MXPA99010821A - Beer and similar light-sensitive beverages with increased flavour stability and process for producing it - Google Patents

Abstract

The invention provides a beer or similar light-sensitive beverage, which has been treated with a protein that binds riboflavin, resulting in a reduced"sunstruck"flavour formation after exposure of the beverage to light and also in an increased overall flavour stability of the beverage during storage. Suitable proteins include riboflavin-binding protein and flavodoxin.

Description

BEER AND SIMILAR SENSITIVE LIGHT BEVERAGES WITH INCREASED FLAVOR STABILITY AND PROCESS FOR PRODUCING FATS DESCRIPTION OF THE INVENTION The invention relates to improvements in the production of beer and similar beverages sensitive to light, in particular with an improvement in flavor stability. of such light sensitive beverages, and with a process to produce such beverages sensitive to light with stabilized flavor.

BACKGROUND OF THE INVENTION AND PREVIOUS TECHNIQUE The implications of exposing beer to the light and the subsequent effect on the taste of beer has long been of interest to beer producers.

The formation of an unpleasant note with scent of skunk "skunky", often called a "sun exposure" taste, when beer is exposed to light, has been a problem that has a lot of time. Many international beer producers see the taste formation generated by sun exposure as a quality flaw, although often unavoidable and market research generally indicates that beers which have a flavor generated by exposure to the sun are less pleasant to the palate compared to those that do not. Therefore, in order to preserve and increase sales volumes, the accepted brewing practice recognizes that the taste generated by sun exposure should be avoided as much as possible. One method to reduce the development of the flavor generated by exposure to the sun is to bottle the beer in dark bottles, although this method is only partially successful. The chemical compound which generates the taste of sun exposure was identified in 1961 by researchers from Kirin Breeries, Japan (Kuroiwa &Haishimoto (1961) reference 1), as 3-methylbut-2-ene-l-thiol , also known as -prenylmercaptan. The subsequent investigation by Hashimoto and. others helped to elucidate the mechanism of formation, which is considered to proceed via separation of free radicals from the prenyl side chain of isoalpha-acids, also known as isolumulones, followed by the combination of the prenyl radical with a thio radical. In turn, the thiol radical is the product of a free radical separation of various sulfur-containing compounds that include hydrogen sulfide and cysteine. Using model systems, several groups have demonstrated the role of riboflavin as a mediator of light energy in the formation of the taste generated by sun exposure (Blockmans et al. (1981) reference 2, and Irwin et al. (1993). ) reference 3).

In addition to working on the flavor generated by exposure to the sun, several groups have also carried out work on the oxidative degradation of isohumulones and the subsequent formation of impaired flavors. This reaction can also be carried out by riboflavin-mediated photolysis (Devreux et al (1981) reference 4, and the various references mentioned in that document). The photolytic separation of isohumulone by means of photosensitization of riboflavin leads, on the one hand, to the taste generated by exposure to the sun, and on the other hand, to the oxidized deteriorated flavors that can induce suggestions to decrease the concentration of riboflavin in drinks. -sensitive to light such as beer to improve its flavor stability during storage (see reference 3). Japanese patent JP 50 019 635B describes a method for preserving sake in which riboflavin is removed by a combination of adding a flavoprotein that does not bind riboflavin and an activated charcoal treatment. The reference does not mention beer or the flavor generated by sun exposure. However, although it has been known for decades that deterioration of beer taste is a problem (see references 1 of 1961) and riboflavin is a major contributor to this problem (see reference 2)., from 1981), until now a solution for this problem has not been known. Therefore, there is still a need to decrease the influence of riboflavin on the taste deterioration of beer and similar light-sensitive beverages. The present invention provides a solution to this problem, old. BRIEF DESCRIPTION OF THE INVENTION The present inventors consider that the development of the flavor generated by sun exposure can be counteracted by adding to beer at least one of the so-called "flavin-binding compounds" capable of binding to riboflavin-or "compounds similar to riboflavin "so as to alter the photosensitizing action of the compounds similar to. riboflavin. In this specification, "riboflavin-like compounds" are defined as compounds that contain a three-ring isoaloxazine moiety. Examples include riboflavin, riboflavin-5'-phosphate (also known as flavin mononucleotide); FMN), flavin adenine dinucleotide (FAD). These compounds are also known as flavin nucleotides; They function as prosthetic groups of the oxidation-reduction enzymes known as flavoenzymes or flavoproteins. Of course, such a compound that binds flavin should be "food grade", which means that it must meet safety requirements in terms of food and should not alter the physical stability of the beer or other beverage.

