of 2.0 (NCYC 1681, B, H, I, J, K). In some cases
Sacch. cerevisiae NCYC 1681 was washed at 4"C with i)
0.6% v/v H3PO4 ii) 0.6% v/v H3PO4 containing 5% v/v
ethanol, iii) 0.6% v/v H3PO4 containing 10% v/v ethanol or
iv) 10% v/v ethanol. Ethanol was added as absolute alcohol
100 (James Burrough (F.A.D.) Ltd., Witham, Essex). The
initial pH values attained in each test immediately after
addition of yeast were respectively, i) 1.86, ii) 1.86, iii) 1.87
and iv) 5.79. All solutions were attemperated to 4°C before
addition of yeast to a final concentration of 10% (wet
wt./vol). Viability was monitored (using methylene blue) over
a 3 hour period.
Assessment of Yeast Flocculation (resting cells)
The ability of cells to flocculate in diluted beer was assessed
using a spectrophotometric method. The yeast was washed
three times in a solution of calcium chloride (lOg Litre'1) and
0.5g wet weight of the yeast resuspended in calcium chloride
solution (10ml, lOg Litre*1). The sample was whirlmixed and
a portion (5ml) mixed with an equal volume of commercial
348 THE RESPONSE OF BREWING YEASTS TO ACID WASHING [J. Inst. Brew.

lager (OG 1036°). The mixture was whirlmixed, 5ml was was fined with isinglass finings at the rate of 7 ml Litre'1 (2
transferred to a sample cell and the absorbancc of the sample pints per barrel). When the finings had been thoroughly
at 600 nm monitored over several minutes. The maximum 'worked into' the beer, the fining jars were kept at a constant
rate of change of absorbance was taken as an indication of the temperature (13°C) for 2 days, after which the haze in the
degree of flocculence. bright beer was assessed. Beer (50ml) was run off and
discarded then 250 ml was collected and the haze measured at
Electron Microscopy of Unwashed and Washed Yeast
20°C in a Radiometer haze meter (90° incident haze measure
A slurry of Sacch. cerevisiae HCYC 1681 (lOg wet weight in
ment). Haze measurements were used as a direct indication
100 ml AnalaR water) from pilot scale (30 Litre) fermenta
of fining performance, high haze values indicating poor fining
tions was washed with phosphoric acid at a pH value of 1.9
performance.
(0.6% v/v H3PO4) or with acidified ammonium persulphate at
a pH value of 2.8. Control samples were washed in water
Results and Discussion
only. The washes were performed at 4°C or 25°C for two
hours. After this time the yeast cell surface was examined by Response of the Yeasts to Acid Washing
means of scanning electron microscopy using cryogenic i) Viability: Cell viability, as assessed by methylene blue
techniques at a temperature of <-130°C; no fixatives were staining, was not significantly affected by any of the washing
used in the preparation of samples. The micrographs treatments when the yeast was washed at 4°C. After two
obtained are thus more likely to be free from artifacts. hours washing the percentage stained cells in both control and
acid washed samples differed by no more than 3% in every
Assessment of Cell Component Excretion
case. This value is within the accepted error of the analysis.
Attempts to assess shock to the yeast by monitoring
Typical sets of experimental results, are shown in Figure 1.
excretion of either material absorbing at 260 nm or nitrogen-
containing compounds (measured using the Kjeldahl method)
were unsuccessful since yeast slurries were found to contain % cells unstained
high background levels of both classes of compounds before
100r
acid washing. However, the firefly-bioluminescence assay28
for adenosine triphosphate (ATP) provided a sensitive
alternative assay for assessment of the rate and extent of
leakage of small molecules from damaged cells. Samples of
yeast slurry (30 ml) were removed at intervals during washing
and centrifuged at 3000g for 10 min. at 4°C. The supernatant
fluid was passed through a 0.22 /xm cellulose acetate
membrane filter (CATHIVEX-GS, Milliporc Prod No.
SOGS 025 05) to remove all micro-organisms. The filtrate
80
was then diluted in 25mM HEPES (N-2-
hydroxyethylpiperazine-N-2-ethane-sulphonic acid) buffer
(pH 7.75) containing 2 mM EDTA (ethylenediamine tet-
raacetic acid) to bring the ATP concentration within the
working range of the assay (max. 3.0 x 10'8M). A Lumac 70
M2010A biocounter was used to measure ATP concentration.
Luciferase-luciferin reagent (100/xL; LUMIT PM; Sonco Ltd,
Batley, West Yorkshire, UK) was added to the diluted filtrate
(100/iL) contained in a polystyrene cuvette and the light
output measured over a ten-second period. The assay was 60
then standardised by addition of 1 pmol ATP (in 10/xL sterile
water) and the light output monitored over a further ten
seconds.

