J. Inst. Brew., March-April, 1992, Vol. 98,/>/>.

133-138 133

THE SENSITIVITY OF DIFFERENT BREWING YEAST STRAINS TO CARBON DIOXIDE INHIBITION:

FERMENTATION AND PRODUCTION OF FLAVOUR-ACTIVE VOLATILE COMPOUNDS

By L. Kruoer, A. T. W. Pickerell and B. Axcell

(The South African Breweries Limited, P.O. Box 782178, Sandton, 2146, South Africa)

Received 29 May 1991

The sensitivity of brewing yeast strains, with different oxygen demands, to carbon dioxide inhibition was
investigated. Laboratory fermentations were performed with, and without, protein-based "yeast foods"
to lower dissolved C02 during fermentation. Differences were observed in yeast fermentative per
formance in the presence and absence of "yeast foods" for all yeast strains tested. Fermentation
performance was improved with the addition of "yeast foods". There was improved carbohydrate
utilisation and amino acid uptake, while acetaldehyde levels at the end of fermentation were decreased.
There was an increase in fusel oil production and acetate ester levels at the end of fermentation. Sulphur
dioxide levels at the end of fermentation were unaffected by "yeast food" addition. Different yeast strains
displayed differing sensitivity to CO2 inhibition for all parameters tested. Sensitivity to C02 was not found
to be related to oxygen demand of the yeast strains.

Key Words: Yeast foods, carbon dioxide, fermentation, yeast
strain, flavour-active volaliles.
Introduction
The inhibitory and regulatory effects of CO, on yeast meta
bolism and fermentation have been extensively studied and a
number of review articles published13-30-34. Much of this work
centred around the use of a deliberately applied top pressure of
CO, to regulate fermentation24161722-24-31-35. More recent work
has looked at the effects of CO2 under milder conditions in the
absence of any deliberately applied top pressure3-l4-15-33.
Some of the effects of CO2 supersaturution that have
been reported include the following: inhibition of cell
growth2-"-14-15-16-22-24-25-32-", loss of cell viability14-22-33-35, reduced
fermentation rate814-24-33, increased cell volume2-6-14-22-35,
approximate doubling of DNA content18-25-35, increased RNA
content, decreased protein content, increased amino acid con
tent"*-" and changes in the lipid content and degree of un-
saturation of the cell membrane fatty acids6.
Knatchbull and Slaughter14 outlined the effects of low CO2
pressures on the production of flavour-active volatiles by yeast.
They showed that CO2 pressures of 0.5 and 1.0 aim (100 kPa)
lead to reduced fusel oil and ester concentrations whereas
acetaldehydc levels were increased. Kruger et /.15, using
protein-based "yeast foods" to lower dissolved CO, concen
trations in the fermenting wort, confirmed these results for their
specific yeast strain. The experiments described in this paper
were carried out to study the sensitivity of different brewing
yeast strains to CO, inhibition and to determine whether
sensitivity to CO2 was related to the oxygen demand of the
yeast strain.
Wort.-All wort used in this study was standard production
wort (original gravity 14.4P, 65% malt:35% maltose syrup)
collected after wort cooling.
Methods
Fermentations.Details of fermentation in 2 litre E.B.C. tall
tubes have been described previously3. Pitching wort contained
0.04 g/litre protein-based "yeast food". Wort containing no
"yeast food" served as a control. All fermentations were run in
duplicate at a constant temperature of 11C for 9 days.
Analytical Measurements.Measurement of yeast counts,
gravities and amino acids have been described beforcJ. Ester,
fusel oil and carbohydrate analyses have been described
previously15. Carbon dioxide was analysed according to the
method of Delente and Gurley9. Levels of sulphur dioxide were
analysed by the para-rosaniline method20. Acetaldehyde was
determined by the method of Delcour et a/.10
Results
% Dissolved CO2
With the addition of "yeast food" the levels of dissolved CO2
during fermentation did not rise much above saturation (0.19%
w/w under these experimental conditions). In contrast, in the
control fermentations the level of CO, reached was well above
saturation, ranging from 15 to 41% higher than control fer
mentations. A typical pattern for all yeast strains is shown in
Fig. 1.

