Research article Institute of Brewing & Distilling

Received: 16 October 2015 Revised: 3 February 2016 Accepted: 23 March 2016 Published online in Wiley Online Library: 27 April 2016

(wileyonlinelibrary.com) DOI 10.1002/jib.331

A new rapid high-throughput method for

prediction of beer colloidal stability
P. Gabriel,1* P. Sladký1 and K. Sigler2
A rapid high-throughput test for prediction of beer colloidal stability has been developed and evaluated. The method is based on
the procedure of aging accelerated by high temperature (60 °C). The aging process is accelerated through decarbonization and de-
fined oxidation of the sample. A significant correlation was observed between the results of this rapid test and a standard accelerated
aging test. The method can be used as a rapid and simple test for checking the efficiency of the stabilization process. The duration
and sensitivity of the test can be modified by adjusting the test parameters. Copyright © 2016 The Institute of Brewing & Distilling

Keywords: colloidal stability; beer stabilization; forced aging test

Introduction of sensitive proteins (13,16). Nephelometric titration tests belong

During storage, oxidizing and reducing reactions take place in beer
samples under the influence of several physical and chemical fac-
tors including temperature, light and dissolved oxygen. Oxidation
reactions change the chemical composition of the sample and
bring about sample aging, which influences colloidal and sensorial
stability. The colloidal system is displaced away from the equilib-
rium, some compounds aggregate and haze develops. The most
common reason for non-biological haze formation in beer during
storage is aggregation of haze-forming polyphenols with
haze-forming proteins (1).
Current technological procedures make it possible to limit the
haze formation over time so that some produced beers can have
a shelf-life of one year or more. Brewery experts in quality control
have long been trying to find methods that will allow them to pre-
dict changes in the colloidal system of beer with high reliability
even before it is shipped from the brewery (2–4). Accelerated or
forced beer maturation that makes use of various methods of heat
shock (5–7) is relatively reliable but time consuming. The length of
the test for beer with a shelf-life of 9–12 months is at least 7 days.
The current requirements for maintaining the beer properties
unchanged for long periods of time and the commonly encoun-
tered transport of beer over long distances call for methods
useful for the fast prediction of colloidal stability. Rapid tests of
colloidal stability include, for instance, haze precipitation tests
in which the addition of suitable agent initiates precipitation of
haze-forming substances, most often proteins, polyphenols or
protein–polyphenol compounds. The resulting turbidity is mea-
sured, and the parameter for assessment is either a value of haze
formed or the amount of added reagents needed to reach a cer-
tain turbidity (8). These tests include a sulphate test (9), a formal-
dehyde test (10), precipitation with magnesium sulphate (6),
Esbach test (11), precipitation with trichloroacetic (6), Chapon’s
chill haze test (12) and others (2). Precipitation tests are very fast
and the results may be obtained within tens of minutes to sev-
eral hours. The correlation of the results with practical shelf-life
is, however, significantly lower than in forcing tests.
A special category of precipitation tests are nephelometric titra-
tion tests. These tests include a test on the content of Chapon’s
tannoids (13,14), its modifications (15) and the test of the content
304
to methods that measure directly the content of beer haze precur-
sors. A specific reagent is titrated to the sample during the course
of the test; this reacts with haze precursors, giving rise to measur-
able haze. Unlike other precipitation tests, a reagent that reacts
with haze-forming precursors in the same manner as individual
haze precursors react with each other during aging is dispensed
into the sample. The detection of haze-forming polyphenols is per-
formed by adding polyvinylpyrrolidone (PVP – protein analogue,
Chapon’s test), while the detection of haze-forming proteins
makes use of tannic acid (test for sensitive proteins). Test results
correlate with natural shelf-life better than the results of other pre-
cipitation tests, hence they are used more and more in practice.
The results of nephelometric titration tests can be used to predict
the colloidal stability of the sample; calculation parameters are dif-
ferent for different types of beer and it is necessary to evaluate
them for each beer type separately (4).
Here we describe an accelerated oxidative aging test as an
alternative or supplement to the nephelometric titration tests. Ac-
celerated aging of the samples in comparison to the standard
bottled forcing test is achieved by oxidation and decarbonization
of defined samples. The test can be performed with part of the
samples prepared for titration nephelometric assays of the con-
tent of haze-forming precursors. A defined volume of the sample
(10 mL) decarbonized in the standard manner is dispensed into a
30 mL sealable test tube, and placed in the thermostabilised wa-
ter bath at 60 °C for a defined period. Accelerated aging of the
samples, which occurs at this temperature, is caused by dissolved
oxygen that enters the sample during decarbonization and dur-
ing the accelerated aging process from the air above the sample.
The maximum attainable dissolved oxygen concentration at
room temperature is about 8 mg/L (8000 ppb). The air in the tube
* Correspondence to: P. Gabriel, Faculty of Mathematics and Physics, Charles
University, 121 16 Prague 2, Czech Republic. E-mail: gabriel@karlov.mff.cuni.cz
1
Charles University, Faculty of Mathematics and Physics, 121 16 Prague 2,
Czech Republic
2
Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20
Prague 4, Czech Republic