Laboratory Scale Fermentations

Duplicate fermentations were carried out in tall tube
fermenters at 18°C for ale yeasts and 12°C for lager yeasts as
described previously16 (pitching rate 2.5 g wet wt. yeast
Litre', all-malt wort, OG 1040° (ale), or containing 20%
adjunct OG 1038" (lager), pH 5.2; air saturated (at 12°C or
18°C respectively) before pitching). Parameters monitored
were, i) specific gravity, (PAAR DMA 45 Digital density
meter), ii) total cell count, (Thoma haemocytometer), iii) cell
viability (methylene-blue staining),7 and iv) pH value (Corn
ing pH meter and combination all-glass electrode).
Assessment of Yeast Flocculation (in vivo)
Flocculation characteristics of washed and unwashed
strains during fermentation were evaluated by plotting the
suspended cell count against % attenuation as suggested by
Stewart & Russell.29
Fining Tests
Fining tests were performed only on ale fermentations,
since, in practice, lagers are not fined in cask. After primary
fermentation, beer was decanted off the yeast sediment using
a glass syphon-tube and nitrogen top-pressure. The use of a
glass syphon tube allowed the beer to be decanted in a
consistent fashion. After discarding the first 150-200 ml, the
beer (800 ml) was transferred into 1 Litre glass vessels and
60
0 20 40 60 60 100 120
Time (mln.) since addition of aold
Fig. 1. Effect of acid washing (H3PO4, pH 2.1,4°C) on the viability of
Sacch. cerevisiae NCYC168 (O), strain L (A), strain H (+) and
strain 1 (D) as assessed by mcthylcne blue staining.
We have presented our data as linear plots (viability vs time)
in order to highlight differences between treatments. High
viability was maintained during washing regardless of
whether the yeast was washed as a thick slurry. (~60%
packed cell volume) or as a dilute suspension (5% packed cell
volume).
ii) Specific oxygen uptake rate (qCb): the qO2 value of
'healthy' Sacch. cerevisiae NCYC 1681 was not significantly
affected by phosphoric acid washing at pH 2.1. 'Unhealthy'
yeasts had lower qO2 values prior to acid washing, but no
change occurred after acid washing even though the fer
mentation performance of this yeast was significantly affected
by the washing procedure. Washing 'healthy' yeast with the
acidified ammonium persulphate method resulted in a small
reduction in qOj but this was not reflected in the subsequent
fermentation performance of the strain.
Vol. 95, 1989] THE RESPONSE OF BREWING YEASTS TO ACID WASHING 349

iii) Acidification Power: Yeasts which responded well to before and after treatment with acidified ammonium persul
acid washing always had satisfactory AP values prior to phate showed that a significant proportion of the extraneous
washing (2.08-2.52). Attempts to assess damage to yeast cells (presumably proteinaceous) material associated with the cells
induced by acid or persulphate, using the AP test produced was removed by the add/persulphate treatment (Figure 3). In
unexpected results in that some stimulation of the yeast, by addition, significant surface blistering, or blebbing occurred.
acid, was indicated. These results will be discussed elsewhere These phenomena were also observed with phosphoric acid
since they are of importance in understanding the mechanism (Figure 4) confirming the observations of Hulse obtained
of resistance of brewing yeast to acid washing. Experiments using lager yeast and phosphoric acid. The severity of this
described below show that the AP test can be used in a blebbing, which was absent in untreated yeast, increased with
predictive role, since yeasts with low AP ('unhealthy') temperature (Figure 4).
ferment poorly and respond poorly to acid washing. When
the AP test is used to select yeast for pitching, the analysis
should be carried out before the yeast is acid washed in order
to avoid erroneous results.16
iv) Excretion of Small Compounds (ATP): In the absence of
added acid the yeasts examined did not release ATP into the
medium (Figure 2) Cells treated with ammonium persulphate

pmole ATP leaked/uL yeast slurry

ao eo eo 120

Tims (mla) since addition of add

Fig. 2. Leakage of ATP from yeast during acid washing. Untreated

yeast (A) did not release ATP at 4°C but cells treated with
acidified ammonium persulphate (D) or phosphoric acid (O)
did. The values shown represent the means of duplicate
determinations.