Experimental

Materials
Yeast strains.The yeast strains employed for this study
were Saccharomyces cerevisiae strains obtained from the
culture collection of Alfred Jorgensen Laboratory,
Copenhagen, Denmark. The strains are referred to by their
catalogue numbers and fall in the following oxygen demand
groups (according to Jakobsen and Thome12).

Strain Oxygen Demand Group

1 4 10
2120, 3012 I
2155,2290 II FfrnanUtlon period (day*)

2091, 2036 III . Fig. I. Dissolved CO, in the presence (D); and absence (AA);
2283, 2298 IV of yeast food.

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 134 FLAVOUR ACTIVE VOLATILE COMPOUNDS
(a) Yeaat atrain 2120
Yul count (x tt>*/ml) Degree plalo
80
18
4 6 7 8
Fermentation parted (day*)
(b) Yeaat strain 2200
Viasl eount (x 107ml)
SO
8 6 7 8
Fermentation period (daya)
Fig. 2. Yeast Count (closed symbols); and attenuation (open symbols):
in the presence (); and absence (AA); of yeast food.
Gross Fermentation Parameters
All the yeast strains tested displayed sensitivity to CO2
inhibition, with regard to attenuation and yeast growth, to a
greater or lesser extent (Fig. 2). With respect to specific amino
acids, the uptake of threonine by all strains was unaffected by
CO2 supersaturation. However the uptake of valine, leucine
and isoleucine was slower and less complete under super
saturated CO2 conditions (Fig. 3). The results for the uptake of
the individual amino acids were combined to produce a pattern
of total amino acid uptake for each yeast strain. These results
show that amino acid uptake sensitivity to CO2 inhibition
varies for different yeast strains (Fig. 4). The uptake of glucose,
fructose, maltose and maltotriose was combined to produce an
overall pattern of carbohydrate utilisation for each yeast strain.
As was demonstrated for amino acid uptake, the relationship
between carbohydrate utilisation and CO2 inhibition is also
strain dependent (Fig. 5).
Referring to the % inhibition due to CO2 in control ferment
ations relative to "yeast food" supplemented fermentations for
the parameters measured (Table I), it is evident that inhibition
of attenuation parallels inhibition of carbohydrate utilisation.
However, there is no direct relationship between inhibition of
yeast growth and retarded or incomplete attentuation. There is
also no direct correlation between inhibition of amino acid
uptake or carbohydrate utilisation and reduced yeast growth.
Sensitivity to CO2 inhibition does not appear to be related to
the oxygen demand of the yeast strain (Table I).
Production of Volatile Compounds
SO2 levels at the end of fermentation were unaffected by
dissolved CO, levels (Table II). SO2 levels were very similar for
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[J. Inst. Brew.
(a) Threonine and leucino
mg/lltre
8 8 7 8 9
Fermentation period (day*)
(b) Vallne and Isoleucine
mo/lltre
28488789
Fermentation period (day*)
Fig. 3. Uptake of threonine (D); leucine (AA); valine (OO);
and isoleucine (VV); in the presence (closed symbols); and
absence (open symbols); of yeast food.
all yeast strains tested. Acetaldehyde levels at the end of
fermentation were generally considerably higher in control
fermentations compared with "yeast food" supplemented fer
mentations. The increases in acetaldehyde were not related to
the oxygen demand of the yeast strains. Different yeast strains
showed a wide range of final acetaldehyde levels (Table II).
Fusel oil and ester production was reduced in control ferment
ations compared with "yeast food" supplemented ferment
ations for all the yeast strains tested. The actual decreases
varied between yeast strains and also between the various fusel
oils and esters but were not related to the oxygen demand of the
yeast strains (Tables III and IV).
Discussion
Improved fermentation performance has been reported due
to agitation/stirring of fermenting wort19-32-36, trub addition33
and the addition of protein-based yeast foods15. Improved yeast
growth and faster attenuation rate were demonstrated under
these conditions. These effects were attributed, in most in
stances1519-3233, to lowering of dissolved CO2 during ferment
ation. The results of this study have also demonstrated im
proved fermentation performance due to lowering of dissolved
CO2 during fermentation for a range of brewing yeast strains.
There have been varying reports in the literature on the
effects of an applied top pressure of CO2 on fermentation rate.
Rice el al.31, Nielsen el al.22 and Knatchbull and Slaughter14
reported little if any effect on fermentation rate. Kumada et
al.17 showed that both the rate and extent of attenuation were
adversely affected by CO2 for fermentations conducted at 9C
under 1 atm of CO2 pressure relative to a control with no CO2
pressure. Arcay-Ledezma and Slaughter2 reported a slower
Vol. 98. 1992] FLAVOUR ACTIVE VOLATILE COMPOUNDS 135