J. Inst. Brew. 2016; 122: 304–309 Copyright © 2016 The Institute of Brewing & Distilling
Method for prediction of beer colloidal stability
above the sample contains an amount of molecular oxygen that
exceeds by an order of magnitude the maximum attainable level
of dissolved oxygen in the sample. Although dissolved oxygen in
the sample is consumed during accelerated aging at elevated
temperature, the air above the sample acts as a reservoir suffi-
cient for its replenishment. Even with standard heat shock test
atmospheric oxygen enters the samples in bottles but only in
ppb quantities.
Following this ‘warm’ phase, the samples are transferred to 0 °C
to induce the formation of a chill haze. The chill haze is measured
after a defined period by a hazemeter. Its intensity corresponds to
the colloidal stability of the system and correlates with the size of
chill haze produced in a bottled sample during the course of the
standard accelerated aging procedure.
It is possible to adjust the duration and the sensitivity of the test
by changing the length of time the sample tubes spend in the cold
and hot bath. Even with beer stabilized to a 12 month shelf-life,
one can get a result within 36 h.
Materials and methods
Apparatus
All measurements of sample haze were performed on a Dual Angle
Turbidimetric and Titrimetric System (DATTS 2000) instrument
constructed at the Faculty of Mathematics and Physics, Charles
University, Prague. It represents a modular system where a wide
range of external modules (dosing peristaltic pumps, dosing piston
pumps, external temperature stabilizing unit, magnetic stirrer, pH
meter, ion-selective meter, etc.) can be used for different haze
measurement tasks.
The fundamental component of the apparatus is the MZN 2009
hazemeter (1-Cube s.r.o.), which measures scattered light intensity
simultaneously at two angles to the incident light beam in a wide
concentration range of scattering particles. The hazemeter is
equipped with an LED light source with 650 nm medium wave-
length according to MEBAK (5). Simultaneous haze measurement
at a 90o angle (nephelometry – H90) and at a forward 12o angle
(forward scattering – H12) provides information about the mean
particle size or shape change during the course of the precipitation
reaction. Samples can be measured in standard glass cuvettes
of 150–250 mL volume, in commercially packed bottles of
0.33–0.5 L volume and, with a special adapter, also in vials of sam-
ple volume from 5 mL. The measuring chamber, which is filled with
distilled water as an immersion liquid, can be temperature stabi-
lized by an external circuit.
The hazemeter communicates with a PC via serial port RS232
and is controlled by the special PC software MZN Control, which
enables the user to perform various titration tests including the
‘Tannoid content test according to Chapon’. A wide variety of set-
ting parameters can be chosen and test results can be evaluated
automatically or semi-automatically from the curve drawn on the
display. One of DATTS 2000 possible configurations is displayed
in Fig. 1.
A 1-Cube BT thermostat was used in the water circuit to main-
tain a temperature of 25 ± 0.2 °C in the measuring chamber. An
adapter for vials of 2 cm diameter was fitted with waterproof mag-
netic stirrer. A software-driven piston dosing pump (accuracy
± 1%) was used for titration agent dosing. A 1-Cube Refrigerated
/Heating Circulator type SU12.1 was used to perform accelerated
aging procedure in bottles.
J. Inst. Brew. 2016; 122: 304–309 Copyright © 2016 The Institute of Brewing & Distilling
Institute of Brewing & Distilling
Figure 1. Dual angle turbidimetric and titrimetric system (DATTS).
Chemicals
PVP K90 and Polyclar Super R were kindly donated by ISP Global
Technologies Deutschland GmbH. Ammonium sulphate and
tannic acid were purchased from Sigma Aldrich. Beer samples
of Pilsner type with various content of haze-forming precursors
in NRW 0.5 L bottles were prepared and donated by Czech
breweries. Some samples were purchased in retail outlets. Water
(conductance below 0.1 μS) was obtained from a reverse osmosis
purification device AQUAL 35 (Miroslav Šustr Co., Brno). A 0.4 g/L
stock solution of PVP K90 was prepared for tannoid content test.
A 0.2 g/L solution of tannic acid (prepared fresh daily) was pre-
pared for conducting the test for sensitive proteins. A sensitive
proteins test was performed according to EBC–Analytica (16),
and a tannoid content test was performed according to a modi-
fied procedure by pre-dosing the sample with saturated ammo-
nium sulphate (15).
Accelerated aging procedure
Accelerated aging of beer samples in bottles was performed by
storage at elevated temperature according to MEBAK–Analytica
(7). Haze was measured on DATTS apparatus in a nephelometric
mode. Samples were aged in 2 day cycles. On the first day of each
cycle (24 h) the samples were placed in a water bath kept at 60
± 0.5 °C. During the second 24 h period the samples were stored
at 0 ± 0.2 °C. Total haze at 0 °C was measured (H0) and the cycle
was repeated until total haze exceeded the original haze
measured at room temperature (H25) by 2 EBC units. The colloidal
lifetime T2 of the sample was defined as the number of warm days
(WD) necessary to reach this point. T2 was calculated by linear
interpolation between the last cycle (denoted i) after which H0(i) is
under the 2 EBC increment limit and the first cycle (denoted
i + 1) in which H0(i+1) exceeds it.