An alternative may be a compound that binds immobilized flavin with which the beer can be contacted for a sufficiently long time to bind the riboflavin-like compound and which can immobilize the flavin-binding compound which is subsequently separated from the beer. When such immobilized compounds that bind flavin are used, the requirements of "food grade" are less severe, although it is preferred that, even in this case, a flavin binding compound, "food grade" immobilized, is used. To demonstrate that this principle can work, the present inventors have developed a method in which riboflavin binds or is inactivated. In this way, the formation of the taste generated by sun exposure can be inhibited or at least postponed for a certain time under conditions in which the taste generated by sun exposure would have formed when the riboflavin had not been inactivated. It has been found that the use of flavodoxin or protein that binds riboflavin retards the formation of the flavor generated by sun exposure. Flavodoxin occurs, among others, in Azotobacter vinelandii. It is an acid stable protein (pl 4.0) with a low molecular weight (P.M. 15-22 kD) which in nature is associated to a single molecule of FMN by a non-covalent binding (the holo form). At low pH, FMN and flavodoxin dissociate and apo-flavodoxin is formed, which can be isolated from the previously associated flavin, where "apo" indicates the shape of the protein without the associated cofactor. The apo-flavodoxin of A. Vinelandii is also capable of binding riboflavin and other flavin analogues. Both the amino acid sequence of (apo) -flavodoxin and the gene encoding it have been published (Tanaka et al, (1975) reference 5 and Bennett et al (1988) reference 6). The flavodoxin of A. vinelandii is susceptible to dimerization due to the formation of intermolecular disulfide bonds between individual flavodoxin molecules, which may result in a loss of biological activity. To avoid this dimerization, a modified flavodoxin is elaborated, in which the cysteine residue of amino acid 69 is replaced by. an alanine residue. This modified flavodoxin is indicated as "C69A flavodoxin". As an alternative, the cysteine residue can be replaced by another amino acid residue, for example, a serine residue, resulting in a C69S flavodoxin. Other flavodoxins can be isolated from, among others, Desulfovibrio vulgaris and Anabaena variabilis, see, for example Pueyo et al. (1996) reference 7. The riboflavin-binding protein can be isolated from the eggs of poultry, preferably chicken eggs, as described in US-PS 4534971 (1985, reference 8), according to which the protein that binds to riboflavin can be obtained homogeneously from chicken egg white by acid treatment, chromatography on DEAE (diethylaminoethyl) Sephadex, and chromatography on sulfopropyl-Sephadex, according to the procedure of J. Becvar (1973) Ph.D. Thesis, University of Michigan (reference 9). The purified protein can be stored, for example if it is frozen at -20 ° C in 10 mM potassium phosphate, 1 mM EDTA buffer pH 7.0 at concentrations between about 1 and 10 mM. The isolated yield is approximately 15 mg (0.37 μmol) per egg. The protein is stable as a sterile solution and as a lyophilized powder. Another method of isolation is described by Hamazume c.s. (1984, reference 10). The riboflavin-binding proteins are also described-by .S.A. Innishitehouse, A.H. Merril Jr., and D.B. McCormick in Chapter 11 (Riboflavin-binding proteins) from Free Chemistry and Biochemistry of Flavoenzymes, Vol. I, edited by F. Müller, CRC Press, Boston, (1991) 287-192 (reference 11), but these proteins that bind Riboflavin have not been suggested as a means to improve the quality of beer and other light-sensitive beverages. Both the apo form and the holo form of the riboflavin-binding protein of chicken egg white are sold by SIGMA, Chemical Company, U.S.A. (1991, reference 12).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of riboflavin on the formation of free radicals, measured by electron spin resonance (ESR) also known as paramagnetic electron resonance (EPR) in a model system at the pH of beer (pH = 4). Figure 2 shows the effect of various riboflavin binding compounds, ie riboflavin-binding protein (RfBP), flavdoxin Fdx), and a non-binding protein, i.e., bovine serum albumin (BSA) on the formation of radicals free measured with electron spin resonance (ESR) in a model system at beer pH (pH = 4). The concentration of riboflavin is 1 ppm = 2.7 μM. Figure 3 shows the effect of various concentrations (0, 0.3, 1.3, 2.7 and 13.5 μM) of riboflavin-binding protein (RfBP) on the formation of free radicals measured by electron spin resonance (ESR) in beer.