(a) Ysast strain 2091 (a) Yaat strain 2283

gmtn

s a t a 9 8 8 T 8 8
FernenUUon period (day*) Fermentation parted (daye)

(b) Yeast strain 2290 (b) \fcast strain 2298

g/tllr*

8 8 r 8 8 8 a 7 8 8
Fermentation period (daya) Fermentation parlod (daya)
Fig. 4. Total amino acid uptake in the presence (D---G); and absence Fig. 5. Carbohydrate utilisation in the presence(DD); and absence
(AA); of yeast food. (A- A); of yeast food.

TABLE I Sensitivity to COi

i Inhibition by CO;

Amino Acid Carbohydrate

Oxygen Uptake Utilisation Attenuation
Yeast Extent
Strain Group Rale Extent Rale Extent Rale Extent Yeast Growth

2I2O I 17 14 40 53 23 35 65
3012 I 27 14 27 1 19 0 See note 3
2155 11 7 14 20 4 15 5 18
2290 II 13 14 0.6 0.3 9 6 48
2036 III 24 15 21 II 18 9 8
39 41 43 4 22 0.8 22
2091 III
2283 IV 30 30 28 5 21 2 49
2298 IV 12 13 24 30 15 19 17

Note 1: All rates calculated on day 4 of fermentation.

Note 2: Extent calculated on day 9 of fermentation.
Note 3: This yeast strain forms chains and clusters and was therefore difficult to quantify.

fermentation rate with CO2 pressure, but suggested that this
was a result of reduced yeast growth. These differences may be
explained by the differing sensitivity of various yeast strains to
inhibition of attenuation due to CO:. Yeast strain 2120 is very
sensitive to CO: inhibition with respect to both the rate and
extent of attenuation whereas strain 2290 is relatively in
sensitive to CO2. With strain 2091, the rate of attenuation is
very sensitive to CO2 inhibition while the extent of attenuation
is virtually unaffected by CCK. These results would suggest that
different effects would be observed for inhibition of attenuation
by CO2, dependent on the sensitivity of the particular yeast
strain employed.
Under the conditions employed in this study retarded attenu
ation cannot be directly attributed to inhibition of yeast
growth. Retarded attenuation parallels the slower and less
complete uptake of carbohydrates during fermentation. Sur
prisingly, inhibition of amino acid uptake and inhibition of
carbohydrate utilisation does not directly correlate with in
hibition of yeast growth, although both factors probably con
tribute to inhibition of yeast growth. It would appear that there

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 136 FLAVOUR ACTIVE VOLATILE COMPOUNDS [J. Inst. Brew.

TABLE II Acctaldehydc and SO > results

Day 9 of fermentationi (mg/litre)

Oxygen Acelaldehyde SO,

V ct

Strain Group + Yeast Food Control + Yeast Food Control

2120 I 16.2 26.6 2.6 2.6

3012 I 8.6 11.7 2.9 2.9

2155 11 10.7 14.5 2.7 3.3

2290 11 12.8 15.4 2.5 2.5

2036 HI 3.9 12.9 2.9 2.8

2091 III 3.6 7.1 2.5 2.7

2283 IV 3.0 10.8 2.8 2.8

2298 IV 20.0 22.8 2.8 2.8

TABLE 111 Higher alcohol and ester results Day 9 of fermentation (nig/litre)

