How to make beer

Abstract

The present invention relates to a method for producing beer and beer-related beverages, comprising adding catalase enzyme and an antioxidant, preferably ascorbic acid, after the mash boil and / or during or after the wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization stages of the brewing process to improve the flavor and / or flavor stability of the finished beer. Catalase and an antioxidant, preferably ascorbic acid, may additionally or alternatively be added to the finished beer itself.

Description

[Technical field]

[0001] The present invention relates to a method for producing beer and beer-related beverages, comprising adding catalase enzyme after mash boiling and / or during or after the wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization stages of the brewing process to improve the flavor and / or flavor stability of the finished beer. Catalase may additionally or alternatively be added to the finished beer itself. The present invention also relates to the use of an antioxidant, preferably ascorbic acid (vitamin C), in addition to catalase during the brewing process. [Background technology]

[0002] The process of making beer involves several stages. In general, the brewing process may involve heating ground malt and other grains with water during mashing to form wort. Yeast is then added to the wort to ferment any fermentable sugars present in the wort into alcohol. The beer may then be further aged and / or stabilized prior to bottling to achieve the desired flavor and flavor stability. The aging and stabilization stages are sometimes referred to as the "lagering" period.

[0003] One problem that can be faced in brewing is the potentially detrimental effect of oxidation on the flavor and stability of the beer produced. Preferably, the flavor of the finished beer should be stable enough to match the desired shelf life of the finished product. Thus, there is a need in the art to provide improved brewing processes that promote increased oxidative stability (and thus reduced free radical production), and thus improved flavor and flavor stability over the shelf life of the final product.

[0004] Enzymes are often added during brewing to contribute to the quality of beer in terms of color, clarification, flavor or texture. The uses of different enzymes are wide-ranging. Catalase is an antioxidant enzyme that catalyzes the decomposition reaction of hydrogen peroxide into water and oxygen. In addition, catalase can act as a peroxidase at low hydrogen peroxide levels. Summary of the Invention [Means for solving the problem]

[0005] The inventors have surprisingly found that adding catalase enzyme and an antioxidant, preferably ascorbic acid, during or after the brewing process has a beneficial effect on beer flavor and flavor stability. The present invention therefore relates to the addition of catalase and an antioxidant, preferably ascorbic acid, after mash boiling of beer and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization or to the final beer itself. Advantageously, a positive effect on flavor due to reduction of Strecker aldehyde and / or flavor stability is achieved in the final beer throughout its shelf life.

[0006] The beneficial effect of catalase and antioxidant, preferably ascorbic acid, may be related to oxidation effects. Oxidation may have a detrimental effect on the flavor and flavor stability of beer. Without wishing to be bound by theory, it is believed that catalase may act on pro-oxidative radical production, for example, only when hydrogen peroxide is formed after consumption of endogenous antioxidant potential (EAP). Frederiksen et al. suggested that application of catalase during mashing into beer may result in a decrease in the rate of radical formation (Frederiksen et al. Journal of Agricultural and Food Chemistry, 56 (2008), 18, pp. 8514-8520).

[0007] Thus, the present invention also describes the synergistic benefits of using additional antioxidants such as ascorbic acid (e.g., in the form of acerola or other natural extracts with antioxidant activity and high EAP) together with catalase for further improved oxidative stability.

[0008] To determine the lag time or intrinsic antioxidant potential (EAP value) of beer as a shelf life indicator, analysis can be performed by electron spin resonance spectroscopy (ESR). ESR imposes forced aging via increasing temperature, which results in an aging process of the beer being tested. With respect to the ESR measurement, the value of the lag time or EAP value determines the intrinsic antioxidant beer stability, which correlates with the flavor stability of the beer.

[0009] The present invention provides a method for producing beer with improved flavor and / or flavor stability, said method comprising adding a catalase composition and an antioxidant, preferably ascorbic acid, after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization, and / or comprising adding a catalase composition and an antioxidant, preferably ascorbic acid, to the final beer.

[0010] It is common practice in the industry to add enzyme compositions during beer production to improve beer brewing, and it is also well known that exogenous enzymes can be added, especially during the mashing step.

[0011] However, when catalase is added at the mashing step, the heat effect from the process leads to denaturation of the enzyme and also to an increase in iron ions, which promote oxygen activation and result in higher ageing susceptibility.

[0012] Advantageously, catalase and an antioxidant, preferably ascorbic acid, may be active after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of beer. In order to avoid inactivation of catalase early in the extraction process, catalase and an antioxidant, preferably ascorbic acid, may be added after mash boiling. In one aspect of the invention, catalase and an antioxidant, preferably ascorbic acid, may be added after wort boiling. In one aspect, catalase and an antioxidant, preferably ascorbic acid, may further be added before wort boiling.

[0013] In one aspect of the invention, the catalase and the antioxidant, preferably ascorbic acid, can be added at more than one stage of the brewing process. For example, the catalase and the antioxidant, preferably ascorbic acid, should be added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization, but can also be added, for example, during mashing and / or before fermentation. The catalase and the antioxidant, preferably ascorbic acid, can also be added directly to the final beer.

[0014] In one embodiment, the catalase is a microbial catalase. In a preferred embodiment, the microbial catalase can be derived from a bacterium or a fungus. In one embodiment, the microbial catalase is derived from Aspergillus sp., Bacillus sp., Saccharomyces sp., Escherichia sp., Pseudomonas sp., Trichoderma sp., or Scytalidium sp. In one embodiment, the microbial catalase is derived from Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Bacillus licheniformis, Bacillus fragilis, Bacillus pumilus, Bacillus halodurans, Saccharomyces cerevisiae, Escherichia coli, Pseudomonas aeruginosa, Trichoderma reesei, or Scytalidium thermophilum. In a preferred embodiment, the microbial catalase is from Aspergillus sp. or Trichoderma sp., preferably A. niger or T. reesei. More preferably, the catalase is from A. niger.

