EP2179283A1

EP2179283A1 - Process for determining bitter substances in beer or beer wort

Info

Publication number: EP2179283A1
Application number: EP08784377A
Authority: EP
Inventors: Ronald Eberl; Jürgen Wilke; Beatrix Parthey
Original assignee: Hochschule Anhalt (FH)
Priority date: 2007-07-28
Filing date: 2008-07-23
Publication date: 2010-04-28
Also published as: WO2009015640A1; DE102007035561B3

Abstract

The invention relates to a process for determining bitter substances in beer and beer wort. The invention relates to a process for determining bitter substances in beer or beer wort based on measurement of visible fluorescence which is emitted by the prominent bitter substances in the presence of certain lanthanoid ions on irradiation with ultraviolet light. The process is characterized in that the sample is highly diluted and one or more certain auxiliary reagents are added which induce the usually suppressed formation of complex compounds from lanthanoid ions and bitter substances and cause a selective amplification of the fluorescence of these complexes which is to be measured.

Description

Method for the determination of bitter substances in beer or wort

The invention relates to a method for the determination of bitter substances in beer and beer wort. This method includes both the qualitative determination, i. the detection, and the quantitative determination, i. the measurement of the concentration of bitter substances in beer and wort, a.

Bitterness is one of the essential quality parameters of beer, which is also perceived and evaluated by consumers. The characteristic bitter taste is caused primarily by the so-called iso-acids (IAS), which are formed during the boiling of the wort from the added Hopfendolden or hop products. In addition, isoacids (IAS) have an influence on the shelf life or stability of beer and the characteristic of a long-lasting froth, which many consumers appreciate.

Within the framework of the Purity Law, only species (hop variety), form (umbels, pellets, extracts), amount and time of hops addition or hops yielding are technologically variable. The concentration of isoacids (IAS) and thus the bitterness of the beer depends not only on the amount of starting materials (the alpha acids), but also, for example, on the temperature-time regime of wort preparation and fermentation, the pH value. Value, the concentration of other substances and the refining and filtering process.

There are formulas for the individual types of beer in which the technological parameters are largely determined, so that under normal conditions the product is produced with the desired bitterness.

For the quality assurance as well as for the process control a fast on-line determination of the bitter substances is therefore of great importance. With such an online determination, it is possible to measure the bitterness of the beer, regardless of the hop dose process close to the process, so that in case of deviations from the normal course of the process appropriate measures can be taken in good time.

Requirements for such a measuring system include u .a. :

- a minimum sample preparation,

- an uncomplicated operation, also by not specially trained personnel, - a reliable and low maintenance operation and

- low chemical consumption and easy disposal.

For the implementation of quality assurance concepts (ISO 9000 ff.), The objective and protocolable analysis of the material to be measured is also important. From an economic point of view, the demand for low acquisition, maintenance and servicing costs is added.

The previously used laboratory methods for determination of bittering are not or only partially suitable for this application. To determine the bittern content in beer and wort only two methods are used:

The first method is based on the extraction of the bitter substances from the acidified sample with isooctane, a non-polar, water-immiscible liquid. After vigorous shaking of the two liquids and the separation of the aqueous and the isooctane phase, the extinction of the isooctane phase is measured with the aid of a quartz glass cuvette in the ultraviolet (UV) wavelength range. For example, this method is mandatory under the analytical regulations of the German (MEBAK), European (EBC) and American (ASBC) brewery associations.

Disadvantages of this method include:

the relatively high time required for pipetting the liquids, shaking, phase separation and transferring the isooctane phase to a quartz glass cuvette, the need for a UV spectrometer or photometer,

- the use of a quartz glass cuvette, which must be kept clean during the measurement and carefully and costly cleaned after the measurement and their handling can cause difficulties in daily life (glass breakage), - the open handling of liquid chemicals and last but not least

- the consumption of chemicals whose disposal is not without problems.