Yeast iStrain

Group 1 Oxygen Demand Group II Oxygen Demand

2120 3012 2155 2290

1-

Yeast Yeast Yeast Yeast

Compound Food Control Food <rontrol Food Control Food Control

n-propanol 22 15 22 21 25 22 24 20
ethylacetate 6 1.8 12 6 12 8 8 6
iso-butanol 21 12 12 II 34 T> n
16
3-methyl-butanol 36 28 54 49 66 58 62 55
2-methyl-butanol 23 12 18 12 23 17 21 19
iso-amyl acetate 0.45 0.14 0.41 0.24 1.2 0.5 0.59 0.31
total higher
alcohols 102 67 106 93 148 119 129 110

TABLE IV Higher alcohol and ester results Day 9 of fermentation (mg/litrc)

Yeast Strain

Group HI Oxygen Demand Group IV Oxygen Demand

2036 2091 2283 2298

Yeast Yeast Yeast Yeast

Compound Food Control Food Control Food Control Food Control

n-propanol 31 24 24 16 22 13 22 18
clhyl acetate 9 8 12 6 12 6 6 3
iso-butanol 30 20 19 7 30 16 23 16
3-methyl-butanol 65 60 55 34 96 50 42 34
2-methyl-butanol 32 24 18 11 29 15 21 15
iso-amyl acetate 1.1 0.62 1.03 0.31 1.0 0.17 0.41 0.17
total higher
alcohols 157 128 116 68 177 94 108 83

is some other rate limiting occurrence due to CO: that is
primarily responsible for reduced yeast growth. Castelli el a/.6
found an increase in total fatty acid content of the cell
membrane from 9.4% to 14% after 6 hours exposure to CO:.
The proportion of unsaturated fatty acids increased from
47.4% to 67.3%. It is possible that these changes in the lipid
composition of the yeast cell membrane contribute to the
inhibition of yeast cell division. It is also possible that different
yeast strains display different sensitivity to lipid compositional
changes in the presence of elevated CO: levels.
Narziss el al.21 showed different patterns of sulphur dioxide
production during fermentation using three different yeast
strains. However, under the conditions employed in this study
no substantial differences were found in the final total SO:
levels for the eight yeast strains tested. SO: production was also
insensitive to CO2 inhibition.
These results support the findings of Brewer and Fenton5
who demonstrated that there was no substantial difference in
end of ferment SO: levels between a fermentation conducted
under CO: pressure relative to a control fermentation at
atmospheric pressure. Dufour el al." have suggested that the
lower the residual amino acid content in green beer, due to