T 2 ðdaysÞ ¼ i þ 2 þ H25  H0 ðiÞ = H0 ðiþ1Þ  H0 ðiÞ
Chill haze Hchill is calculated as a difference between total haze
measured at 0 °C (H0) and the original haze measured at room tem-
perature (H25)
Hchill ¼ H0  H25
305
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Institute of Brewing & Distilling
Accelerated oxidative aging procedure
(1) Samples were decarbonized in a standard manner, for exam-
ple by 20 min shaking.
(2) A 10 mL sample was injected into a tube, which was then
closed. Any tube with closure could be used for the measure-
ments (we used 30 mL closed tubes with screw caps, 20 mm
in diameter).
(3) Turbidity was measured at room temperature  H25.
(4) Samples were placed for a Thot (h) time period into a liquid
thermostat at 60 °C.
(5) Samples were transferred for a Tchill (h) period into a liquid
thermostat at 0 °C.
(6) Turbidity is measured at 0 °C  H0.
(7) Calculate chill haze Hchill = H0  H25.
Results and discussion
Basic parameters of the proposed oxidative accelerated aging test
were verified on 15 lager beer samples of several brands from
one brewery. Two brands of beer were divided into five batches
(‘Brand 1’, ‘Brand 2’ in Figs 2 and 4); other brands were only divided
into one or two batches (‘Other brands’). Samples of different
brands and batches differed in gravity and alcohol content. Forced
aging of bottled samples was performed according to the MEBAK
methodology (7) and T2 (number of warm days at 60 °C until chill
haze of 2 EBC is reached), was determined and the tannoid
content test with addition of ammonium sulphate (TC) and a test
on the content of sensitive proteins (SP) were performed.
Decarbonized samples prepared for titration assays were also used
to carry out the accelerated aging test in tubes. Three tubes were
prepared from each sample.
The tannoid content in tested beer samples ranged from 14.4 to
27.1 mg/L (mean 22.5 mg/L); the content of sensitive proteins var-
ied from 5.2 to 10 EBC (mean EBC 7.4). Differences in contents of
haze-forming precursors corresponded to the difference in the col-
loidal stability measured in bottled samples (T2) in the range from
chill
Figure 2. Comparison of chill haze measured in bottled samples after five warm days (H
chill
306
60 °C (H OXI 5 h).
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P. Gabriel et al.
3.7 to 13.0 WD (average 6.4 WD). The dissolved oxygen content in
the samples was in the range from 30 to 80 ppb.
To determine the effect of oxidation and decarbonization of the
sample on accelerated aging, we compared the chill haze mea-
sured in bottled samples during the course of standard acceler-
ated aging procedure with that measured in decarbonized and
oxidized samples in test tubes. Figure 2 shows a comparison of
chill haze measured in bottled samples after 5 WD (Hchill 5WD) with
chill haze arising in decarbonized and oxidized samples in tubes
after 5 h at 60 °C (Hchill OXI 5 h) in 15 batches of beer. The chill haze
values correlated, with the slope of the fitted linear line being 0.85.
One hour at 60 °C for decarbonized and oxidized sample in the
tube induced the formation of chill haze almost equal to that in
a bottled sample after 24 h at 60 °C (1 WD). Sample decarbonization
plus oxidation accelerated beer aging approximately 20 times.
‘Hchill 5WD’ values can be predicted from ‘Hchill OXI 5 h’ values
using the linear relation with good accuracy (standard
deviation ± 0.5 EBC).
To determine the effect of time of exposure of samples to 60 °C
on the chill haze intensity, one sample of decarbonized beer was
dispensed into eight tubes. The tubes were placed for varying pe-
riods (from 1 to 24 h) in a thermostat at 60 °C. They were then
transferred for 24 h to 0 °C and chill haze was measured. The results
are shown in Fig. 3. The chill haze intensity (Hchill) increased linearly
with the time of sample exposure to the elevated temperature. Un-
like the time course of increase of chill haze in bottled samples (4)
there was no lag phase. A similar experiment was repeated with
beer samples with different contents of haze-forming precursors.
The slope of the chill haze increase was influenced by the content
of haze-forming precursors and overall colloidal stability of the
sample. Extending the exposure time of the sample at 60 °C in-
creased the sensitivity of the assay.
Figure 4 shows a comparison of chill haze measured in bottled
samples after 5 WD (Hchill 5 WD) with chill haze arising in
decarbonized and oxidized samples in the test tubes after 24 h at
60 °C (Hchill OXI 24 h). Chill haze measured in decarbonized samples
after 24 h at 60 °C was approximately 4–5 times higher than after
5 h at 60 °C; hence the extension of the warm phase of the cycle
5WD) with chill haze arising in decarbonized and oxidized samples in tubes after 5 h at
J. Inst. Brew. 2016; 122: 304–309
Method for prediction of beer colloidal stability
Institute of Brewing & Distilling