DETAILED DESCRIPTION OF THE INVENTION In its broadest sense, the present invention relates to a process for increasing the flavor stability of beer or other light sensitive beverages, which process comprises inactivating the riboflavin-like compound or compounds present in the beverage by allowing bind to flavin-binding compounds present in a sufficient amount and for a sufficient time to obtain a measurable improvement in the flavor stability of the beverage as compared to a similar beverage which has not been contacted with the compound that binds flavin. In this specification, the expression "inactivation of the riboflavin-like compound or compounds present in that beverage" means to prevent riboflavin-like compounds from generating free radicals in that beverage by allowing such "riboflavin-like compounds to bind to a linking compound. Flavor A first embodiment of the invention is a process in which the flavin-binding compound is incorporated into the beverage in an amount sufficient to observe a measurable improvement in the flavor stability of the beverage during storage as compared to a beverage similar in which the flavin binding compound has not been added A second embodiment of the present invention is a process in which inactivation of the riboflavin-like compound or compounds is obtained by: (a) contacting the beverage with a sufficient amount of immobilized flavin-binding compound in a carrier material compatible with the beverage, followed by (b) allowing the riboflavin-like compound or compounds to form a complex with the immobilized flavin-binding compound, and (c) subsequently removing the immobilized complex from the beverage. The process of the invention is related to an improvement in the flavor stability of beer. It is considered that 3-methyl-but-2-ene-1-thiol, which gives rise to the taste generated by sun exposure, is derived from isohumulones. These isohumulones are present in the hops and therefore the process of the present invention is particularly suitable for use with beverages produced by adding hops during brace. Preferably, the beverage contains up to 10 weight percent alcohol and, more preferably, at least 0.1 weight percent alcohol. Most preferably, the beverage contains between 0.5 and 7 weight percent alcohol. Preferably, the flavin binding compound is a protein. Examples include flavodoxin and riboflavin-binding protein. It is also contemplated that other flavin binding compounds may be used, which include antibodies against riboflavin or a riboflavin-like compound, especially against the active site in the light thereof, or a fragment of such an antibody as (Fab ') 2; Fab, Fv, scFv or variable heavy chain fragments (VH originating from a "classical" or HC-V antibody that originate from a "heavy chain" antibody). The experiments described in the examples below show, that approximately 1.3 μM of riboflavin or flavodoxin binding protein effectively decreases the formation of radicals in beer or beer model systems containing approximately 1 ppm riboflavin. Therefore, a useful decrease in radical formation is obtained by the addition of at least 0.5 moles of a flavin-binding compound per mole of riboflavin-like compound to riboflavin. Preferably, the amount of flavin binding compound is greater, for example, at least equimolar, a-two, or even greater than five times, on a molar basis to the amount capable of binding a riboflavin-like compound, is promoted the formation of free radicals. Of course, the amount of flavin binding compound depends on the affinity of the flavin-binding compound to riboflavin-like compounds. The higher this affinity, the lower the amount of flavin-binding compound that is required to bind most of the riboflavin present in the light-sensitive beverage. Such affinity can be measured by general techniques known to those familiar in the art (see, for example, Druggeman et al. (1995) reference 13). When the affinity of a flavin binding compound is determined subsequently the amount of flavin binding compound, or simply by testing the various amounts of flavin binding compound, for example on a scale of 1: 10: 100: 1000, etc. , the final test of an improvement in the flavor stability of the beer or beverage and / or the absence of flavor formation generated by sun exposure is evaluated by a trained test team to judge the taste of the beverage by smell or taste, or both. The minimum amount of flavin-binding compound to be added is related to the amount of riboflavin present, but is generally at least 1 ppm by weight, and preferably more than 10 ppm by weight with respect to the beverage to which it will be added. More preferably, 20 ppm, and possibly more than 50 ppm by weight, relative to the beverage is added per phase. The upper limit of the compound that binds added flavin is determined by practical conditions such as the price of the binding compound, the influence on taste, etc. In general, excessive amounts of any compound will not be added to a consumable product such as beer and other light sensitive beverages. Therefore, a practical upper limit is 0.5% by weight of flavin-binding compound with respect to the weight of the beverage. Usually, the amount will not exceed 0.3% by weight and, normally, satisfactory results are obtained with less than 0.1% by weight with respect to the weight of the beverage.

