colloidal stability of beer

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The Possibilities of Particle Analysis Demonstrated by the Measurement of the

Colloidal Stability of Filtered Beer

Jean Titze1,*, Manuel Christian2, Fritz Jacob2, Harun Parlar3 and Vladimr Ilberg4

ABSTRACT

J. Inst. Brew. 116(4), 405412, 2010 Long-term stability is one of the most important quality criteria of beer. Three groups of measuring methods are available for its determination: real time tests, predictive tests and indicative tests. One of the most common methods is the predictive forcing test, which is a time-consuming method for accelerating beer ageing, e.g., at 0C and 60C. Two ways exist to perform this test: (1) follow-up of haze development and determination of the lag phase or (2) the measurement of haze values after several days. The first option was evaluated by performing a long-term Forcing test over a period of 4 months by analyzing a bottom-fermented beer. It was shown that the haze curve followed a typical course with a lag phase, an increase phase and a station-ary phase. Significant differences between the measurement after the cooling and the warm period were shown. In search of quicker methods and more accurate predictive indicators, the charge titration method was developed as an alternative to deter-mine the particle charge of filtered beers, whereupon the rela-tionship was elucidated between the increase in hazing and de-crease of potential along with the advanced aging of beer. The results showed that with increased particle size due to agglom-eration, the total charge decreased. In this array of trials, two differently stabilized beers were examined. Although both beers showed different haze values in the beginning (0.32 EBC and 1.30 EBC), the less stabilized beer had only 10 warm days and in contrast the beer with the good stabilization had over 20 warm days. With the help of the total charge, predictions were possible regarding the long-term stability of the beer. Key words: colloidal stability, haze, particle analysis, predictive forcing test, real time test, total charge.

INTRODUCTION Nowadays many existing problems in the beverage in-

dustry, such as premature hazing of filtered beverages or gushing of carbonated beverages11,17,31 are caused by in-sufficient physico-chemical stability and surface-active substances3,8. In a further development of gushing analy-sis4,5,13,19 the feasibility of the combined particle analysis to prevent gushing was demonstrated by applying it to beverage products6.

The most frequent quality criterion of beer is the long-term stability. Once a filtered beer shows hazing, it is not marketable according to EU regulation no. 178/2002. Due to increased globalization, distribution routes have be-come longer, which requires the beer to stay stable for a longer time. In order to produce a brilliant, clear and bright beer, the use of a stabilization agent is inevitable. Because of the numerous factors that influence the forma-tion of haze, the prediction of the colloidal stability of a beer is somewhat difficult to achieve. Without an adequate prediction model as to the expected stability, breweries are forced to stabilize virtually blind, which often re-sults in an over-stabilization of the beer, a process that is not only unwanted because of impaired nutrition, but which can also lead to an increased financial burden.

Until now, examinations only enabled one to predict the tendency for hazing of beer after filling, such as the forcing method15. This method refers to the physico-chemical stability of beer, however, it requires days to be performed. The haze has to be determined visually or with apparatuses according to the EBC formazin units. Estab-lished forcing tests deliver acceptable results, but do not allow any interventions in the filtration and stabilization process during the beers production.

Another way of analyzing for colloidal stability can be found in particle analysis, which includes the determina-tion of surface charge, first introduced by Gerdes12, and is based on the polyelectrolyte titration method developed by Schempp and coworkers20,21. In order to make faster predictions on the stability of filtered beers after filling, this method has been developed, taking up the following principle: dipoles are being built due to the interaction of particles and ions, causing a measurable voltage. After polyelectrolytic titration, the surface net charge of parti-cles is changed irreversibly, so the surface potential de-creases. Once the potential reaches zero, the titration vol-ume serves as an equivalent to the whole net charge of the particles in the beer. The initial potential provides addi-

1 Deloitte, Rosenheimer Platz 4, 81669 Mnchen, Germany. 2 Research Centre Weihenstephan for Brewing and Food Quality,Technische Universitt Mnchen, Alte Akademie 3, 85354 Freis-ing-Weihenstephan, Germany.

3 Department for Chemical-Technical Analysis and Chemical Food Technology, Technische Universitt Mnchen, WeihenstephanerSteig 23, 85354 Freising, Germany.

4 University of Applied Sciences (Hochschule Weihenstephan-Tries-dorf), Am Hofgarten 4, 85354 Freising, Germany.

* Corresponding author. E-mail: [email protected] Parts of this paper were presented at the 2nd International Sympo-sium for Young Scientists and Technologists in Malting, Brewingand Distilling, May 1921, 2010 in Freising Weihenstephan, Ger-many.