[0015] In a preferred embodiment, the catalase has or comprises a sequence according to any of SEQ ID NOs: 1, 2, 3 or 4 or a sequence having at least 70% identity thereto. For example, the catalase may have a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. In one embodiment, the catalase may comprise or consist of a sequence according to SEQ ID NOs: 1, 2, 3 or 4 or a sequence having at least 70% identity thereto. Most preferably, the catalase according to the invention has the sequence of SEQ ID NO: 1. Catalases may be produced by recombinant techniques well known to those skilled in the art.

[0016] In one aspect of the invention, the catalase composition further comprises glucose oxidase.

[0017] In one aspect of the invention, antioxidants can be used in addition to catalase during the brewing process. Any suitable antioxidant can be used, including both synthetic and natural antioxidants. The antioxidant can be added at any stage of the brewing process. In one aspect, the antioxidant can be added at the beginning of fermentation. In a preferred aspect, the antioxidant can be added at the end of fermentation. In a more preferred aspect, the antioxidant can be added during the stabilization and / or maturation stages described herein. Most preferably, the antioxidants described herein can be added to the finished beer or beverage product.

[0018] In one aspect, ascorbic acid can be used in addition to catalase. The ascorbic acid described herein can be added at any stage of the brewing process. In one aspect, the ascorbic acid can be added at the beginning of fermentation. In a preferred aspect, the ascorbic acid can be added at the end of fermentation. In a more preferred aspect, the ascorbic acid can be added during the stabilization and / or maturation stages described herein. Most preferably, the ascorbic acid described herein can be added to the finished beer or beverage product.

[0019] In a further aspect according to the invention, ascorbic acid may be added in the form of a natural antioxidant. In this regard, the natural antioxidant may be added during the brewing process and / or may be added to the finished beer. Any suitable natural antioxidant may be used according to the invention.

[0020] By way of example, the natural antioxidant may be selected from tocopherols, polyphenols (tannins, gallic acid, etc.), acerola extract (naturally occurring ascorbic acid), pomegranate extract, rosemary extract (E392 or spice extract), lemon extract, green tea extract, blend of rosemary and lemon extract, rosemary extract and blend of green tea and rosemary extract. In a preferred embodiment, said natural antioxidant is acerola extract.

[0021] The synthetic antioxidants may be selected from BHA (E320), BHT (E321), TBHQ (E319), ascorbyl palmitate (E304), synthetic tocopherols, ethoxyquin (E324), sodium ascorbate (E301), EDTA (E385), propyl gallate (E310), ascorbic acid (E300) and KMS / NaMS (E224 / E223).

[0022] The present invention also encompasses a beer obtained or obtainable by the method according to the invention. In one aspect, the final beer comprises a catalase as described herein and an antioxidant, preferably ascorbic acid. The final beer may also or alternatively comprise glucose oxidase.

[0023] In one aspect of the present invention, the oxidative stability of beer is improved.

[0024] In one aspect of the invention, the production of Strecker aldehyde in beer is reduced, including the production of benzaldehyde and / or trans-2-nonenal.

[0025] The present invention also encompasses the use of the catalase composition and an antioxidant, preferably ascorbic acid, described herein in the production of beer, wherein said catalase composition and an antioxidant, preferably ascorbic acid, are added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization, and / or are added to the final beer.

[0026] In a preferred embodiment, the catalase composition has a catalase activity of at least 0.1 U / ml.

[0027] In a preferred embodiment, the catalase composition has a catalase activity of at least 1 U / ml.

[0028] In a preferred embodiment, the catalase composition has a catalase activity of at least 10 U / ml.

[0029] In a preferred embodiment, the catalase composition has a catalase activity of at least 100 U / ml.

[0030] In a preferred embodiment, the catalase composition has a catalase activity of at least 1,000 U / ml.

[0031] In a preferred embodiment, the catalase composition has a catalase activity of at least 10,000 U / ml.

[0032] In a preferred embodiment, the catalase composition has a catalase activity of at least 100,000 U / ml.

[0033] In a preferred embodiment, the catalase composition provides a catalase activity of at least 0.1 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0034] In a preferred embodiment, the catalase composition provides a catalase activity of at least 1 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0035] In a preferred embodiment, the catalase composition provides a catalase activity of at least 10 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0036] In a preferred embodiment, the catalase composition provides a catalase activity of at least 100 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0037] In a preferred embodiment, the catalase composition provides a catalase activity of at least 1,000 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0038] In a preferred embodiment, the catalase composition provides a catalase activity of at least 10,000 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0039] In a preferred embodiment, the catalase composition provides a catalase activity of at least 100,000 U / ml in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0040] In one aspect, the production of Strecker aldehyde in the final beer is reduced.

[0041] In one aspect, trans-2-nonenal production is reduced in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0042] In one aspect, trans-2-nonenal production in the final beer is reduced.

[0043] In one embodiment, trans-2-nonenal production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably trans-2-nonenal production is reduced by at least 50%.

[0044] In one aspect, trans-benzaldehyde production is reduced in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0045] In one aspect, trans-benzaldehyde production in the final beer is reduced.

[0046] In one embodiment, benzaldehyde production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably at least 50% benzaldehyde production is reduced.