Due to the high material costs, the second frequently used method can only be used in large breweries or brewery laboratories. Here, the bitter substances are separated by chromatography (HPLC), from the other beer or wort components and also detected by UV spectroscopy. The possible separate determination of the individual bitter substances is for routine analysis generally not required. Disadvantages of this method for the aforementioned task include:

- The very high material costs for the basic equipment of the HPLC system, - A relatively large amount of time, especially for the preparation and post-processing of the HPLC method and the sample preparation (degassing, filtration),

- the consumption of high-quality chemicals (HPLC-eluent),

- high maintenance and installation costs, especially if the method is required only occasionally, and that - qualified operators and maintenance personnel are required.

Only very few have been described in the literature on the state of the art other methods for the detection or for the determination of bitter substances in beer, such as by thin-layer chromatography, but these methods are also not for fast and operational evidence, the so-called online Detection of bitter substances are suitable because they have similar disadvantages as the two methods described.

WO 2005/045421 A1 includes the qualitative determination of bitter substances in beer or intermediate products of beer production by means of UV / VIS spectroscopy. Bittern content can not be directly determined from the UV / VIS absorbance measurement, as the bitterness contributes less than 1% to the total absorption and its spectrum is greatly overshadowed by other UV / VIS absorbents. The composition of the material matrix and thus also its influence on the measurement varies between the different beers in some cases very strong, but also in one and the same beer it comes through the influence of process variables and the natural variation of the vegetable source materials to different matrix signals. The very small proportion of bitter substances in the total signal should be able to be separated according to the teaching of WO 2005/045421 A1 by means of chemometric methods. The high computational cost of such evaluation requires a computer that must be coupled with the actual measurement system. The resulting total effort, as well as the necessary care in dealing with sensitive measuring and computing technology, is opposed to an online use in harsh everyday operations, especially in small breweries. Concrete information on the reliability and measurement accuracy of the overall system described are not yet available.

The determination of the bittern content by means of fluorescence measurements is briefly mentioned in the description of the fluorescence meter BioView. However, the isohululones themselves show practically at the concentrations that occur no fluorescence, suggesting that it is an indirect process that exploits the correlations of the bittern content with other fluorescent substances. Apart from a correlation factor (0.92), which was not further defined, no further information was provided on these measurements, even on request. Basis of this application is the teaching of the patent US 5,716,850, in which, inter alia, the determination of the bitterness of beer is described by means of a fluorescence measurement. The method is no longer used in practice after serious incorrect measurements, which speaks for the above-mentioned assumption of an indirect, non-causal correlation of fluorescence and bitter substance concentration. Another attempt to determine the bitterness of beer based on the autofluorescence of the samples was undertaken by Christensen et al., Journal of the Institute of Brewing, Vol. 111, 2005, pp. 3-10. For this purpose, a three-dimensional fluorescence matrix (intensity of the fluorescence signal as a function of excitation and emission wavelength) for each sample was taken with great effort and subjected to a chemometric evaluation. Although the achieved accuracy could be sufficient for orienting online measurements under certain circumstances, the enormous effort for measuring and evaluating a practical application clearly stands in the way. JB Tomlinson et al., Journal of the Institute of Brewing, Vol. 101, 1995, pages 113 to 118, described a method in which europium ions were introduced into beer, which complexed with the bitter compounds Irradiation with ultraviolet light showed visible fluorescence. Thus, the isohumulone concentration in pure bitter solutions could be well determined. In contrast, the intensity of the generated fluorescent light in the beer proved to be extremely weak. Matrix effects, in particular the color of the beer, have been blamed for the large deviations between the fluorescence detected bitterness values and the corresponding reference values.

In Christensen et al., Journal of the Institute of Brewing of, Vol. 111, 2005, p.3 to 10 the results of a chemometric analysis were also the fluorescence ^¬ zenzspektren presented by beer after addition of europium. Again, the required effort is out of all proportion to the achievable result, in particular, with this method, an online use on site hardly to realize.

This also applies to a method for detecting and measuring the concentration of bitter substances in beer or wort, which is described in DE 10360302 A1. In this method, the liquid sample must first be foamed. Subsequently, the lanthanoid-ion foam, for example a europium Solution, offset. In the presence of bitter substances, a strong fluorescence occurs, which is visually recognizable and can also be detected by measurement.