Publication no. G-2011-0111-113 2010 The Institute of Brewing & Distilling

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406 JOURNAL OF THE INSTITUTE OF BREWING

tional information on colloidal stability. With the obtained value of charge, a definite prediction can be made towards long-term stability, when hazing takes place, and when stability decreases.

In previous studies it was shown, that the potential of the particles in beer has a linear relationship to the zeta potential28,29,30. As the physical stability of the dispersions decreases, the zeta potential also decreases (high zeta po-tential = high stability). The amount of the surface charge (= titrated volume), as an indirect measure of the number of particles, should not be too high. Otherwise, due to thermal movement (Brownian motion), there is a higher probability that the particles will agglomerate (lower number of particles = better stability).

Charge analyzing systems have become an efficient, flexible and manageable technology, especially for routine analysis and quality assurance, for example in the paper industry16,26, or for the characterization of latices10. It has also been widely and successfully applied in food analy-sis, e.g., for the fining of juices9, gushing control of bever-ages2, investigations of the effect of isinglass related to the particle net charge of beer and other applications7. Measurement of the colloidal stability

During storage, filtered beer becomes hazy due to col-loidal haze formation. This turbidity is caused predomi-nantly by interacting substances that form visible colloids. Most substances present in this respect are polyphenols and proteins, but also associations of polypeptides and polysaccharides, or of polypeptides with minerals1,2225. The polyphenol flavon-3-ole is one of the compounds primarily responsible for haze formation, by complexing with proline rich proteins18.

There are three groups of methods available to charac-terize colloidal stability14: 1. actual colloidal stability follow-up (real time tests), 2. predictive forcing tests (e.g., MEBAK method

2.15.2.115), 3. indicative tests.

For the first method, the beer is stored at room tem-perature during its entire shelf life. The typical curve of the haze formation during that storage period is shown in Fig. 1 (according to the real time test). The hazing process is divided into three phases14. In the first phase, the lag or latency phase, nearly no haze formation occurs. The haze values stay stable. In dependence of the quality of the stability of the beer, this stage can last for a couple of months (the longer the lag phase, the better the colloidal stability of the beer). In the second phase, called the in-crease phase, a rapid and apparently linear increase up to 2 EBC Formazin units can be seen. The rate of the haze formation is also dependent on the beer type14. The sec-ond phase shifts to the stationary phase, where the haze development rate slows down and finally reaches a maxi-mum value. The maximum value is also beer type depend-ent.

MATERIALS AND METHODS Course of action

In the first part of this investigation bottom-fermented and filtered beer of one filling was analyzed over a period of 4 months (27.11.2009 02.04.2010) using a modified forcing test according to MEBAK method 2.15.2.115.

In the second part, two bottom-fermented filtered beers, one sample with a strong stabilization, which

Fig. 1. Hazing curve of a filtered beer measured with 90 scattered light during shelf life (real time test)14.

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means a longer shelf life with no visible hazing formation, and the other sample with less stabilization treatment, were examined by a forcing test (forcing haze). Addition-ally, both beers were analyzed with a particle charge titra-tion method to measure the surface potential and the surface charge, measured as the titrated volume V neces-sary for the neutralization of the entire particle surface charge ( = 0 mV).

Forcing method

The forcing test used was a slightly modified method adapted from MEBAK method 2.15.2.115. For this, each beer bottle was subjected to temperatures of 60C and after that cooled to 0C for 24 h. The MEBAK method was modified as follows. After the heating period, as well as after the cooling period, the total haze of the beer was measured at room temperature (20C). Thus, one warm day was defined as the time of one warm and one cooling period, i.e., 48 h in total.

For the forcing test, an incubator, model UL 40 (Mem-mert GmbH & Co. KG, Schwabach, Germany) and a re-frigerator model KT 5050 (Colora Messtechnik GmbH, Lorch, Germany) were used. The turbidity was measured with a dual-angle turbidimeter (LabScat Sigrist-Photome-ter AG, Ennetbrgen, Switzerland) applying the following conditions: direct measurement in a closed 0.5 L beer bottle that had been freed from all labels before examina-tion; 90 scattered light at 650 nm. The haze was meas-ured in EBC formazin units.