chill
Figure 3. Effect of exposure time of a decarbonized and oxidized sample to 60 °C on the formation of chill haze (H ).

chill
Figure 4. Comparison of chill haze measured in bottled samples after five warm days (H 5WD) with chill haze arising in decarbonized samples in the test tubes
chill
after 24 h at 60 °C (H OXI 24 h).

from 5 to 24 h increased the sensitivity of the test. The chill haze
values in Fig. 4 correlate with the correlation factor 0.89, which is
higher than the correlation factor in Fig. 2. ‘Hchill 5WD’ values can
again be predicted from ‘Hchill OXI 24 h’ values using the linear rela-
tion with good accuracy (standard deviation ± 0.5 EBC). The chill
haze values for beer samples of one brand (marked ‘Brand 1’, ‘Brand
2’ in Fig. 4) correlate with an even higher correlation factor (>0.91).
Figure 5 compares the colloidal stability of bottled samples mea-
sured by the standard accelerated aging procedure (T2) with the
chill haze arising in decarbonized samples in the test tubes after
24 h at 60 °C (Hchill OXI 24 h). The results closely correlate and the
colloidal stability can thus be predicted from the chill haze of
decarbonized samples using the fitting of a power law curve with
an accuracy of ±0.62 WD.
Figure 6 compares the colloidal stability of bottled samples mea-
sured by the standard accelerated aging procedure (T2) with the
content of haze-forming precursors. According to Vaag (4) we used
the product of tannoid content (TC) and sensitive proteins (SP) for
the prediction of colloidal stability from haze-forming precursors
content. The results correlate with a much lower coefficient than
in Fig. 5. The correlation with T2 results did not increase even when
using a different formula for predicting the colloidal stability from
the results of haze-forming precursor test. The content of haze-
forming precursors is not sufficient to predict colloidal stability
with an accuracy better than ±1.5 WD. This is due to the fact that
the experiment was conducted with beer samples of different
brands, which varied in their composition and original gravity
values (9.5–12°) and the alcohol content (3.0–4.5).
With the majority of accelerated aging procedures a sufficient
formation of chill haze in bottled samples was achieved by exposing
them to 0 °C for 24 h. In the present method, we used small-volume
samples in tubes that experienced a much faster heat transfer and
307