The publication Eberl, R .; Parthey, B .; Wilke, J .: Fluorimetric rapid determination of bitter substances in beer and wort, Brauwelt (2006) No. 27, pages 788 to 790 relates to the development of a process-applicable fluorescence method for the determination of bitter substances in beer and wort. In particular, it deals with the compensation of both optical and chemical disturbances, which are mainly due to the material matrix of the beer samples. For this purpose, various variants are shown, which should enable an application of the method in operational practice. In particular, a time-resolved fluorescence measurement using a xenon flash lamp is proposed, whereby the proportion of the self-fluorescence of the sample is eliminated and the influence of ambient light is almost completely excluded. The disadvantage of this method, however, are the higher price, the larger size, a more complicated control and the greater energy requirements.

DE 10233077 A1 discloses a rapid detection of beer and hop bitter substances in a liquid with the aid of carrier systems, the carrier systems being brought into contact either with or without pretreatment with an analyte. The analyte is isolated thereon or in it. Subsequently, the evaluation is carried out with a reagent integrated in the carrier material or a subsequent reaction step by means of optical methods. The support systems consist of a porous matrix, such as a polymer, in which sorptive and reactive particles, for example of silicic acid-based material, are incorporated, or to which a coating which has been added with a silica material is applied. These silicic acid-based polymer materials are intended to impart the desired specific absorption properties to the carrier system. The reagent required for detection can either be integrated directly into the carrier material or used in a subsequent step, the reagent consisting inter alia of a salt of the lanthanides dysprosium, samarium, europium or terbium. However, this method too complicated and costly for an intended online application.

The object underlying the invention is to provide a cost-effective method that allows both the qualitative and a quantitative online determination of bitter substances in beer and wort. The object of the invention is achieved by a method for the determination of bitter substances in beer or wort, which is based on the measurement of visible fluorescent light, which is emitted by the relevant bitter substances in the presence of certain lanthanide ions upon irradiation with ultraviolet light. According to the invention, the sample is diluted to strong and added a specifically acting auxiliary reagent.

On the one hand, the concept of the invention consists in attenuating the disturbing matrix effects on the fluorescence light measurements by dilution so that their influence on the measurement result can be neglected. In internal investigations, those at J.B. Tomlinson et al., Journal of the Institute of Brewing, Vol. 101, 1995, pp. 113-118. In an internal continuation of this work, the causes of the serious differences between the measurements in pure IAS solutions and beer were examined in more detail. It turned out that the material matrix of the beer actually plays a key role here. The influence of the matrix is mainly due to the strong absorption of the excitation radiation and not due to the absorption of the emitted radiation, as erroneously in J.B. Tomlinson et al., Journal of the Institute of Brewing, Vol. 101, 1995, pp. 113-118.

In classical fluorescence spectroscopy, the emitted fluorescence is measured at an angle of 90 ° to the excitation beam. This is generally based on highly diluted solutions whose absorption at the wavelength of the excitation radiation is negligible. For highly absorbent samples, the method of "front face fluorescence" is used, as in the measurement methods according to JB Tomlinson et al., Journal of the Institute of Brewing, Vol. 101, 1995, pp 113-118 and Christensen et al , Journal of the Institute of Brewing, Vol. 111, 2005, pp. 3-10, where the excitation and detection of fluorescence are on the same side of the cuvette, so that the stimulating light does not have to penetrate the absorbing sample Internal measurements on model solutions show that the influence of an absorbent beer-like matrix is similar to that of front-face fluorimetry as it is in a 90 ° array.