at a pH value of 2.8 leaked or excreted ATP at a significant

rate. Moreover, phosphoric acid washing (pH value 2.1)
induced a leakage rate 4-5 times faster than that induced by
ammonium persulphate. These results suggest that at 4°C,
brewers' yeast is damaged by acid washing. However, such
damage does not affect the fermentation performance of
these yeast(s) in the fermentation immediately after washing.
v) Flocculation: In the presence of acid, yeast cells of all
strains dcflocculated. However, if the cells were washed in
buffer and resuspended in beer, or buffer containing suffi
cient calcium ions, then flocculence was only slightly dimi
nished. In contrast, treatment with ammonium persulphate
resulted in a loss of flocculence that could not be reversed by
these procedures. However, flocculence loss induced by
ammonium persulphate is not of great signficance to the
brewing process since the ability to flocculate is regenerated
during the fermentation following the wash (see below, and
Figure 6). Solutions of ammonium persulphate decompose to
form ammonium sulphate, sulphuric acid and ozone. Since
similar effects were not induced by ammonium sulphate or
sulphuric acid, it is possible that the surface properties of
Sacch. cerevisiae NCYC 1681 were changed by ammonium
persulphate due to the release of ozone, a stong oxidising
reagent. Some oxidising agents such as periodate13 are known
to modify the flocculation properties of brewing yeast. But
others, such as potassium permanganate or hydrogen perox
ide failed to affect flocculence in our experiments. Thus the
accessibility of yeast surface moieties to the oxidising agent
may be important in addition to the chemical composition
and strength of the oxidising agent.
Electron micrographs of Sacch. cerevisiae NYCY 1681
Flo. 3. Scanning electron micrographs of Sacch. cerevisiae NCYC
1681 before (a) and after (b) washing with acidified ammonium
persulphate, at 4°C. Scale bars represent lOjim.
Acid washing makes brewing yeast 'sticky'. After washing,
yeast from centrifuged pellets can be pulled out into short
(1-2 cm) strands; untreated yeast does not exhibit this
phenomenon (Figure 5). This also shows that acid washing
modifies the surface properties of the yeast cell, although, as
discussed above, it appears that regeneration of these
properties during fermentation minimises their process sig
nificance.
Laboratory-Scale Fermentations
No significant differences in the fermentation behaviour
(lag phase, attenuation rate, flocculation, pH profile) be
tween unwashed and washed yeasts were observed providing
the yeast was washed under ideal conditions. Typical fer
mentation profiles are shown in Figure 6. Fermentation
performance of Sacch. cerevisiae NCYC 1681 was unaffected
even when this yeast was washed with phosphoric acid (pH
value of 1.88, 4°C) for 22 hours.
The top-cropping ale yeasts formed a significant head
during fermentation in the tall tube fermenters; the appear
ance of which was the same in fcrmenters pitched with acid
washed or untreated yeast.
350 THE RESPONSE OF BREWING YEASTS TO ACID WASHING [J. Inst. Brew.

Speolflo gravity
1.04,

1.03

1.02

1.01

20 40 60 60

Time (hours)

-6/
Yeast count (x10 7ml_.)

Fig. 4. Scanning electron micrographs of control and acid treated

Sacch. cerevisiae NCYC 1681. (a) control (unwashed) cells; (b)
20 40 60 80 100
acid washed cells (4°C); (c) acid washed cells (25°C). Scale bars
represent 1 /un. % attenuation

Fig. 6. Fermentation performance of unwashed (D), phosphoric acid

washed (A) and acidified ammonium persulphate washed (O)
Sacch. cerevisiae; NCYC 1681 with respect to (a) fermentation
profile and (b) decollation profile (yeast count/% attenuation).
Fermentation rates and flocculation behaviour were unaffected
by acid washing at 4°C. The values shown represent the mean of
duplicate fermentations.