[0047] In one aspect, the shelf life of the beer is extended.

[0048] In one embodiment, the shelf life of the beer is extended by at least about 4 months, or by at least about 6 months, or by at least about 9 months, or by at least about 12 months, or by at least about 24 months.

[0049] The present invention also provides the use of a catalase composition in the production of beer, wherein said catalase composition is added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization, and / or is added to the final beer.

[0050] In one aspect, in the use according to the invention, the catalase composition is a catalase composition according to the invention.

[0051] The present invention also provides a beer obtained or obtainable by the process according to the invention.

[0052] The present invention also provides a beer comprising the catalase.

[0053] The present invention also provides a beer comprising catalase and glucose oxidase.

[0054] In one embodiment, the shelf life of the beer is extended to at least about four months.

[0055] In one embodiment, the shelf life of the beer is extended to at least about six months.

[0056] In one embodiment, the shelf life of the beer is extended to at least about 9 months.

[0057] In one embodiment, the shelf life of the beer is extended to at least about 12 months.

[0058] In one embodiment, the shelf life of the beer is extended to at least about 24 months.

[0059] In one embodiment, the shelf life of the beer is extended by at least about four months.

[0060] In one embodiment, the shelf life of the beer is extended by at least about six months.

[0061] In one embodiment, the shelf life of the beer is extended by at least about 9 months.

[0062] In one embodiment, the shelf life of the beer is extended by at least about 12 months.

[0063] In one embodiment, the shelf life of the beer is extended by at least about 24 months.

[0064] In one aspect, the flavor stability of the beer is improved. [Brief description of the drawings]

[0065] [Figure 1] Shown are the results of EAP and T600 determinations with POBN on oxidized wort stability with the addition of catalase shown in SEQ ID NO:1 by forcing the test at 60° C. and 3.5 mM POBN using 0.0, 4.4, 8.8, 22.1 and 44.2 U / ml as indicated in the legend. [Diagram 2] Shown are the results of EAP and T600 determinations with POBN on oxidized wort stability with the addition of catalase shown in SEQ ID NO:1 by forcing the test at 60°C and 3.5 mM POBN using 0.0, 0.9, 1.8, 2.7, 3.5, 4.4 and 6.2 U / ml as indicated in the legend. [Diagram 3] Shown are the results of EAP and T600 determinations with POBN on oxidative low EAP beer (pilsner type) stability with the addition of catalase shown in SEQ ID NO:1 by forcing the test at 60°C and 3.5 mM POBN using 0.0, 0.2, 0.4, 0.8, 1.6, 2.4, 3.1 and 3.9 U / ml as shown in the legend. [Figure 4] The results of EAP and T600 determination with POBN in oxidation medium EAP beer (pilsner type) stability with the addition of catalase shown in SEQ ID NO: 1 are shown by forcing tests at 60°C and 3.5 mM POBN using 0.0, 0.2, 0.39, 0.79 and 1.97 U / ml as shown in the legend. [Diagram 5]FIG. 1 shows standard MEBAK beer analysis of Tuborg and Odense Pilsner (Denmark) used in storage stability studies. [Figure 6] FIG. 1 shows ESR measurement storage tests using (Odense Pilsner fresh, Odense with 650 μl HO and 1 month storage, Odense with 650 μl catalase shown in SEQ ID NO:1 and 1 month storage; Tuborg fresh, Tuborg with 650 μl HO and 1 month storage, Tuborg with 650 μl catalase shown in SEQ ID NO:1 and 1 month storage). [Figure 7] 1 shows the mashing program used for the laboratory scale malt-based mashing trials. [Figure 8] FIG. 2 shows the wort analysis (extract, pH and color) of dark beer wort with and without the catalase shown in SEQ ID NO:1. [Figure 9] FIG. 2 shows ESR measurement mashing tests of dark beer wort with or without the addition of catalase shown in SEQ ID NO: 1 during mash preparation (dark beer wort with 0.63 U / ml catalase (A. niger), dark beer wort with 1.26 U / ml catalase (A. niger), dark beer wort with 0.63 U / mL catalase (T. reesei), dark beer wort with 1.26 U / ml catalase (T. reesei), boiled dark beer wort with 0.63 U / mL catalase (A. niger), boiled dark beer wort with 0.63 U / ml catalase (T. reesei) and control / reference dark beer wort). [Figure 10]GCMS-SPME relative quantification of the following Strecker aldehyde off-flavors in the accelerated beer oxidation test is shown: 2-methylfuran, hexanal, 2-butenal, trans-3-penten-2-one, 4-hexen-3-one, furfural, trans-2-nonenal, benzaldehyde, phenylacetaldehyde, and ethyl nicotinate as well as two unidentified substances (ions, RT: 10.21; ions: 95.00 / unidentified and RT: 9.25; ions: 81.00 / unidentified). Control beer was unopened and kept at room temperature (RT) for 14 days. Headspace oxidized beer samples were incubated at either 30°C or 60°C for up to 14 days to accelerate oxidation as indicated by the label. [Figure 11a] Beer freshness in terms of free radical generation measured in uM for beer freshness testing with the following additives: Foodpro CAT A. niger catalase, FL002_GC119 T. reesei catalase, sodium metabisulfite (Na2S2O5) and FL003 Guardian Acerola acerola juice concentrate, which were added to bottles stored in a refrigerator for 14 days with a headspace air flush or at 30°C for 14 days. [Fig. 11b-11d] The same data as in Figure 11a, but with samples with similar free radical generation grouped together. Figure 11b shows samples O1FA22 and O1FA22_no with no freshness protection and slower oxidation rates. Figure 11c shows samples with acceptable freshness performance but poor oxidation. Figure 11d shows samples with low to acceptable freshness protection but better oxidation performance. [Figure 11e] The oxidation rates of the beer samples ranked from lowest to highest are shown, with a lower oxidation rate being preferred for beer. [Figure 12a]ESR extract studies are shown using a control beer along with dark beer spiked with 0.25% acerola extract, 0.02% powdered rosemary extract, 0.03% rosemary & lemon extract blend, 0.12% liquid rosemary extract, and 0.009% green tea and rosemary extract blend. [Figure 12b] ESR extract studies are shown using a control beer along with pilsner beer spiked with 0.25% acerola extract, 0.02% powdered rosemary extract, 0.03% blend of rosemary and lemon extracts, 0.12% liquid rosemary extract, and 0.009% blend of green tea and rosemary extracts. [Figure 13] Figure 2 shows the results of EAP and T1000 determination with POBN on oxidative low EAP beer (pilsner type) stability with addition of natural antioxidant extracts as described in the legend by forcing test at 60°C and 3.5 mM POBN. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The process of beer brewing is well known to those skilled in the art. Standard methods may include providing malted barley and / or unmalted adjuncts, called grist. The next stage of the process may involve mashing the grist with water (with heating and stirring) so that carbohydrates are broken down into fermentable sugars using enzymes naturally present in the malt. The influence of enzymes in the brewing process plays an important role. Malt usually contains many enzymes, which are most active when the grains come into contact with the wort (wort filtration is the process of separating the wort from the solids). This may be called the mashing phase. Temperatures during the later stages of the mashing process may exceed 75°C.