In order to be able to carry out fluorescence measurements largely undisturbed through the sample matrix, the sample is diluted according to the invention. The absorptive influence of the matrix weakens with increasing dilution, so that at a sufficiently high dilution the influence of the matrix is sufficient. cheπd can suppress. The concomitant reduction in the fluorescence intensity can be compensated by a corresponding sensitivity of the measuring arrangement. Measurements of low fluorescence intensities (and thus correspondingly low fluoride concentrations) can be realized with relatively little effort with today's device technology. Surprisingly, however, it has been shown in the present case that the measurement of the fluorescence of low fluorophore concentrations has no non-limiting limits. If one dilutes an IAS model solution successively and measures the fluorescence after addition of Europium (Eu) - or some other Lanthanoid ions, one finds a threshold value concentration of approximately 5 mg / l IAS, below which the fluorescence on values returns close to zero. This behavior is almost independent of Eu concentration and other factors and is likely due to a concentration-dependent inhibition of complex formation.

In order to reduce the UV absorption of beer samples sufficiently for fluorescence measurements, a dilution of at least 1:20 is required. At the usual IAS concentrations of 20 to 40 mg / l, the IAS concentrations are already well below the experimentally found threshold. Thus, there is another essential part of the concept of the invention, which consists in that in the method according to the invention the observed

Inhibition of complex formation and the resulting strong and non-linear decrease in the measured fluorescence of these complexes is repealed by an auxiliary reagent. Surprisingly, it has been found that the observed unfavorable threshold behavior can be effectively suppressed by adding certain auxiliary substances to the IAS lanthanoid mixture. While the addition of substances, also referred to as "synergistic reagents", to enhance weak fluorescence has already been described several times, for example in BS Panigrahi et al., Spectrochimica Acta Part A 51, 1995, pp. 2289-2300 and N. Arnaud et al., Analyst, Vol. 123, 1998, pp. 261-265, has not previously disclosed that such substances are apparently also able to overcome inhibition of complex formation.

Based on this discovery, the fluorimetric determination of bitter substances in a beer matrix can be performed by

1. diluting a sample so strongly that the disturbing influence of the absorbent matrix becomes negligibly small,

2. Added lanthanide ions and a small amount of one or more certain auxiliary reagents, which on the one hand induces the formation of bitter lanthanide complexes at low concentrations and on the other hand increase the fluorescence by increasing the quantum yield, 3. measures the resulting fluorescence, and 4. from the measured value via a simple, preferably linear relationship determined the desired bitter value.

As synergistic auxiliary reagents according to the invention organic compounds such as trioctylphosphine oxide (TOPO) and compounds of the compound classes organic phosphates and / or sulfoxides use.

Preferably used as lanthanides are europium (Eu), dysprosium (Dy), terbium (Tb) and / or samarium (Sm).

Particular preference is given to embodiments of the invention with dysprosium (Dy) or europium (Eu) as lanthanoid, in which case trioctylphosphine oxide (TOPO) is used as the synergistic organic auxiliary reagent in these embodiments.

It has proved to be particularly advantageous if the lanthanides are present in acidic solution, with a pH between pH 1 and pH 4 being preferred.

The dilution of the sample is preferably carried out in a volume ratio of 1:20 to 1: 500.

In a particularly advantageous embodiment of the invention, the auxiliary reagent is added simultaneously with the dilution of the sample.

Further details, features and advantages of the invention will become apparent from the following description of exemplary embodiments with reference to the figures. Show it:

Figure 1: the representation of the fluorescence of IAS solutions different

Concentration after addition of Eu ions and the additional use of the auxiliary reagent trioctylphosphine oxide,

FIG. 2 shows the representation of the fluorescence signals of 20 different beer samples measured according to the invention as a function of the actual IAS contents, FIG. 3 shows the representation of the IAS concentrations of 20 beer samples determined according to the invention from the fluorescence signals as a function of the actual IAS concentrations.