P Poor fining manifests itself in the production of hazy beers.
Fig. 5. •Stickiness' of acid washed Sacch. cerevisiae; NCYC 1681.
Control (unwashed) yeast (left) cannot be pulled out in strands;
acid washed yeast (right) is sticky and can be pulled out in short
strands.
Fining Performance
In no case, however, did add washing affect fining perform
ance as judged by haze values (Table 1). Indeed, in some
««*the beer produced by the washed yeast fined better than
«« control. Parallel experiments earned out using respira-
y
tory-defident variants of NCYC 1681 ((one a spontaneous
p
variant, one induced by the mutagen ethidium bromide),
Vol. 95, 1989] the response op brewing yeasts to acid washing 351

TABLE I. Effect of Add Washing (at 4°C) on Fining Performance

Haze(°EBC)

Sacch.
cerevisiae
Sacch. NCYC 1681 Sacch. Sacch.
cerevisiac (Respiratory cerevisiae cerevisiae
NCYC1681 deficient variant) NCYC 1236 strain F

control:
no treatment 1-6 10-2 2-2 1-6

phosphoric acid
(PH21) 1-9 0-7 1-2

acidified ammonium
persulphate (pH 2-8) 1-7

The data shown represent the mean values from duplicate experiments.

which fine poorly, showed that the fining test employed could
detect differences between yeasts which fined well and those
which fined poorly (Table 1). The material responsible for the
haze in beers produced by respiratory-deficient variants
appeared to be proteinaceous since it was soluble in strong
alkali and stained pink with eosin yellow. Although these
results provide no evidence for inferior fining performance by
acid-washed yeasts, they should be treated with a degree of
caution since surface-associated phenomena can be greatly
influenced by scale effects. Only experiments carried out at
brewery scale can fully resolve the question of the effect of
acid washing on fining performance.
had been washed at 4°C, performed satisfactorily but those
inoculated with yeast which had been washed at 24°C
performed poorly (Figure 7).
Death rates of both ale and lager yeasts during acid
washing were strongly influenced by temperature:- the
greater the temperature, the faster the rate of death (Figure
8). Death rates under 'ideal conditions' (4°C) were insignifi
cant.
NCYC 1681
% oells unstained

The Influence of Washing Temperature on the Response of

Yeast to Acid Washing
Viability of Sacch. cerevisiae NCYC 1236 was unaffected by
acid washing provided that the wash was performed at 4°C
(Figure 7). Washes carried out at 24°C however, brought
about a dramatic reduction in viability confirming the results
of Ogden23 who washed Sacch. cerevisiae NCYC 1236 at
room temperature. Fermentations pitched with yeast, which