[0067] After mashing, the liquid extract, called wort, is separated from the solids (spent grain particles and adjuvants) to obtain a clear wort. Hops can then be added to the wort. After adding the hops, the wort is boiled and cooled to facilitate the addition of yeast. The fermentation stage involves the fermentation of fermentable sugars into alcohol by yeast. After main fermentation, which typically lasts for 5-10 days, most of the yeast is removed and the so-called "green beer" is formed.

[0068] The "green" beer resulting from this primary fermentation still contains some unsettled yeast and relatively high levels of undesirable flavour components, such as diketones, e.g. diacetyl and acetylaldehyde. During the maturation phase, these undesirable flavour components are converted into bland tasting compounds. For example, during maturation, the amount of diacetyl, a compound with a buttery off-flavour, can be converted into tasteless acetoin. A subsequent stabilisation phase can increase the colloidal stability of the beer. In this regard, the beer can be held to promote the formation of protein-polyphenol aggregates, after which the precipitated aggregates can be removed, e.g. by filtration / sedimentation. The phase incorporating both the maturation and stabilisation phases of beer production can be called the lagering period.

[0069] In accordance with the present invention, an enzyme composition comprising catalase and an antioxidant, preferably ascorbic acid, is added during the brewing process at a point after the mashing step and / or after the wort boiling.

[0070] Catalase can be added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization, and / or is added to the final beer.

[0071] The beer produced by the method according to the invention can be any type of beer. The term "beer" as used herein is intended to cover at least beer prepared from a mash prepared from unmalted cereals as well as all mashes prepared from malted cereals and all mashes prepared from a mixture of malted and unmalted cereals. The term "beer" encompasses not only bottom-fermented beers, but also top-fermented beers and beers prepared with adjuncts and beers with all possible alcohol contents.

[0072] Preferred types of beer include beer, strong beer, stout, porter, dark beer, pilsner, regier, bitter, export beer, malt liquor, happoshu, high alcohol beer, low alcohol beer, low calorie beer or light beer. The invention also extends to non-alcoholic or low alcohol beer. In a preferred embodiment, the beer may be an IPA, a pilsner beer or a low alcohol or no alcohol beer.

[0073] In one aspect of the invention, the shelf life of beer can be improved or extended, in one aspect the shelf life can be extended up to 4, 6, 9, 12 or 24 months.

[0074] In one aspect of the invention, the production of Strecker aldehyde can be reduced, including a reduction in the production of trans-2-nonenal and / or benzaldehyde of at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. Preferably, the production of trans-2-nonenal and / or benzaldehyde is reduced by at least 50%.

[0075] Advantageously, the present invention can facilitate reducing the amount of sulfites used in the brewing process or present in the final beer product.

[0076] The beer obtained according to the invention is typically packaged. Any suitable packaging can be used, for example bottles, kegs or cans. The beer prepared according to the invention can be packaged in bulk tanks. Thus, the invention provides a packaged product comprising the beer obtained according to the invention, for example a bottle, keg or can comprising such a beer.

[0077] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges include the numbers that define the range.

[0078] The headings provided herein are not intended to limit the various aspects or embodiments of the present disclosure, which can be understood by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole. As used herein, the term "polynucleotide" is synonymous with the term "nucleotide sequence" and / or the term "nucleic acid sequence." Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction.

[0079] The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the term "polypeptide" and / or the term "protein". In the present disclosure and claims, amino acid names, conventional one-letter codes and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Unless otherwise indicated, any amino acid sequence is written in amino to carboxy orientation from left to right.

[0080] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, which, of course, may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting. Because the scope of the present disclosure is limited only by the appended claims.

[0081] Where a range of values ​​is given, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed to the first decimal place of the unit of the lower limit. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either limit is included, neither is included, or both are included in the smaller range, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0082] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0083] As used herein, the terms "comprising," "including," "having," "having" and "comprising" are synonymous with "including," "comprising" or "containing," "comprising" and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps. The terms "comprising," "including," "having," "having" and "comprising" also include the term "consisting of."