In the first embodiment of the process of the invention it is not necessary to remove the compound that binds flavin before the beverage is consumed. Processes such as filtration or treatment with absorbers such as activated charcoal which have been used to reduce unpleasant flavors but which may also affect the desired flavor can be avoided. An effective carrier material for the immobilization of the flavin-binding compound is a silica-type material because such material is already in use in the production of beer, but other carrier materials can also be used which are preferably "grade" food ", - an example of which is AV1CELMR (cellulose food grade). When antibodies or fragments thereof are used as a flavin-binding compound, they can be coupled to a carrier material according to known techniques. For example, European patent EP-B1-0434317 (1997 and 1991, reference 14) claims immunoabsorbent materials comprising a specific binding agent (SBA) immobilized on a porous solid phase carrier material. The SBA can have a molecular weight of up to about 25,000 and can be composed of one or more variable domain proteins (VH and / or VL, where corresponding VH and VL are held together only by natural interactions), or it can be a fragment Natural Fv or a Dab fragment or a scFv. The SBA can be attached to the porous solid phase carrier material by a binder, preferably hydrophobic, having, for example, 5-20 amino acid residues. Immobilized immunoabsorbents can be used in an affinity purification process. Examples are agarose and porous silica (both silica activated by glutaraldehyde and epoxy silica converted to the diol derivative) as the porous carrier material and Fv antilisozyme and Dab (= anti Vl lysozyme with a peptide "myc" which binds VH to the carrier material of silica) as an antibody fragment. Another way of immobilizing a flavin, proteinaceous linking compound is by producing a transformed yeast having on its outer surface a flavin-proteinaceous binding compound, according to the method described in PCT application WO 94/18330 ( 1994, reference 15), in which recombinant DNA techniques are used to produce such binding protein or a functional part thereof. The binding protein is immobilized by producing it as part of a chimeric protein that also comprises an anchor part derivable from the C-terminal part of an anchoring protein, whereby it is ensured that the binding protein is located inside or outside of the cell wall of the host cell. Suitable anchoring proteins are yeast agglutinin FL01 (a protein associated with the flocculation phenotype in S. cerevisiae), the main cell wall protein of lower eukaryotes, and a proteinase of lactic acid bacteria. For secretion, the chimeric protein may be comprised of a signal peptide including yeast coupling factor, yeast agglutinin, Saccharomyces invertase, Kluyveromyces inulinase, Bacillus α-amylase and lactic acid bacteria proteinase. Reference 15 also provides a process for carrying out an isolation process by using such a transformed host, wherein the medium containing the specific compound is contacted with such host cell to form a complex, separate the middle complex and, optionally, releasing the specific compound from the. binding protein. Such a flavin proteinaceous compound may be a flavin binding protein or an antibody against a riboflavin-like compound or a fragment of such an antibody. If heavy chain antibodies or fragments thereof are desirable, antibodies and antibody fragments against a riboflavin-like compound similar to those described in PCT application WO 20 94/04678 (1994, reference 16) and application may be used. PCT WO 94/25591 (1994, reference 17), heavy chain antibodies or fragments thereof which lack light chains. A third embodiment of the invention is a beer or other light-sensitive beverage stabilized against the formation of A flavor generated by exposure to the sun either by incorporation into beverages of a sufficient amount of a flavin-binding compound capable of binding to the riboflavin-like compound or compounds present in the beverage, or by removing the riboflavin-like compound or compounds treat the drink with a compound that binds immobilized flavin. Such a beverage may be the result of a process according to the first or second embodiments of the invention. If the riboflavin is not removed from the beverage, the latter preferably contains a sufficient amount of the flavin-binding compound so that the taste generated by sun exposure is not detectable if the. The beverage is packaged in a light-permeable container, preferably a clear glass bottle, exposed to light at 40 ° C for half an hour. This exhibition is an accelerated test for the storage stability of the drink. Preferably, as the flavin binding compound, a protein that binds riboflavin or a flavotoxin or both is used, which can be isolated from natural sources. The addition of a flavin binding compound will result in a beverage having an increased flavor stability and less sensitivity to light. It has been found that the use of a flavin-binding compound not only suppresses the formation of a flavor generated by exposure to light, but also retards the formation of a damaged flavor. The invention is illustrated with some examples, in which experiments are described with flavodoxin and riboflavin-binding protein to eliminate riboflavin in beer and beer model systems. The examples are preceded by a "materials and methods" section that includes the preparation of both the flavodoxin and the riboflavin-binding protein used in the examples.

Materials and methods A. Purification of C69A flavodoxin from E. coli (containing a plasmid comprising a gene encoding the C69A mutant of the flavodoxin from Azotobacter vinelandii).

The flavodoxin of A. vinelandi i is able to dimerize due to the formation of mtermolecular bisulfide bonds between individual flavodoxin molecules, which can result in a loss of biological activity. To avoid this dimerization, a mutant flavodoxin is produced in which the single cysteine residue is replaced by an alanine residue which results in a modified flavodoxin indicated as C69A flavodoxin.

Reagents Buffer A contains 100 mM Tris-HCl, pH 8.0, 0.5 mM EDTA and 0.5 mM DTT (dithiothreitol).

Buffer B contains 50 mM KPi, pH 7.0 (K2HP04 / KH2P04) with 150 mM KCl and 0.5 mM EDTA.

Sodium acetate shock absorbers of pH 3.0 and pH 6.0.

Ty medium, containing casein hydrolyzate (10 g / 1), yeast extract (5 g / 1) and KCl (5 g / 1) Protein purification E. coli cells that grew for 24 hours at 37 ° C in an 8 1 fermenter filled with Ty medium at pH 7.4. The E. coli used is elaborated before by transformation of E. coli by introduction of an expressible gene coding for the C69A mutant of flavodoxin A. vinelandii using conventional recombinant DNA techniques. Collection of the cells by centrifugation (15 minutes, 8,000 rpm) 1: 1 resuspension with buffer A, together with DNAse, 1.5 mM MgCl2 and 0.75 mM EDTA. Perform press braking (1000 bar) in order to break the cells (do this twice). Sediment the remaining cell walls by centrifugation for 15 minutes at 15,000 rpm, collecting the cell-free extract (CFE).

Stage 1 Precipitation with protamine sulfate Protamine sulfate (from Sigma) is a strong basic protein which binds to DNA and large biomolecules by electrostatic interactions. Gently add protamine sulfate to the CFE (on ice, continuous agitation) until the concentration is 0.5% (w / v). The precipitated proteins are sedimented (15 minutes, 15,000 rpm) and the supernatant is collected.

