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

Yeast Effects of Ethanol

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

60
Duplicate fermentations were carried out in tall tube
0 20 40 60 60 100 120
fermenters at 18°C for ale yeasts and 12°C for lager yeasts as
described previously16 (pitching rate 2.5 g wet wt. yeast Time (mln.) since addition of aold
Litre', all-malt wort, OG 1040° (ale), or containing 20%
adjunct OG 1038" (lager), pH 5.2; air saturated (at 12°C or Fig. 1. Effect of acid washing (H3PO4, pH 2.1,4°C) on the viability of
18°C respectively) before pitching). Parameters monitored Sacch. cerevisiae NCYC168 (O), strain L (A), strain H (+) and
were, i) specific gravity, (PAAR DMA 45 Digital density strain 1 (D) as assessed by mcthylcne blue staining.
meter), ii) total cell count, (Thoma haemocytometer), iii) cell
viability (methylene-blue staining),7 and iv) pH value (Corn We have presented our data as linear plots (viability vs time)
ing pH meter and combination all-glass electrode). in order to highlight differences between treatments. High
viability was maintained during washing regardless of
Assessment of Yeast Flocculation (in vivo)
whether the yeast was washed as a thick slurry. (~60%
Flocculation characteristics of washed and unwashed
packed cell volume) or as a dilute suspension (5% packed cell
strains during fermentation were evaluated by plotting the
volume).
suspended cell count against % attenuation as suggested by
ii) Specific oxygen uptake rate (qCb): the qO2 value of
Stewart & Russell.29
'healthy' Sacch. cerevisiae NCYC 1681 was not significantly
Fining Tests affected by phosphoric acid washing at pH 2.1. 'Unhealthy'
Fining tests were performed only on ale fermentations, yeasts had lower qO2 values prior to acid washing, but no
since, in practice, lagers are not fined in cask. After primary change occurred after acid washing even though the fer
fermentation, beer was decanted off the yeast sediment using mentation performance of this yeast was significantly affected
a glass syphon-tube and nitrogen top-pressure. The use of a by the washing procedure. Washing 'healthy' yeast with the
glass syphon tube allowed the beer to be decanted in a acidified ammonium persulphate method resulted in a small
consistent fashion. After discarding the first 150-200 ml, the reduction in qOj but this was not reflected in the subsequent
beer (800 ml) was transferred into 1 Litre glass vessels and 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 Flo. 3. Scanning electron micrographs of Sacch. cerevisiae NCYC
these yeast(s) in the fermentation immediately after washing. 1681 before (a) and after (b) washing with acidified ammonium
v) Flocculation: In the presence of acid, yeast cells of all persulphate, at 4°C. Scale bars represent lOjim.
strains dcflocculated. However, if the cells were washed in
buffer and resuspended in beer, or buffer containing suffi
Acid washing makes brewing yeast 'sticky'. After washing,
cient calcium ions, then flocculence was only slightly dimi
yeast from centrifuged pellets can be pulled out into short
nished. In contrast, treatment with ammonium persulphate
(1-2 cm) strands; untreated yeast does not exhibit this
resulted in a loss of flocculence that could not be reversed by
phenomenon (Figure 5). This also shows that acid washing
these procedures. However, flocculence loss induced by
modifies the surface properties of the yeast cell, although, as
ammonium persulphate is not of great signficance to the
discussed above, it appears that regeneration of these
brewing process since the ability to flocculate is regenerated
properties during fermentation minimises their process sig
during the fermentation following the wash (see below, and
nificance.
Figure 6). Solutions of ammonium persulphate decompose to
form ammonium sulphate, sulphuric acid and ozone. Since Laboratory-Scale Fermentations
similar effects were not induced by ammonium sulphate or No significant differences in the fermentation behaviour
sulphuric acid, it is possible that the surface properties of (lag phase, attenuation rate, flocculation, pH profile) be
Sacch. cerevisiae NCYC 1681 were changed by ammonium tween unwashed and washed yeasts were observed providing
persulphate due to the release of ozone, a stong oxidising the yeast was washed under ideal conditions. Typical fer
reagent. Some oxidising agents such as periodate13 are known mentation profiles are shown in Figure 6. Fermentation
to modify the flocculation properties of brewing yeast. But performance of Sacch. cerevisiae NCYC 1681 was unaffected
others, such as potassium permanganate or hydrogen perox even when this yeast was washed with phosphoric acid (pH
ide failed to affect flocculence in our experiments. Thus the value of 1.88, 4°C) for 22 hours.
accessibility of yeast surface moieties to the oxidising agent The top-cropping ale yeasts formed a significant head
may be important in addition to the chemical composition during fermentation in the tall tube fermenters; the appear
and strength of the oxidising agent. ance of which was the same in fcrmenters pitched with acid
Electron micrographs of Sacch. cerevisiae NYCY 1681 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.

Fining Performance
P Poor fining manifests itself in the production of hazy beers.
In no case, however, did add washing affect fining perform
ance as judged by haze values (Table 1). Indeed, in some
Fig. 5. •Stickiness' of acid washed Sacch. cerevisiae; NCYC 1681. ««*the beer produced by the washed yeast fined better than
Control (unwashed) yeast (left) cannot be pulled out in strands; «« control. Parallel experiments earned out using respira-
acid washed yeast (right) is sticky and can be pulled out in short y
tory-defident variants of NCYC 1681 ((one a spontaneous
p
strands. 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 had been washed at 4°C, performed satisfactorily but those
detect differences between yeasts which fined well and those inoculated with yeast which had been washed at 24°C
which fined poorly (Table 1). The material responsible for the performed poorly (Figure 7).
haze in beers produced by respiratory-deficient variants Death rates of both ale and lager yeasts during acid
appeared to be proteinaceous since it was soluble in strong washing were strongly influenced by temperature:- the
alkali and stained pink with eosin yellow. Although these greater the temperature, the faster the rate of death (Figure
results provide no evidence for inferior fining performance by 8). Death rates under 'ideal conditions' (4°C) were insignifi
acid-washed yeasts, they should be treated with a degree of cant.
caution since surface-associated phenomena can be greatly
influenced by scale effects. Only experiments carried out at NCYC 1681
brewery scale can fully resolve the question of the effect of
% oells unstained
acid washing on fining performance.