Particle charge titration analysis After the haze measurement, the surface potential and

the surface charge of each sample were determined using a Charge Analyzing System (CAS) (AFG Analytic GmbH, Leipzig, Germany). First, the beer bottles were opened at room temperature. Afterwards, the samples were poured into vials and sonicated for a minimum of 5 min in a bath sonicator (model Branson 1200 Sonic Bath; Heinemann, Schwbisch Gmnd, Germany), until visible CO2-formation took place. After the foam disinte-grated (foam that clung on the bottle wall was redissolved by gentle shaking), the samples were measured without further pre-treatment. Polyelectrolytic titration took place during the measurements using a computer-controlled dosing pump. For this, 0.001 N polydiallyl dimethyl am-monium chloride (Poly-DADMAC) was used.

Prior to each measurement of the beer samples with the CAS, an adjustment of the potential intensity was per-formed. As the examined beer samples contain colloids with a negative particle surface net charge, a negatively charged polyelectrolyte standard solution was used for the adjustment. The potential of the standard solution was -1,200 mV.

RESULTS AND DISCUSSION Long term forcing test

Figure 2 shows two curves of the long-term forcing test. For this, nine bottles of the same batch of beer were

Fig. 2. Long-term forced haze formation curves of bottom-fermented beer, measured after each warm period (triangles) and cooling period (diamonds).

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measured and the mean value was calculated. The first curve (triangles) shows the results after the warm period, the second curve (diamonds) after the cooling period. In other words, the second curve gives the results after each completed warm day. According to MEBAK, the forcing test would have been finished after 10 warm days, be-cause on the 11th of December the value was higher than 2 EBC Formazin units (Fig. 2). However, as the haze was measured at 20C, and not as according to MEBAK at 0C, the end of the test would have been reached even earlier.

The difference of the two curves is that after each warm period the haze value was lower than after a cooling period, although the measurement was conducted at the same temperature (20C). This phenomenon is because after the long cooling period of 24 h, the relatively short time employed to heat the beer to room temperature be-fore the measurement takes place (approx. 40 min) is not sufficient for the complete disappearance of the chill-haze.

It can be observed that both curves increase nearly linearly in the first two months (approx. up to the 22.01.2010). After that point, it appears that the curves increase more slowly and finally (after three months) pass into the stationary phase.

It is noticeable that after passing ~10 EBC units, the calculated confidence intervals of each value are larger, which indicates that the measurement system has reached its detection limit for repeatable measurements. The accu-racy of the system can be influenced by the fact that after 2 months a slight deposit (sediment) developed. Depend-ing on the handling of the sample, parts of this deposit could be swirled up during the haze measurement.

The investigation showed that the course of the meas-ured haze of the accelerated aged beer by the forcing test was similar to the phases in the prior described real time test (Fig. 1). Particle charge titration analysis

During the second part of the investigation, two differ-ently stabilized, filtered and bottom-fermented beers were analyzed.

Figure 3 shows the haze values of the less stabilized beer that was measured after each warm day at 20C. In this case, the forcing test was conducted only until there was a difference of 2 EBC formazin units, measured as the difference between the initial haze (start; 0 warm days) and the measured haze at the corresponding time. For the less stabilized beer, the haze value was around 0.34 EBC and according to the forcing test, the trial ended after 11 warm days by reaching a haze value of 2.77 EBC. A haze increase of 2 EBC was thus reached after 10 warm days. From the curve it can be clearly deduced that the haze values remained almost constant during the first days (lag phase) and started to increase later (increase phase).

Parallel to the determination of haze, the surface poten-tial as well as the surface charge were measured via titra-tion. A typical titration curve is presented in Fig. 4.

The titration curves for each beer were measured over the whole period of the forcing test. For each beer and each warm day, a curve was obtained to characterize the potential and the titrated volume. A compilation of the titration curves of the beer sample is presented in Fig. 5. All measurements revealed a typical course. The courses of the titrations were approximated in Fig. 5 with the help of a characteristic function. The theory and its derivation

Fig. 3. Hazing curve of a less stabilized beer sample measured after each warm day using a 90 scattered light measurement method27.

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Fig. 4. Typical titration curve of the less stabilized beer27.

Fig. 5. Compilation of characteristic titration curves of a less stabilized beer sample (*, number of warm days of each titration curve,0 = start)27.


have been already introduced in previous publications27. It can be clearly seen, through the calculation of and V for each curve, that with increased aging, both the po-tential and the surface charge determined by the titrated volume decrease.

In other investigations, it was shown that with the help of the calculation of and V, that the decreases of both the surface potential as well as the titrated volume were in a linear relationship27.

In total, five samples of the strongly stabilized beer were examined by measuring the haze values. The mean value of these five samples at the beginning was 1.30 EBC. The measured start values were unusually high in comparison with the starting values of the less stabilized beer, which were only 0.32 EBC27. However, a visible haze could not be observed in both cases.