[0084] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that any of such publications constitutes prior art to the claims appended hereto.

[0085] As used herein, the term "pasteurization" refers to the destruction of microorganisms in an aqueous solution by heating. The implementation of pasteurization in the brewing process is typically through the use of a flash pasteurizer or a tunnel pasteurizer. As used herein, the term "pasteurization unit or PU" refers to a quantitative measure of pasteurization. One pasteurization unit (1 PU) of beer is defined as one minute at 60 degrees Celsius. It is calculated as follows: PU=t×1.393^(T), in the formula, t = time at pasteurization temperature in the pasteurizer (min) T = temperature in the pasteurizer (in degrees Celsius) - here 60°C [^(T) represents the exponent of (T)]

[0086] Depending on the type of beer, the raw materials and microbial contamination, the brewer and the perceived effect on the beer flavor, different minimum PUs can be used. Typically, 14-15 PUs are required for pasteurization of beer. Depending on the pasteurization equipment, pasteurization temperatures typically range from 60 to 72 degrees Celsius and pasteurization times are calculated accordingly. Further information can be found in "Technology Brewing and Malting" by Wolfgang Kunze, Research and Teaching Institute of Brewing, Berlin (VLB), 3rd edition fully updated, 2004, ISBN 3-921690-49-8.

[0087] Catalase The catalase enzyme catalyzes the first order reaction. 2H2O2 → 2H2O+O2

[0088] Catalases for use according to the present invention may belong to EC 1.11.1.6.

[0089] In a preferred embodiment, the catalase is a microbial catalase, such as a catalase isolated or derivable from a fungus or a bacterium. In one aspect, the microbial catalase is derived from Aspergillus sp., Bacillus sp., Saccharomyces sp., Escherichia sp., Pseudomonas sp., Trichoderma sp., or Scytalidium sp. In one embodiment, the microbial catalase is derived from Aspergillus niger, Aspergillus oryzae, Bacillus subtilis, Bacillus licheniformis, Bacillus fragilis, Bacillus pumilus, Bacillus halodurans, Saccharomyces cerevisiae, Escherichia coli, Pseudomonas aeruginosa, Trichoderma reesei, or Scytalidium thermophilum.

[0090] In a preferred embodiment, the catalase is derived from an Aspergillus sp. or Trichoderma sp., preferably from Aspergillus niger or Trichoderma reesei. More preferably, the catalase is derived from Aspergillus niger. The catalase may be produced by recombinant techniques, such methods being well known in the art. In one embodiment, the catalase may be expressed in Aspergillus niger or Trichoderma reesei.

[0091] Catalases suitable for use according to the present invention can be recovered from recombinant cell cultures by methods well known to those of skill in the art, including, for example, ammonium sulfate or ethanol precipitation, acid extraction and chromatographic methods, such as high performance liquid chromatography (HPLC).

[0092] Catalases suitable for use according to the invention can be naturally occurring purified products, products or chemically synthesized products produced by recombinant techniques from prokaryotic or eukaryotic hosts, such as bacteria, yeast, fungi, higher plants, insects and mammalian cells.

[0093] The catalase may be mixed with a carrier or diluent that does not interfere with the intended purpose of the catalase. In one aspect, the catalase suitable for use according to the invention may be in a substantially purified form. In one aspect, the catalase may not be purified. As discussed further below, the present invention advantageously allows for the presence of glucose oxidase side activity in the catalase composition, i.e., increased tolerance to the presence of glucose oxidase. This is economically beneficial, as it means that glucose oxidase does not need to be purified from the catalase composition, saving the cost of further purification.

[0094] Catalase activity can be measured in Baker units / g, with one Baker unit being defined as the amount of catalase that decomposes 264 mg of hydrogen peroxide in a "double exhaust" analysis based on simultaneous inactivation of catalase with hydrogen peroxide and decomposition of hydrogen peroxide by catalase after incubation at pH 7.0 and 25.0°C for 60 minutes. In one embodiment of the invention, catalase is added at a dose of at least about 7000 U / l of wort. In one embodiment, catalase is added at a dose of at least about 5000 U / l of wort. In one embodiment, catalase is added at a dose of at least about 2000 U / l of wort. In one embodiment, catalase is added at a dose of at least about 1000 U / l of wort. In one embodiment, catalase is added at a dose of at least about 500 U / l of wort. In one embodiment, catalase is added at a dose of at least about 7000 U / l of wort.

[0095] Alternatively, catalase activity may be measured in CIU. The decomposition of hydrogen peroxide can be monitored spectrophotometrically at 240 nm. 濃 The time taken for a specific decrease in absorbance at 25°C is the expression of catalase activity. One CIU is defined as the enzyme activity that decomposes 1 μM H2O2 per minute at pH 7.0 and 25°C, lowering the H2O2 concentration from 10.3 mM to 9.2 mM.

[0096] Reaction conditions: Enzyme concentration approximately 100CIU / ml Substrate concentration 10.3mM H2O2 Buffer 50mM phosphate Temperature 25℃ pH 7.0

[0097] detection: Wavelength 240nm Absorbance range: 0.450~0.400 Time range: 0.267~0.400 minutes (16~24 seconds)

[0098] In a preferred embodiment, the catalase comprises the sequence SEQ ID NO: 1 or SEQ ID NO: 2 or a sequence having at least 70% identity thereto. In a preferred embodiment, the catalase comprises a variant, fragment, derivative or homologue of the sequence SEQ ID NO: 1 or SEQ ID NO: 2 as defined herein below. The variant, fragment, derivative or homologue according to the invention has catalase activity. The catalase may be in isolated form.