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

washing. Viability as assessed by methylene blue staining, was
Fig. 7. Response of Sacch. cerevisiae NCYC 1236 to acid washing at determined over a period of two hours at 4°C (D); 10°C (+);
4°C and 24°C. Unwashed yeast (O); phosphoric acid washed 16°C (O); 20°C (A) and 25° (V). One ale strain (Sacch.
yeast, pH 2.1,4°C D; 24°C(A). The values shown represent the cerevisiae NCYC 1681) and one lager strain (Sacch. cerevisiae
mean of duplicate fermentations. NCYC 1324) were examined.
352 THE RESPONSE OF BREWING YEASTS TO ACID WASHING [J. Inst. Brew.

Effect of Ethanol Spec!do gravity

Acid or ethanol alone have little effect on viability of (a)
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
1.02
physiological condition (see below).

cells unstained
100
1.01
40 80 120 ISO

Time (hours)

Specific gravity

(b)

BO 120

Time (hours)

40 80 120 160 Fig. 10. The effect of yeast condition on response of Sacch. cerevisiae
strain L 10 add washing (phosphoric acid pH2.1; 4°C, 2h).
Tims (mln.) since addition of acid Fermentations were carried out with washed (A) and unwashed
(D) yeast, (a) and (b) represent data obtained from "unhealthy"
and "healthy" yeast respectively.
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 Yeast propagated using the normal protocol16 was unaffected
(+), (c) 0.6% v/v H3PO4 containing 10% v/v ethanol (A) or (d) by acid washing but that propagated by the stressful protocol
10% ethanol (0). Viability was monitored, using methylene fermented poorly after acid washing (Figure 11). In particu
blue, at intervals up to an including three hours. Ethanol acted
lar, the lag phase was extended. Interestingly acid washing
synergistically with the acid to kill the cells.
did not influence the viability or qO2 value of the stressed
yeast, although low values were obtained for both these
Influence of Physiological Condition on the Response of Yeast parameters prior to acid washing.
to Acid Washing.
Ale yeast obtained from brewery L responded poorly to Conclusions
both phosporic acid and acidified ammonium persulphate. The results presented here indicate that sensitivity of yeast
Fermentation rates were reduced by both treatments (Figure to acid washing is governed predominantly by environmental
10a). In addition, the viability and qOj value of the yeast (phenotypic) factors rather than inherited (genotypic) fac
dropped during washing. Significantly, this particular batch of tors. Brewing yeasts, both ale and lager, are highly resistant
yeast had a low viability and poor fermentative activity before to acidic environments but the extent of this resistance is
commencing the wash. However, when this yeast was reduced by increased temperature and the presence of
propagated by out standard protocol16 the viability, fermenta ethanol. In addition, 'healthy' cells (i.e. those capable of
tive activity and response to acid washing (Figure 10b) all performing satisfactorily in fermentation) are more resistant
improved significantly. to the effects of acid than are 'unhealthy' cells (i.e. those
Confirmation that the physiological condition of the yeast incapable of performing satisfactorily in fermentation). From
is important in resistance to acid washing was obtained as these conclusions, practical guidelines for acid washing of
follows. Pilot brewery ale yeast, Sacch. cerevisiae NCCY yeast can be derived (Table 2).
1681, which is resistant to the effects of acid washing was The choice of acid to acidify the yeast is determined by
propagated using conditions likely to stress the organism. The several factors. These included acceptability of the acid as an
stress involved exposure to low levels of nitrite (such as would additive or processing aid, cost of the acid, handling
occur if the fermentation was contaminated with nitrate- characteristics of the acid (with particular regard to safety),
reducing bacteria), prolonged storage under beer at high and effectiveness of the acid as a yeast washing agent.
(23°C) temperature and underpinning of the fermentation. 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

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
40 80 120 Caiderbank for assistance in the preparation of this paper and
the Director of the Brewing Research Foundation for
Time (hours) permission to publish. In addition, the contribution of the
Fig. 11. The response of "healthy" and "unhealthy" Sacch. cerevlsiac many breweries which provided yeast samples for this study is
NCYC 1681 to acid washing. "Healthy" (A) or "unhealthy" (D) gratefully acknowledged.
Sacch. cercvisiae NCYC 1681 prepared as described in the text
was washed at 4°C with phosphoric acid (pH 2.1). Fermentation Reference
profiles were plotted for unwashed yeast (solid line) and for 1. Anon Allgemelne Braumelster Zcttung, 1942, 58, 1. Abstracted
washed yeast (broken line). The fermentation rate of the Journal of the Institute of Brewing, 1942, 48 266.
"healthy" yeast was unaffected by acid washing, but that of the 2. Brewing Research Foundation, Annual Report. Journal of the
"unhealthy" yeast was significantly reduced by the treatment. Institute of Brewing, 1967, 73, 120.
The viability, as assessed by methylene blue staining, was no 3. Brewing Research Foundation, Annual Report. Journal of the
significantly affected by the washing treatment (inset). Institute of Brewing, 1968, 74, 126.

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 [J. Inst. Brew.

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