Stage 2 Precipitation with 50% ammonium sulfate The supernatant is carefully adjusted to 50% saturation by gradually adding powdered ammonium sulfate (on ice, with stirring). The yellow / white precipitate is removed by centrifugation (15 minutes, 15,000 rpm) and the green supernatant is collected.

Stage 3 Precipitation with 75% ammonium sulfate The green supernatant is carefully adjusted to 75% saturation by gradually adding powdered ammonium sulfate (on ice, agitation). The white precipitate is removed by centrifugation (15 minutes, 15,000 rpm) and a bright yellow supernatant is collected.

Stage 4 Anion exchange chromatography (DEAE-Sepharose) A DEAE-sepharose column connected to a rapid protein liquid chromatography (FPLC) system is used. HE equilibrates with 75% ammonium sulfate in buffer A with 3 ml / minute. The yellow supernatant is applied on the column (3 ml / minute). It is positively charged to DEAE and this is capable of binding negatively charged ions. However, at 75% ammonium sulfate, all binding sites are occupied by S04. • 10 Because of this, the proteins which are applied to the column have no charged interaction but only hydrophobic interaction. In this case, DEAE serves as a hydrophobic interaction column. The column is washed with buffer A + 75% ammonium sulfate, for several hours. The flavodoxin is binds strongly to the column while most other proteins elute. The flavodoxin is eluted from the column using a 1 M KCl solution (3 ml / minute). Protein extracts are dialysed against buffer A.

Step 5 Anion exchange chromatography (high load K-Sepharose) A high-loading K-sepharose column (from Pharmacia) to an FPLC system. The column is balanced cushion A (3 ml per minute). The yellow protein extract is applied on the column and the following salt gradient is used; 0-16 minutes; 0.1 M KCl in buffer A, 16-96 minutes: KCl 0.1 M-KC1-0.7 M in buffer A, 96-100 minutes: KCl 0.7 M-KC1 1.0 M in buffer A, 100-108 minutes: KCl 1.0 M in buffer A, 108-113 minutes; KCl 1.0 M - 0.1 M KCl in buffer A, 113-128 minutes: 0.1 M KCl in buffer A The flavodoxin begins to elute at 0.49 M. The yellow fractions are collected and concentrated using an Amicon YM-10 filter (final volume 5 ml), dialyzed against buffer B.

Stage 6 Gel filtration A column of Superdex 75PG 26/600 (from Pharmacia) with buffer B (0.5 ml / minute). Flavodoxin extract (concentrate) is applied to the column. A flow rate of 1.0 ml / minute is used. The yellow fraction is collected.

Step 7 Preparation of Apo protein Cold trichloroacetic acid (TCA, dissolved in Tris 0.3 M + 0.3 mM EDTA) to provide a final concentration of 5% (w / v) TCA. It is allowed to sit for 5 minutes in the dark, and then centrifuged (10 minutes, ,000 rpm). The yellow supernatant is discarded. The white precipitate is resuspended in TCA 5% (in Tris 0.3 M, EDTA 0.3 mM). The mixture is centrifuged as above and the precipitate is dissolved in a minimum volume of 0.3 M Tris, pH 7 and 0.3 mM EDTA. It slides against 0.1 M KPi, pH 7, and 0.3 mM EDTA and is stored at 4 ° C. The volume of the supernatant is measured from all the purification steps and samples of 0.5 ml are taken for analysis by SDS-PAGE and protein determination. To obtain an indication of the purity of the different fractions, the absorbance ratio A280 / A455 can be measured.

B. Purification of riboflavin-binding protein (RfBP) from chicken egg white The protein that binds chicken riboflavin is a stable and acid phosphoglycoprotein. It binds to a riboflavin molecule with a dissociation constant of 1.3 nM. It is produced in the liver and oviduct of laying hens, and they are deposited in the egg yolk and egg white. The yellowish coating of a typical white egg is due to the riboflavin bound to this protein (the native egg white RfBP is usually only about 30-35% saturated with riboflavin).

Although a single gene that responds to estrogen controls the synthesis of the protein in the liver and oviduct, the protein isolated from yolk has a more complicated composition of carbohydrates and seems to lose seven amino acids in the carboxyl terminal part present in the protein of egg.

Reagents 50 mM sodium acetate, pH 4.5 25 mM sodium acetate, pH 3.0 25 mM sodium acetate, pH 5.6 100 mM KPi, pH 6.0 10 mM riboflavin (5 mg of riboflavin dissolved in 1 ml of dimethylsulfoxide (DMSO).