[0099] Sequence identity In addition to the specific amino acid sequences and polynucleotides referred to herein, the present invention encompasses variants, homologues, derivatives and fragments thereof. The term "variant" is used to mean a nucleotide or amino acid sequence that differs from the wild-type sequence.

[0100] For example, variants may contain substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more positions compared to the wild-type sequence. Variants can be made using methods known in the art, such as site-scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, and using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences can be readily synthesized using methods known in the art.

[0101] In some aspects, a variant is a naturally occurring nucleotide or amino acid sequence that differs from the wild-type sequence. For example, a variant can be a naturally occurring genetic variant.

[0102] The term "isolated" means that the sequence is naturally occurring, naturally associated, and at least substantially free of at least one other component found in nature. In one aspect, an "isolated variant" or isolated active fragment thereof as used herein refers to a polypeptide that is at least 30% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure as determined by SDS-PAGE.

[0103] In some aspects, the variant is an engineered variant, e.g., the variant can be engineered by recombinant methods.

[0104] In one embodiment, the "wild type sequence" is SEQ ID NO:1.

[0105] The term "homolog" refers to an entity having a certain homology with a subject nucleotide sequence, where the term "homology" can be considered the same as "identity."

[0106] In this context, a homologous sequence is taken to include an amino acid or nucleotide sequence that may be at least 70%, 75%, 80%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Typically, a homologue will contain, for example, the same active site as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), it is preferred in the context of the present invention to express homology in terms of sequence identity.

[0107] Suitably, the homologue of catalase according to the present invention is an active catalase. That is, the homologue of the variant of the present invention has catalase activity. In one aspect, a homologous sequence is understood to include an amino acid sequence or a nucleotide sequence that has one or several additions, deletions and / or substitutions compared to the subject sequence. Homology comparison can be performed by eye or, more commonly, by using a readily available sequence comparison program. This commercially available computer program can calculate the % homology between two or more sequences.

[0108] Percent homology can be calculated over contiguous sequences, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0109] This is a very simple and consistent method, but it potentially results in a large reduction in the % homology when performing a global alignment, since it cannot take into account that, for example, in a normally identical pair of sequences, an insertion or deletion may cause subsequent amino acid residues to be removed from the alignment. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0110] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) will achieve a higher score than one with many gaps. Typically, an "affine gap cost" is used that imposes a relatively high cost on the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will naturally produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is preferred to use the default values.

[0111] Calculation of maximum % homology or maximum % identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties.

[0112] Relationships between protein sequences are usually found by aligning them together and assigning a score to this alignment. There are two main types of sequence alignment: pairwise sequence alignment only compares two sequences at a time, and multiple sequence alignment compares many sequences. Two important algorithms for aligning pairs of sequences are the Needleman-Wunsch algorithm and the Smith-Waterman algorithm. Common tools for sequence alignment include BLAST for pairwise alignment and ClustalW, MUSCLE or MAFFT for multiple alignment.

[0113] There are two types of pairwise alignment: local alignment and global alignment. Local alignment is the alignment of two subregions of a sequence pair. This type of alignment is appropriate when aligning two sequences that may have local regions of similarity embedded in a background of non-homologous sequences. For local alignment, the Smith-Waterman algorithm is the most commonly used. Other pairwise alignment tools include BLAST. Global alignment is the sequence alignment across the entire length of two or more protein sequences. In global alignment, the sequences are assumed to be homologous along their entire length. An efficient algorithm for global alignment is described in Needleman and Wunsch 1970. To assess the similarity across multiple protein sequences, multiple protein sequence alignment tools such as CLUSTALW, MAFFT and MUSCLE are applied.

[0114] A suitable computer program for carrying out such an alignment is available in the Vector NTI (Invitrogen) software package, which uses an algorithm comparable to the CLUSTAL W iterative multiple sequence alignment algorithm (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Examples of software capable of carrying out sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999, Short Protocols in Molecular Biology, 4th Ed-Chapter 18), BLAST2 (see FEMS Microbiol Lett, 1999, 174(2):247-50; FEMS Microbiol Lett, 1999, 177(1):187-8), FASTA (Altschul et al., 1990, J. Mol. Biol., 403-410) and AlignX. At least BLAST, BLAST 2 and FASTA are available for offline and online searching (see Ausubel et al., 1999, pages (7)58 to (7)60).

[0115] Although % ​​homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a numerically scaled similarity score matrix is ​​generally used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). Vector NTI programs generally use the public default values ​​or a custom symbol comparison table if one is supplied (see user manual for further details). For some applications, it is preferred to use the default values ​​for the Vector NTI package.

[0116] Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity, which the software typically does as part of the sequence comparison to generate a numerical result.

[0117] When determining sequence identity, gap penalties may be used. Examples of parameters used for pairwise alignments are shown in Tables 1 and 2 below.

[0118] [Table 1]

[0119] [Table 2]

[0120] In one embodiment, CLUSTAL can be used with gap penalties and gap extensions set as defined above.

[0121] Suitably, the degree of identity for nucleotide sequences is determined over at least 20 contiguous nucleotides, preferably over at least 30 contiguous nucleotides, preferably over at least 40 contiguous nucleotides, preferably over at least 50 contiguous nucleotides, preferably over at least 60 contiguous nucleotides, preferably over at least 100 contiguous nucleotides.