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Vol.98, 1992) FLAVOUR ACTIVE VOLATILE COMPOUNDS
improved uptake of Group II and Group III amino acids, the
lower would be the levels of SO2 in green beer. Results of this
study show that although amino acid uptake is inhibited by
CO2 there is no corresponding increase in SO, values. Aeration
of pitching wort and lipid addition to pitching wort have been
shown to reduce the formation of SO2'-5-21-26. In the experiments
reported by Dufour el /." reduced amino acid uptake and
increased SOs production occurred in worts of limited lipid
content. Pickerell ct /.28 reported reduced Group II and III
amino acid uptake in fermentations with yeast of sub-optimal
physiological status. This was ascribed to malfunction of the
cell membrane initialed by inadequate pitching yeast glyco-
gen reservesthe same rationale was used to explain over
production of sulphur dioxide. The work reported in this study
would suggest that the primary reason for reduction of SO: in
the experiments conducted by Dufour el/." was the increased
lipid content of (he wort which stimulated yeast membrane
activity. Under conditions of identical aeration, wort lipid
composition and good yeast physiological status SO: pro
duction was unrelated to amino acid uptake in all the yeast
strains tested.
CO2 pressure has been reported to result in an increase in
acctaldchyde over control fermentations2-17-22. In this study
increased acetaldchyde production under supersaturated CO;
conditions has been confirmed. All the yeast strains tested
showed increases in acetaldehyde levels in control ferment
ations relative to yeast food supplemented fermentations. The
magnitude of the increases was yeast strain dependent. Accord
ing to Jones and Greenfield" the reaction pyruvate-*
acctaldehyde is insensitive to CO? inhibition. However, the
reaction pyruvale-acetyl-CoA. gluconale-6-P-ribulose-5-P
and isocitrate-oxoglutarate are sensitive to CO2 inhibition.
The increased acetaldehyde in control fermentations could
therefore be explained by the accumulation of pyruvate due to
inhibition of the formation of ribulose-5-P. oxoglutarale and
acelyl-CoA by the repressive effects of CO2. Accumulation
of pyruvate could lead to increased acctaldehyde formation.
Slaughter31 has suggested that CO: renders the cell membrane
more permeable to acetaldehyde so the concentration in the
medium increases co-incidcnlally. Changes in membrane
properties of the cell are likely to contribute to most of the
effects that have been demonstrated due to CO:. Prasad &
Rose29 have reported altered solute transport in yeast cells
resulting from changes in cell membrane lipids.
Nordlov33 demonstrated that addition of SO2 at the begin
ning of fermentation led to increased acctaldehyde production.
He suggested that acetaldehyde production depends on the
amount of SO2 formed. Pickcrell et al.s demonstrated that
yeast in poor physiological condition produced high levels of
sulphur dioxide during fermentation, which was accompanied
by high acetaldehyde levels. It was suggested that high levels of
sulphur dioxide determined the level of acetaldehyde remaining
at the end of fermentation (through the formation of stable
adducts). The results of this study suggest that in fermentations
employing yeast strains in good physiological condition, that
produce low levels of SO:, acctaldehyde production is in
dependent of SO: production. All SO: values at the end of
fermentation were very similar for the yeast strains tested.
However, acetaldehyde values range from 3.0 to 26.6 mg/litre.
It would appear that yeast strains with poor fermentation
efficiency produce high levels of acetaldehyde.
It is well established that CO2 inhibits the synthesis of higher
alcohols and acetate esters2ui5l7"24"M"1. Knalchbull and
Slaughter14 stated that it is unlikely that CO: affects fusel oil
production through its effect on amino acid uptake, as threo-
nine, the precursor of n-propanol, was unaffected by CO2. The
absorption of the branched chain amino acids, precursors of
isobutanol and iso-amyl alcohols, was markedly reduced. How
ever, considering the relative contribution of the Ehrlich and
biosynthetic pathways to (he formation of fusel alcohols7 it is
probable that the increased production of fusel oils is as a result
of a combination of improved amino acid uptake and carbo-
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137
hydrate utilisation in yeast food supplemented fermentations
relative to control fermentations. Although Ihreonine uptake
for all yeast strains tested was unaffected by CO2, only 30% of
n-propanol is derived from this amino acid7. Increased valine
and carbohydrate uptake could account for the increased
iso-butanol and n-propanol production. Increased 3-methyl-
butanol and 2-methyl-butunol would be primarily due to
increased leucine and isolcucine uptake respectively. The
primary acetate esters, ethyl acetate and iso-amyl acetate are
formed by a reaction between acelyl-CoA, cthunol and 3-
melhylbulanol respectively. Reduced levels of acetyl-CoA due
to CO: inhibition and decreased clhanol and 3-methylbutano!
would be expected to lead to a decrease in their corresponding
esters in control fermentations. Alternatively, or additionally,
CO: membrane effects may retard activity of acelyl alcohol
transferase (AAT) which is believed to be a controlling factor in
ester formation27. The results of this study have confirmed that
the formation of higher alcohols and acetate esters is inhibited
by CO2 for all the yeast strains tested. The actual inhibition is
yeast strain dependent and also varies between the different
higher alcohols and esters. In general iso-amyl acetate pro
duction appears more sensitive to CO; inhibition than ethyl
acetate production.
Conclusions
The results of this research have demonstrated that different
lager brewing yeast strains have different sensitivities to CO2
inhibition. Sensitivity to CO: inhibition could in no way be
related to the oxygen demand of the yeast strain.
Acknowledgements: The authors would like to thank the
Directors of the South African Breweries Limited for
permission to publish this paper. They would also like to
acknowledge the technical assistance of the Analytical
Chemistry Department, Research and Development, South
African Breweries; particular thanks to Mrs R. Luck, Mrs A.
Frascr and Mrs M. Phawcni.
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