Protein Purification Stage 1 Preparation of egg white 12 eggs are broken and the egg white is carefully separated from the egg yolk. The egg whites are collected in a beaker and stored on ice. The egg white is diluted to 1 liter with 50 mM sodium acetate, pH 4.5. Add 1 ml of 10 mM riboflavin and stir gently for 30 minutes on ice. The insoluble precipitate is removed by centrifugation (20 minutes, 14,000 rpm).

Stage 2 Anion exchange chromatography One column (50 ml bed volume) is packed with DEASE-Sepharose (from Pharmacia). It is equilibrated with 50 mM sodium acetate, pH 4.5. A flow rate of 1 ml / minute is used. The egg white solution is carefully applied on the column and washed with 50 M sodium acetate to remove unbound protein. RfBP is eluted with 50 mM sodium acetate + 1 M NaCl. The yellow band is harvested.

Stage 3 Precipitation with ammonium sulfate Carefully adjust to a 55% saturation by gradually adding powdered sulfate (stirring on ice). The white precipitate is removed by centrifugation (10 minutes, 10,000 rpm) and the supernatant is carefully collected. The volume of the supernatant is measured and RfBP is precipitated by gradually adding pulverized ammonium sulfate to 85% saturation (stirring on ice). The precipitate is dissolved in 5 ml of 0.1 M KPi buffer, pH 6.0, and the yellow protein solution is dialysed against the same buffer.

Stage 4 Gel filtration A Superdex-200 PG column (from Pharmacia) is equilibrated in 50 mM KPi buffer, pH 6.0. Carefully apply the partially purified protein on the column. A flow rate of 1.0 ml / minute is used. The yellow fraction is collected.

Step 5: Preparation of apo-protein by chromatography on CM-sephar The binding of riboflavin is pH dependent (pKa = 3.5). The pH of the protein solution is adjusted to pH = 3. Riboflavin completely dissociates from RfBP. A column of CM (= carboxymethyl) sepharose (from Pharmacia) (20 ml) is packed and equilibrated with 25 mM sodium acetate, pH 3.0. The solution of RfBP (pH = 3) is applied on the column. Apo-RfBP will join the column. Wash with 25 mM sodium acetate, pH 3, to remove riboflavin. Apo-RfBP is eluted with sodium acetate. 25 mM, pH 5.6 + 200 mM NaCl. Pure apo-RfBP is dialysed with 25 mM sodium acetate, pH 3.0 and stored at 4 ° C. The volume of supernatant is measured from all purification steps and samples of 0.5 ml are taken for analysis by SDS-PAGE and determination of protein concentration. To obtain an indication of purity of the different fractions, the absorbance ratio A, 80 / A455 can be measured.

Model System Studies ESR analysis A sample of beer / beer model (light-treated plus a spin trap) is placed in an open-end capillary electron spin (ESR) resonance tube (external radius - = 3 mm, inner radius = 1.5 mm) which is placed in a larger ESR tube (radius = 5 mm). Both tubes are of Suprasil 1 quartz (from Heraeus, Nijmegen). The smaller ESR tube has a rubber tube sealed at the end so that it works like an udder. The sample column in the smaller tube is 1 cm above the bottom and at least 2 cm long. Free radicals are detected in a Bruker ER 200 D electronic paramagnetic resonance spectrometer under the following conditions Field 3485 Gauss Width of sweep 100 Gauss Attenuation 10 dB Modulation amplitude 2.5 Gauss Gain 1.25E6 Frequency 9.74 GHz Sweep time 200 s Time constant 0.2 S Temperature Ambient temperature Comparative Example 1 Effect of riboflavin on the formation of free radicals Riboflavin (0, 0.5, 1, 2, 3, 4, 5, 6, 1, 8, 9 and 10 μg / ml) is suspended in 1 ml of 5% (v / v) ethanol containing 50% sodium acetate. mM adjusted to pH 4.0 with glacial acetic acid. The spin trap: Nt-butyl-a-phenylnitrone (PBN) is added to aliquots of 0.25 ml in each 1 ml of rivoflavin solution so that the final PBN concentration is 25 mM Each solution is exposed to the light (8 W fluorescent tube in a closed light box) in a 1.5 ml sealed clear glass GC bottle for 4 hours, during which time the sample temperature does not exceed 20 ° C. The resulting light-treated sample is analyzed for free radicals using electron spin rasonance.The results are presented in Figure 1, which shows that with an increasing level of riboflavin, the ESR signal is also increased to approximately 5 ppm of riboflavin, after which no further increase in the ESR signal was observed Additional experiments were carried out with model systems using a standard level of riboflavin of 1 ppm.This accords with a typical level of 'riboflavin' in beer of 0.1-0.8 ppm.