* aril* uratalnad

20 40 60 80 100

Tims (mln.) since addition of add

NCYC 1324
% cells unstained

O 10 «0 «0 M ISO MO
Ttma (mln.) ■Ino* addition of aold

1.01

20 40 eo eo 100 120

100 Tims (mln.) since addition of add

Tims (hours) Fig. 8. Effect of temperature on the response of yeast to acid

Fig. 7. Response of Sacch. cerevisiae NCYC 1236 to acid washing at
4°C and 24°C. Unwashed yeast (O); phosphoric acid washed
yeast, pH 2.1,4°C D; 24°C(A). The values shown represent the
mean of duplicate fermentations.
washing. Viability as assessed by methylene blue staining, was
determined over a period of two hours at 4°C (D); 10°C (+);
16°C (O); 20°C (A) and 25° (V). One ale strain (Sacch.
cerevisiae NCYC 1681) and one lager strain (Sacch. cerevisiae
NCYC 1324) were examined.
352 THE RESPONSE OF BREWING YEASTS TO ACID WASHING
Effect of Ethanol
Acid or ethanol alone have little effect on viability of
Sacch. cerevisiae NCYC 1681 but together they act synergisti-
cally to kill cells even at 4°C (Figure 9). These results show
that yeast which are acid washed in the presence of ethanol
are more likely to suffer damage than those washed in its
absence. In particular, dangers may exist in washing yeast
from very high gravity fermentations or in washing stored
yeast, which has produced ethanol by fermentation of
internal glycogen reserves. In addition, stored yeast is
potentially more sensitive to acid due to its changed
physiological condition (see below).
cells unstained
100
40 80 120 160
Tims (mln.) since addition of acid
Fig. 9. Influence of %thanol on response of yeast to acid washing.
Sacch. cerevisiae NCYC 1681 was washed at 4°C with, (a) 0.6%
v/v H3PO4 (□), (b) 0.6% v/v H3PO4 containing 5% v/v ethanol
(+), (c) 0.6% v/v H3PO4 containing 10% v/v ethanol (A) or (d)
10% ethanol (0). Viability was monitored, using methylene
blue, at intervals up to an including three hours. Ethanol acted
synergistically with the acid to kill the cells.
Influence of Physiological Condition on the Response of Yeast
to Acid Washing.
Ale yeast obtained from brewery L responded poorly to
both phosporic acid and acidified ammonium persulphate.
Fermentation rates were reduced by both treatments (Figure
10a). In addition, the viability and qOj value of the yeast
dropped during washing. Significantly, this particular batch of
yeast had a low viability and poor fermentative activity before
commencing the wash. However, when this yeast was
propagated by out standard protocol16 the viability, fermenta
tive activity and response to acid washing (Figure 10b) all
improved significantly.
Confirmation that the physiological condition of the yeast
is important in resistance to acid washing was obtained as
follows. Pilot brewery ale yeast, Sacch. cerevisiae NCCY
1681, which is resistant to the effects of acid washing was
propagated using conditions likely to stress the organism. The
stress involved exposure to low levels of nitrite (such as would
occur if the fermentation was contaminated with nitrate-
reducing bacteria), prolonged storage under beer at high
(23°C) temperature and underpinning of the fermentation.
[J. Inst. Brew.
Spec!do gravity
(a)
1.02
1.01
40 80 120 ISO
Time (hours)
Specific gravity
(b)
BO 120
Time (hours)
Fig. 10. The effect of yeast condition on response of Sacch. cerevisiae
strain L 10 add washing (phosphoric acid pH2.1; 4°C, 2h).
Fermentations were carried out with washed (A) and unwashed
(D) yeast, (a) and (b) represent data obtained from "unhealthy"
and "healthy" yeast respectively.
Yeast propagated using the normal protocol16 was unaffected
by acid washing but that propagated by the stressful protocol
fermented poorly after acid washing (Figure 11). In particu
lar, the lag phase was extended. Interestingly acid washing
did not influence the viability or qO2 value of the stressed
yeast, although low values were obtained for both these
parameters prior to acid washing.
Conclusions
The results presented here indicate that sensitivity of yeast
to acid washing is governed predominantly by environmental
(phenotypic) factors rather than inherited (genotypic) fac
tors. Brewing yeasts, both ale and lager, are highly resistant
to acidic environments but the extent of this resistance is
reduced by increased temperature and the presence of
ethanol. In addition, 'healthy' cells (i.e. those capable of
performing satisfactorily in fermentation) are more resistant
to the effects of acid than are 'unhealthy' cells (i.e. those
incapable of performing satisfactorily in fermentation). From
these conclusions, practical guidelines for acid washing of
yeast can be derived (Table 2).
The choice of acid to acidify the yeast is determined by
several factors. These included acceptability of the acid as an
additive or processing aid, cost of the acid, handling
characteristics of the acid (with particular regard to safety),
and effectiveness of the acid as a yeast washing agent.
Experiments which will be reported in detail elsewhere, have
Vol. 95, 1989] THE RESPONSE OF BREWING YEASTS TO ACID WASHING 353