The production of sample solutions for fluorescence measurement will first be described with reference to the following exemplary embodiment. From 20 different types of beer (pils), 100 ml were taken and degassed by the action of ultrasound (2 min). 1 ml each of the samples was diluted 1: 250 with water. 1 ml of the diluted sample was pipetted into a fluorescence cuvette. Another pipette was used to add 100 μl of a 20 mM dysprosium (Dy) -

Solution in 1 normal hydrochloric acid (1 N HCl) in the cuvette. Finally, 50 μl of a 0.1% strength solution of trioctylphosphine oxide (TOPO) were added to the cuvette. The three components were mixed thoroughly in the cuvette. After a waiting time of 1 min, the fluorescence was measured with a fluorescence spectrometer (CARY ECLIPSE, Varian.). To excite the fluorescence, a wavelength of 283 nm was chosen; the intensity of the emitted radiation was measured at a wavelength of 575 nm. The reference method used to determine the bitterness of the samples was the IAS analysis by high performance liquid chromatography (HPLC). In this standard chemical analysis method, the substance to be measured is separated from the sample by chromatography and its concentration is determined using a suitable detector, in this case a UV detector. Under the same conditions, a standard substance with a precisely known concentration of the substance sought is then analyzed. From the comparison of the two detector signals, the sought concentration can be determined.

The international IAS standard ICS-11 (Labor Veritas, Zurich, Switzerland) was used to determine the concentration of iso-alpha-acids (IAS) as the main bitter substance.

Fig. 1 includes a graph showing the fluorescence of IAS solutions of different concentrations prepared similarly as in the previous procedure. In contrast to the previous rule, however, europium (Eu) ions were added as lanthanide ions. The values obtained without the addition of auxiliary reagents and those of the samples in which trioctylphosphine oxide (TOPO) was used as an additional auxiliary reagent can be compared with one another at specific concentration values using the diagram. It shows at the lower concentration values below the threshold concentration of about 5 mg / l IAS, that there without Addition of the auxiliary reagent which returns fluorescence scores close to zero. This strong and nonlinear decrease in fluorescence is due to inhibition of complex formation. The addition of trioctylphosphine oxide (TOPO) abolishes this inhibition and, as is clear from FIG. 1, leads to the occurrence of evaluable linear concentration-dependent fluorescence signals even in these areas of low concentration of IAS.

FIG. 2 shows the results of the fluorescence measurement as a function of the values of the reference method (HPLC). The line is the result of a linear regression of the measured values. The statistical evaluation gives a linear correlation coefficient of 0.945 and a standard deviation of the regression line of 1.7 mg / l.

Using the relationship thus found, one can conclude from individual fluorescence readings on the IAS content of the sample. Finally, FIG. 3 shows the relationship between the IAS concentrations determined on the basis of fluorescence and those analyzed by means of HPLC in the examined samples.

Claims

claims

1. A method for the determination of bitter substances in beer or wort, based on the measurement of visible fluorescent light, which is emitted by the relevant bitter substances in the presence of certain lanthanide ions when irradiated with ultraviolet light, characterized in that a dilution of the sample with Water in a ratio of at least 1: 20 is added and one or more organic auxiliary reagents are added, selected from the compounds / compound classes trioctylphosphine oxide (TOPO), organic phosphates and / or sulfoxides, which from normal at low concentrations of bitter substances suppressed formation of complex compounds Lanthanoid ions and bitter substances induce and cause a selective amplification of the measured fluorescence of these complexes.

2. The method according to claim 1, characterized in that as lanthanide europium (Eu), dysprosium (Dy), terbium (Tb) and / or samarium (Sm) is used.

3. The method according to any one of claims 1 or 2, characterized in that as lanthanide dysprosium (Dy) or europium (Eu) and as an auxiliary reagent trioctyl phosphine oxide (TOPO) is used.

4. The method according to any one of claims 1 to 3, characterized in that the added lanthanide is present in acidic solution.

5. The method according to claim 4, characterized in that the pH of the acidic solution is between pH 1 and pH 4.

6. The method according to any one of claims 1 to 5, characterized in that the dilution of the sample in a volume ratio of 1: 20 to 1: 500 takes place.

7. The method according to any one of claims 1 to 6, characterized in that the addition of the auxiliary reagent takes place simultaneously with the dilution of the sample.

8. The method according to any one of claims 1 to 7, characterized in that the concentrations of the bitter substances are determined from the measured values via a linear relationship.