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 P. Gabriel et al.
Institute of Brewing & Distilling

chill
Figure 5. Correlation of shelf-lifetime prediction by accelerated aging [T2 (WD)] with the chill haze arising in decarbonized samples in the test tubes after 24 h at 60 °C (H OXI 24 h).

Figure 6. Correlation of the content of haze-forming precursors (TC*SP; tannoid content * sensitive proteins) with shelf-life prediction by accelerated aging (T2).

chill
Figure 7. Effect of exposure time of a decarbonized and oxidized sample to 0 °C on chill haze (H ) formation (after 24 h at 60 °C).

temperature change during cooling and heating. We measured
the time course of increase of chill haze in a decarbonized oxidized
sample in the tube (after 24 h at 60 °C) depending on the time of
exposure to 0 °C. Figure 7 shows a typical time course. Chill haze
308
(Hchill) initially sharply increased but the increase gradually slowed
down. After 10 h at 0 °C, the haze increase was very slow, 1–2%/h.
A 12 h exposure to 0 °C is sufficient to reach 85% of the value
achieved after 24 h. The exposure of samples to 0 °C can thus be

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Method for prediction of beer colloidal stability
shortened to 12 h without a significant decrease in the sensitivity
of the method.
Conclusions
A method was designed for the rapid determination of the
colloidal stability of beer samples. The method is based on the
principle of accelerated aging of samples at elevated temperature
(accelerated aging test). Compared with standard methods of
forced aging, the process is accelerated by defined decarbonization
and oxidation of the sample.
Using this procedure, decarbonized and oxidized samples after
5 h at 60 °C reached the same chill haze as standard bottled sam-
ples after 5 days at 60 °C (Fig. 2). Extension of the exposure of
decarbonized samples to 60 °C for from 5 to 24 h increased the
chill haze 4–5 times (Fig. 4) whereas shortening of the exposure
to 0 °C to 12 h did not significantly reduce the chill haze intensity
(Fig. 7). The duration and sensitivity of the test can be set by
adjusting the parameters. It is possible to obtain the result within
36 h even for the most stabilized beer.
The chill haze intensity generated in decarbonized samples in
test tubes after 24 h at 60 °C correlates with the results of the col-
loidal stability of bottled samples measured by the standard forc-
ing procedure (Fig. 5). Their relationship can be fitted by a power
curve that can be used for prediction of the colloidal stability with
an accuracy of ±0.62 WD in the range of 3-13 WD.
According to our results, the proposed method is more accurate
than the methods measuring the content of haze-forming precur-
sors and the formula can be used to calculate and predict colloidal
stability for different brands of beers.
The proposed test requires no excessive time, labour, material
equipment and laboratory instrumentation. It allows the parallel
preparation and measurement of large numbers of samples. When
multiple (3–5) tubes are prepared from one sample, it is possible to
increase the measurement accuracy and reduce the standard error
of the result. Owing to its velocity and ease of use, the method can
be used to control the operation and function of the stabilization
process before shipment of the product from the brewery.
Another possibility is to use the method for laboratory testing of
the efficiency of stabilizing agents and evaluation of the relation-
ship between the content of haze-forming precursors and colloidal
stability of the final beer species.
J. Inst. Brew. 2016; 122: 304–309 Copyright © 2016 The Institute of Brewing & Distilling
Institute of Brewing & Distilling
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