Examples 1-2 and Comparative Example 2 Effect of flavin binding proteins and bovine serum albumin (BSA) on the formation of free radicals 0 Riboflavin (1 ppm or 2.7 μM) is suspended in 1 ml of 5% ethanol (v / v) containing sodium acetate 50 mM adjusted to pH. 4.0 with glacial acetic acid. The spin trap is added: N-t-butyl-a-phenylnitrona (PBN) in aliquots of 0.25 ml in each 1 ml of riboflavin solution so that the final concentration "of 'PBN is 25 mM. To this model system is added the protein to be tested in a range of concentrations (0, 0.5, 2. 0, 4.0 and 20.0 μM) for each protein that binds riboflavin (RfBP, Example 1), flavodoxin (Fdx, Example 2) and, as a comparison, 0 bovine serum albumin (from Sigma, Comparative Example 2). Each solution is exposed to light (8 W fluorescent tube in a closed light box) in a sealed, clear glass GC bottle for 4 hours. During this time, the temperature of the sample does not exceed 20 ° C. The resulting solution treated with light is analyzed to determine free radicals using electron spin resonance. The results are presented in Figure 2, which shows that by increasing the concentration of flavin binder, the amount of free radicals is reduced by both the riboflavin-binding protein and flavodoxin. In the samples treated with BSA, the amount of free radicals does not change when increasing the BSA concentration, although the level of free radical initiation is lower than without the addition of BSA. These results suggest that the specific flavin binding properties of RfBP and Fdx suppress the tendency of riboflavin to generate free radicals when exposed to. light. Apparently, it is not protein per se that suppresses the formation of free radicals, because BSA does not produce a similar result.

Examples 3-4 Effect of flavin-binding proteins on flavor formation generated by sun exposure in model beer systems Riboflavin (1 ppm or 2.7 μM) is suspended in 1 ml of 5% (v / v) ethanol containing 50 mM sodium acetate, adjusted to pH 4.0 with glacial acetic acid. Cysteine hydrochloride (from Sigma) and Isohopcon (from English Hop Products, UK) are added to the model system to provide final concentrations of 10 ppm and 20 ppm, respectively. To this model system is added flavin binder in a range of concentrations (0, 0.3, 1.3, 2.7 and 13.5 μM) for each of the riboflavin binding proteins (RfBP; Example 3) and flavodoxin (Fdx, Example 4). Each solution is exposed to light (8 W fluorescent tube in a closed light box) in a sealed clear glass GC bottle for 4 hours. During this time, the temperature of the sample does not exceed 20 ° C. The resulting solution treated with light is evaluated for flavor or flavor determination (generated by sun exposure) by a trained panel of tasters.

Table I: Taste evaluation of model system samples treated with RfBP / Fdx The results shown in the previous table, which show that by increasing the concentration of both RfBP and Fdx, the flavor "generated by sun exposure" decreases. Fdx appears to be more efficient at reducing the taste formation "generated by sun exposure" than RfBP. The results of examples 1-4 show that the flavin binding property of both the riboflavin-binding protein and flavodoxin in the riboflavin binding has resulted in a reduced formation of free radicals and taste "generated by exposure to sun "in a beer model system.

Example 5 Effect of flavin-binding proteins on flavor formation "generated upon sun exposure" in beer systems A very pale aged beer (1 ml) with riboflavin-binding protein (RfBP) is treated to provide a final protein concentration of 0, 0.3, 1.3, 2.7 and 13.5 μM. The spin trap is added: N-t-butyl-a-phenylnitrona (PBN) in aliquots of 0.25 ml in each 1 ml of beer so that the final concentration of PBN is 25 mM. Each solution is exposed to light (8 W fluorescent tube in a closed light box) in a sealed, clear glass GC bottle for 4 hours. During this time, the temperature of the sample does not exceed 20 ° C. The resulting solution treated in the light is analyzed to determine free radicals by spin resonance of electrons. The results are presented in Figure 3, which shows that the free radical signal decreases with increasing RfBP concentration. This experiment confirms that the binding capacity of the riboflavin-binding protein for flavin photosensitizers reduces the formation of free radicals in beer.