[0122] Suitably, the degree of identity for nucleotide sequences is determined over at least 100 contiguous nucleotides, preferably over at least 200 contiguous nucleotides, preferably over at least 300 contiguous nucleotides, preferably over at least 400 contiguous nucleotides, preferably over at least 500 contiguous nucleotides, preferably over at least 600 contiguous nucleotides, preferably over at least 700 contiguous nucleotides, preferably over at least 800 contiguous nucleotides.

[0123] Suitably, the degree of identity with respect to protein (amino acid) sequences is determined over at least 100 contiguous amino acids, preferably over at least 200 contiguous amino acids, preferably over at least 300 contiguous amino acids.

[0124] For example, the degree of identity with respect to the amino acid sequence of the fragment may be determined over the length of the fragment sequence, in other words, the degree of identity with respect to the amino acid sequence of the fragment may be determined over the length of a shorter sequence.

[0125] Preferably, the degree of identity with respect to amino acid or protein sequences may be determined over the entire mature sequence taught herein, for example over the entire SEQ ID NO:1 disclosed herein.

[0126] In this context, the term "query sequence" refers to a homologous or foreign sequence that is aligned with a subject sequence to determine whether it falls within the scope of the present invention. Thus, such a query sequence can be, for example, a prior art sequence or a third party sequence.

[0127] In a preferred embodiment, sequences are aligned by a global alignment program and sequence identity is calculated by determining the number of exact matches identified by the program divided by the length of the subject sequence.

[0128] In one embodiment, the degree of sequence identity between a query sequence and a subject sequence is determined by: 1) aligning the two sequences with any suitable alignment program using a default scoring matrix and default gap penalties, 2) determining the number of exact matches (an exact match is when the alignment program identifies an identical amino acid or nucleotide in the two aligned sequences at a given position in the alignment), and 3) dividing the number of exact matches by the length of the subject sequence.

[0129] In a further preferred embodiment, the global alignment program is selected from the group consisting of CLUSTAL and BLAST (preferably BLAST), and sequence identity is calculated by determining the number of exact matches identified by the program divided by the length of the subject sequence.

[0130] A sequence such as a variant sequence or a catalase-active fragment thereof may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0131] Conservative substitutions can be made, for example, in accordance with the following table: As shown in Table 3, amino acids in the same block in the second column and preferably in the same line in the third column can be substituted for each other.

[0132] [Table 3]

[0133] The invention also encompasses possible homologous substitutions (substitution and replacement are both used herein to mean the replacement of an existing amino acid residue with an alternative residue), i.e., basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitutions may occur, i.e., from one class of residue to another, or instead involve the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine.

[0134] Substitutions can also be made with synthetic amino acids (e.g., unnatural amino acids), such as alpha * and alpha disubstituted * Amino acids, N-alkyl amino acids * , lactic acid * , halide derivatives of natural amino acids, e.g., trifluorotyrosine * , p-Cl-phenylalanine * , p-Br-phenylalanine * , pI-phenylalanine * , L-allyl-glycine * , β-alanine * , La-aminobutyric acid * , Lg-aminobutyric acid * , La-aminoisobutyric acid * , Le-aminocaproic acid # , 7-aminoheptanoic acid * , L-Methionine Sulfone#*, L-Norleucine * , L-Norvaline * , p-nitro-L-phenylalanine * , L-hydroxyproline # , L-thioproline * , methyl derivatives of phenylalanine (Phe), e.g., 4-methyl-Phe * , Pentamethyl-Phe * , L-Phe(4-amino) #, L-Tyr(methyl) * , L-Phe(4-isopropyl) * , L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) * , L-diaminopropionic acid # and L-Phe(4-benzyl) * Examples include:

[0135] symbol * is used for the purposes of the above discussion (regarding homologous or non-homologous substitutions) to indicate the hydrophobicity of the derivative, while # is used to indicate the hydrophilicity of the derivative, and # * exhibits amphiphilic characteristics.

[0136] The variant amino acid sequence may include a suitable spacer group that may be inserted between any two amino acid residues of the sequence, examples of which include alkyl groups, such as methyl, ethyl or propyl groups, in addition to amino acid spacers, such as glycine or b-alanine residues. A further form of variation includes the presence of one or more amino acid residues in peptoid form, as will be appreciated by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues in which the a-carbon substituent is present on the nitrogen atom of the residue, rather than on the a-carbon. Processes for preparing peptides in peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0137] The nucleotide sequences used in the present invention may contain synthetic or modified nucleotides therein. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it should be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of the nucleotide sequences of the present invention.

[0138] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein.

[0139] Other variants of the sequences described herein can be obtained, for example, by probing DNA libraries made from a wide range of individuals, for example individuals from various populations. In addition, other homologues can be obtained, and such homologues and fragments thereof can generally selectively hybridize to the sequences shown in the sequence listing herein. Such sequences can be obtained by probing cDNA libraries made from other animal species or genomic DNA libraries from other animal species, probing such libraries under medium to high stringency conditions with a probe comprising all or part of any one of the sequences in the attached sequence listing. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the present invention.

[0140] Variants and strain / species homologs can also be obtained using degenerate PCR with primers designed to target sequences within the variants and homologs that code for conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning amino acid sequences from several variants / homologues. Sequence alignment can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.

[0141] The primers used in degenerate PCR contain one or more degenerate positions and are used under lower stringency conditions than those used for cloning sequences with a single sequence primer against a known sequence.

[0142] Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of characterized sequences. This can be useful, for example, when silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequence is to be expressed. Other sequence changes, such as introducing restriction enzyme recognition sites or altering the properties or functions of the polypeptides encoded by the polynucleotide, may be desired.