In Fig. 6, the haze curve of the stabilized beer is shown. Although the beer had a very high haze value in

Fig. 7. Compilation of characteristic titration curves of a strongly stabilized beer sample (*, numberof warm day of each titration curve, 0 = start).

Fig. 6. Formation of haze curve of the strongly stabilized beer sample measured after each warmday using a 90 scattered light measurement method.

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the beginning, a haze increase of 2 EBC was observed only after 23 warm days. According to internal analytical experience of the brewery, average values for this beer type are 15 warm days. The forcing test showed that the beer had a very high colloidal stability.

Parallel to the hazing measurement, a particle charge titration was performed. In Fig. 7, the characteristic func-tions of the analyzing days (0 warm days, 13 warm days and 24 warm days) are shown.

As seen in Fig. 5, a decrease of the potential as well as a decrease of the titrated volume was observed. The sur-face potential of both beer samples was similarly high for zero warm days.

For the less stabilized beer ~ 450 mV (Fig. 5) and ~ 410 mV for the strongly stabilized sample (Fig. 7) were measured. According to the theory, the high values of the surface potential of the particles in both beers should be an indication of high stability.

However, examining the entire particle surface charge measured via the titrated volume, a significant difference between the less and the strongly stabilized beer can be seen (~ 5 mL for the less stabilized beer and only 0.7 mL for the strongly stabilized beer). The titrated volume is an indirect measurement parameter for particle concentra-tion. When more particles are present in the samples, the probability for particle agglomeration is higher due to Brownian movement. Therefore, the number of particles and thus the total particle surface charge, measured by the titrated volume for total charge neutralization ( = 0 mV), is an indicator for long-term stability. These relationships were experimentally shown in this work using different stabilized beers. A sample with more particles corre-sponds to a less stabilized beer, for which a higher titrated volume is necessary. In comparison to that, for the strongly stabilized beer samples with fewer particles, a lower titrated volume is sufficient.

According to the method of the characteristic function, the parameters and V of the curves in Fig. 7 were calculated. By the comparison of both parameters over the time, it can be seen that and V are in a linear rela-tionship, with a coefficient of determination of R2 = 0.9896 (Fig. 8). This proves the practicability of the method.

The linear relationship of and V was previously shown with beer samples with a coefficient of determina-tion of R2 = 0.99027.

CONCLUSIONS With the help of a long-term forcing test over a period

of 4 months, set up as described, it was shown that the course of the hazing curve was in accordance to the real time haze behaviour of beer with a lag, an increase, and a stationary phase. In addition, a significant difference be-tween the measuring point after the warm period and the cooling period could be observed and explained.

In the second part of this work, the method of particle analysis was shown as an alternative for the prediction of long-term stability of beer. This was conducted using two different stabilized beers. The results showed that the sur-face potential, as well as the surface charge of the parti-cles in beer, could be used as an indicator for colloidal stability. It was shown that a distinct relationship existed between haze increase (at 90) and the surface potential decrease or the titratable charge decrease.

Although the initial surface potential of both beer sam-ples was similarly high (for the less stabilized beer ~ 450 mV and for the strongly stabilized sample ~ 410 mV), the amount of particle surface charge was significantly higher in the less stabilized beer sample (Fig. 7). This difference in the amount of surface charge could be the reason for the different stabilities. Furthermore, it

Fig. 8. Correlation between and V ( and V were calculated by the characteristic titration curves of the strongly stabilized beer (see Fig. 7).

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was shown that it was not possible to draw conclusions on the long-term stability of beer using only the haze value at the beginning of the measurement, as the strongly stabilized beer had a higher start value of 1.3 EBC, in contrast to the less stabilized beer with a value of 0.32 EBC.

With this measuring method it is possible to predict the colloidal stability of a beer in a storage tank before the filling operation. This should allow for stabilization as needed before filtration of the beer.

ACKNOWLEDGEMENTS

The authors thank Mr. Markus Schmidt from the Research Center Weihenstephan for Brewing and Food Quality for the helpful assistance in the daily measurement of the haze over the four month time period.

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(Manuscript accepted for publication October 2010)

Zachary Smolsky

INTRODUCTIONMeasurement of the colloidal stabilityCourse of actionForcing methodParticle charge titration analysis

RESULTS AND DISCUSSIONLong term forcing testParticle charge titration analysis

CONCLUSIONS

colloidal stability of beer

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