REFERENCES 1. Y. Kuroiwa amd N. Hashimoto, - American Society of Brewing Chemists (ASBC) Proceedings (1961) 28-36; Composition of Sunstruck Flavor Substance and Mechanism of Its Evolution. 2. C. Blockmans, J. vande Meersche, C. A. Massechelein and A, Devreux, - Proceedings of the European Brewery Convention (EBC), 18th Congress, Copenhangen (1981) 347-357; Photodegradation amd formation of carbonyl - and sulphur compounds in beer. 3. A. J. Irwin, L. Bordeleau, and R.L. Barker; ASBC Journal 51 (1993, No. 1) 1-3; Model Studies and Flavor Threshold Determination of 3-Mehthyl-2-Butene-l-Thiol in Beer. 4. A. Devreux, C. Blockmans, and J. vande Merereche; EBC Monograph VII, Copenhagen - November (1981) 191-201; CARBONYL COMPOUNDS FORMATION DURING AGING OF BEER 5. M. Tanaka, M. Harris, and K.T. Yasunobu; Biochem Biophys. Res. Commun. 66. (1975) 639-644; The amino acids sequence of the AzotoiDacter vinelandii flavodoxin 6. L.T. Bennett, M.R. Jacobson, and D.R. Dean; J. Biol. -Chem. 263 (1988) 1364-1369; Isolation, Sequencing, and Mutagenesis of the nifFin gene encoding flavodoxin from Azoto acter vinelandii. 7. J. J. Pueyo, G.P. Curley and S.G. Mayhew; Biochem. J. 313 (1996) 855-861; Kinetics and thermodynamics of the binding of riboflavin, riboflavin 5'-phosphate and riboflavin 3 ', 5'-biphosphate by apoflavodoxin 8. US-PS 4534971; published 13.08.1985; J.F. Fisher (Regents of the University of Minnesota, USA, expired 15.08.1993; Complexation of anthracycline and anthraquinone antibiotics by the apo riboflavin binding protein from eggs 9. J. Becvar (1973) Ph.D. thesis, University of Michigan 10. Y Hamazume, T. Mega, and T. Ikenaka, J. Biochem 95. (1984) 1633-1644, Characterization of Heng Egg White- and Yolk-W Riboflavin Binding Proteins and Amino Acid Sequence of Egg White- Riboflavin Binding Protein 5 11 .SA Innisitehouse, AH Merrill Jr., and DB McCormick in Chapter 11 (Riboflavin Binding Proteins) of the book Chemistry and Biochemistry of Flavoenzymes, Vol. 1, edited by F. Muller, CRC press, Boston, (1991) 287-292 12. SIGMA Chemical Company, Catalog (1991) Biochemicals 10 Organic Compounds for Research and Diagnostic Reagents, page-894 13. YE Bruggeman, RG Schoenmakers, A. Schots, EHW, Pap A. van Hoek, AJ. Visser and R. Hilhorsy; Eur. J. Biochem, 234 (1995) 245-250; Monoclonal antibodies against two electron reduced and a quantification of affinity constants for this oxygen-sensitive molecule 14. European rranted patent EP-B1-0434317; CROSFIELD LIMITED; published on 05.03.1997 and as application on 26.06.1991; Immunoadsorbents 20 15. PCT application WO 94/18330; UNILEVER; published on 18.08.1994; Process for immobilizing biuding proteins to the cell wall of a microbial cell by producing a fusion protein 16. PCT Application WO 94/04678; C. Casterman & R. Ha ers; published on 03.03.1994; Immunoglobulens devoid of light chains

Claims (12)

REI INDICATIONS

1. A process for increasing the flavor stability of beer or other light-sensitive beverage, which process comprises inactivating a riboflavin-like compound or riboflavin-like compounds present in the beverage by allowing them to bind to a flavin-binding compound present in the beverage. a sufficient amount, and for a sufficient time to obtain an improvement in the flavor stability of the beverage as compared to a similar beverage, which has not been put on. contact with the compound that binds flavin.

2. The process as described in claim 1, wherein the flavin binding compound is incorporated in the beverage in an amount sufficient to observe a measurable improvement in the flavor stability of the beverage during storage compared to a beverage similar to that of the beverage. which has not been added the compound that binds flavin.

3. The process as described in claim 1, wherein inactivating the riboflavin-like compound or compounds is obtained by: (a) contacting the beverage with a sufficient amount of immobilized flavin-binding compound in a compatible carrier material with the beverage, followed by (b) allowing the riboflavin-like compound or compounds to form a complex with the immobilized flavin-binding compound, and (c) subsequently separating the immobilized complex from the beverage.

4. The process as described in claim 3, wherein the carrier material is a silica type material.

5. The process as described in any of claims 1 to 4, wherein the flavin-binding compound is a protein.

6. The process as described in claim 5, wherein the flavin, proteinaceous binding compound is selected from the group consisting of riboflavin-flavodoxin binding protein.

7. The process as described in any of claims 1 to 6, in which the improvement of the flavor stability of the beverage is evaluated by using a trained taster team to judge the taste of the beverage when smelling or tasting, or both Things, in order to compare the drinks with a similar drink which has not been in contact with the compound that binds flavin.

8. A beer or other light-sensitive beverage, stabilized to prevent the formation of a flavor generated by sun exposure either by incorporating into the beverage an amount of a flavin-binding compound capable of binding a riboflavin-like compound or compounds present in beverages, or by removing the riboflavin-like compound or compound when treating the beverage with a flavin-immobilized compound.

9. The beverage, as described in claim 8, wherein the taste generated by sun exposure is not detectable if the beverage packaged in a light-permeable bottle is exposed to light at 40 ° C for half an hour.

10. The beverage, as described in claim 8 or 9, which contains a protein that binds riboflavin or flavodoxin, or both.

11. A beer or other light-sensitive beverage, with increased flavor stability, due to the incorporation of at least one flavin-binding compound capable of binding riboflavin-like compounds.

12. The beverage, as described in claim 11, wherein the flavin binding compound is a protein that binds riboflavin or flavodoxin, or both.