C oells unstained shown that the critical control parameter in this respect is
100
wash pH value, regardless of which acid is used to acidify the
Specific gravity slurry. In principle, any acid could be used, however, several
1.04
other criteria must be satisfied, i) the acid must be of
food-grade quality (or better) to ensure product safety, ii) the
acid should not cause processing difficulties or exacerbate
existing difficulties, iii) the acid should preferably not be
excessively hazardous to the operator at the in-use concentra
tion (concentrated suphuric add requires special care), iv) the
1.03 cost should not be prohibitive. While several acids satisfy the
above criteria to varying degrees, phosphoric and citric acids
deserve special mention. Since they arc weak acids, control of
yeast pH is easily accomplished; strong acids, such as
sulphuric acid can cause difficulties in this area, a slight
over-dosing of add resulting in an excessively low yeast pH
Tims (mln.) tlnoe iddltlon or sold value. Weak acids also lower wort pH slightly when the yeast
1.02 is pitched thus restricting the opportunity for re-
contamination. However, since they are weak acids, their
influence on final beer pH is negligible.
The ability of acidified ammonium persulphate to modify
the flocculation properties of brewing yeast suggests that this
method should be approached cautiously in cases where the
surface properties of the yeast play a dedsive role in
1.01
determining beer clarity. This method is, however, satisfac
tory under most conditions.

Acknowledgements: The authors thank Jacqueline Fernandez

40 80 120
Time (hours)
Fig. 11. The response of "healthy" and "unhealthy" Sacch. cerevlsiac
NCYC 1681 to acid washing. "Healthy" (A) or "unhealthy" (D)
Sacch. cercvisiae NCYC 1681 prepared as described in the text
was washed at 4°C with phosphoric acid (pH 2.1). Fermentation
profiles were plotted for unwashed yeast (solid line) and for
washed yeast (broken line). The fermentation rate of the
"healthy" yeast was unaffected by acid washing, but that of the
"unhealthy" yeast was significantly reduced by the treatment.
The viability, as assessed by methylene blue staining, was no
significantly affected by the washing treatment (inset).
and Sheila Lee for technical assistance, Nigel Davies and
Robert Muller for preparing the electron micrographs, Bo
Kara for assistance in the early part of this work, Jill
Caiderbank for assistance in the preparation of this paper and
the Director of the Brewing Research Foundation for
permission to publish. In addition, the contribution of the
many breweries which provided yeast samples for this study is
gratefully acknowledged.
Reference
1. Anon Allgemelne Braumelster Zcttung, 1942, 58, 1. Abstracted
Journal of the Institute of Brewing, 1942, 48 266.
2. Brewing Research Foundation, Annual Report. Journal of the
Institute of Brewing, 1967, 73, 120.
3. Brewing Research Foundation, Annual Report. Journal of the
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TABLE II. The Do's and Don'ts of Acid Washing

The Do's of Add Washing are:

1. Use food grade acid; preferably phosphoric or citric acid.

2. Chill the acid and the yeast slurry before use (<5°C).
3. Wash the yeast as a beer slurry or as a slurry in water.
4. Ensure constant stirring whilst the add is added to the yeast and preferably
throughout the wash. ""
5. Ensure that the temperature of the yeast slurry does not exceed 5°C during washing.
6. Check the pH value of the yeast slurry,
7. Pitch the yeast immediately after washing.

The Don'ts of Add Washing are:

8. Don't allow the temperature of the yeast to exceed 5°C during washing.
9. Don't wash yeast for more than two hours.
10. Don't store washed yeast.
11. Don't wash 'unhealthy' yeast (i.e. yeast which has been stored for long periods,
heavily contaminated yeast, or yeast from slow fermentations). If it is essential that
such yeast is washed, use a higher pH value in the wash and/or shorter contact times
until the yeast has been used to pitch one or more fermentations and recovers full
activity.
12. Avoid washing yeast from very high gravity fermentations (>8% v/vethanol.)
 354 THE RESPONSE OF BREWING YEASTS TO ACID WASHING
4. Bruch, C. W., Hoffman, A., Gosine, R. M. & Brenner, M. W.
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Brewing, in preparation.
6. Daoud, I. S. & Searle, B.A. Yeast Vitality and Fermentation
Performance. In: European Brewery Convention Monograph-
XII. EBC Symposium on Brewers' Yeast. Vuoranta (Helsinki),
Finland 1986. Verlag Hans Carl (Brauwett-Verlag), Nurnberg,
1987, plO8.
7. European Brewery Convention Analytica Microbiologica. Jour
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8. Feurstein, G. Wochenscrift fur Braurehe, 1911, 28, 16. Ab
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J. R. A. Pollock. Academic Press, London, 1981, p.l.
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11th Congress, Madrid 1967, Elscvicr Publishing Company,
Amsterdam, pl87.
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