[0143] Glucose oxidase activity In one aspect of the invention described herein, the catalase composition may also include glucose oxidase (GOX) activity.

[0144] In one embodiment, GOX represents a side activity to catalase, for example, when catalase is expressed in a host cell, such as T. reesei, the GOX side activity remains in the catalase composition.

[0145] In one aspect, the ratio of glucose oxidase:catalase activity may be greater than 0.001, more preferably greater than 0.0001, and even more preferably greater than 0.00001.

[0146] In one aspect, the GOX can have the sequence of SEQ ID NO:5 (T. reesei GOX) described herein, or a variant, derivative, homologue, or active fragment thereof.

[0147] A glucose-oxidase unit (GODU) is the amount of enzyme that oxidizes 1 μmol of β-D-glucose per minute. Glucose oxidase (β-D-glucose:oxygen-1-oxidoreductase, EC 1.1.3.4.) oxidizes β-D-glucose to δ-glucono-lactone and hydrogen peroxide in the presence of oxygen. The hydrogen peroxide produced oxidizes ABTS-R (2,2-azino-di-(3-ethylbenzthiazoline)-6-sulfonate) in the presence of peroxidase (POD). This produces a green-blue color that is measured photometrically at 405 nm. β-D-glucose + O2GOD, 30℃, pH5.6 → δ-gluconolactone + H2O2 H2O2+ABTS red POD → 2H9O + ABTS r

[0148] Reaction conditions: Substrate glucose 90mM (16.2g / l) ABTS 1.25mM (688mg / l) Glucose oxidase 0.0061~0.0336GODU / ml Peroxidase (POD) 2930U / l Buffered Acetate, 100mM PH5.60±0.05 Temperature: 30℃±1 Response time: 36 seconds (8×4.5 seconds) Wavelength 405nm

[0149] Antioxidants: In one aspect of the invention, antioxidants can be used in addition to catalase during the brewing process. Any suitable antioxidant can be used, including both synthetic and natural antioxidants. The antioxidant can be added at any stage of the brewing process. In one aspect, the antioxidant can be added at the beginning of fermentation. In a preferred aspect, the antioxidant can be added at the end of fermentation. In a more preferred aspect, the antioxidant can be added during the stabilization and / or maturation stages described herein. Most preferably, the antioxidants described herein can be added to the finished beer or beverage product.

[0150] In one aspect, ascorbic acid can be used as an antioxidant. The ascorbic acid described herein can be added at any stage of the brewing process. In one aspect, the ascorbic acid can be added at the beginning of fermentation. In a preferred aspect, the ascorbic acid can be added at the end of fermentation. In a more preferred aspect, the ascorbic acid can be added during the stabilization and / or maturation stages described herein. Most preferably, the ascorbic acid described herein can be added to the finished beer or beverage product.

[0151] In a further aspect according to the invention, ascorbic acid may be added in the form of a natural antioxidant. In this regard, the natural antioxidant may be added during the brewing process and / or may be added to the finished beer. Any suitable natural antioxidant may be used according to the invention.

[0152] By way of example, the natural antioxidant may be selected from tocopherols, polyphenols (tannins, gallic acid, etc.), acerola extract (naturally occurring ascorbic acid), pomegranate extract, rosemary extract (E392 or spice extract), lemon extract, green tea extract, blend of rosemary and lemon extract, rosemary extract and blend of green tea and rosemary extract. In a preferred embodiment, said natural antioxidant is acerola extract.

[0153] The synthetic antioxidants may be selected from BHA (E320), BHT (E321), TBHQ (E319), ascorbyl palmitate (E304), synthetic tocopherols, ethoxyquin (E324), sodium ascorbate (E301), EDTA (E385), propyl gallate (E310), ascorbic acid (E300) and KMS / NaMS (E224 / E223).

[0154] As described herein, the method according to the present invention may also include the addition of ascorbic acid (vitamin C). Ascorbic acid can be added in any suitable form. Ascorbic acid is a powerful antioxidant and is usually added as a food ingredient. When used in low doses, it is flavorless and has only a minimal effect on the pH of the product to which it is added.

[0155] The ascorbic acid described herein can be added at any stage of the brewing process. In one aspect, the ascorbic acid can be added at the beginning of fermentation. In a preferred aspect, the ascorbic acid can be added at the end of fermentation. In a more preferred aspect, the ascorbic acid can be added during the lagering period, i.e., during stabilization / aging as described herein. Most preferably, the ascorbic acid described herein can be added to the finished beer or beverage product.

[0156] According to the present invention, ascorbic acid may be used at a dosage of more than about 0 to about 1% by weight, for example, more than about 0 to about 0.5% by weight, more than about 0 to about 0.25% by weight, more than about 0 to about 0.1% by weight, or more than about 0 to about 0.05% by weight, 0.04% by weight, 0.03% by weight, 0.02% by weight, or 0.01% by weight. In a preferred embodiment, the dosage may be more than about 0 to about 0.1% by weight. More preferably, the dosage may be more than about 0 to about 0.25% by weight.

[0157] Ascorbic acid can be added in liquid or powder form. Vitamin C and any preparations thereof can be used in accordance with the present invention.

[0158] If ascorbic acid is not recognized as a processing aid, it may be labeled as E300-E304. The names are as follows: E300 ascorbic acid; E301 sodium ascorbate; E302 calcium ascorbate; E303 potassium ascorbate; E304 fatty acid esters of ascorbic acid, such as ascorbyl palmitate. Any of these may be used in the present invention.

[0159] In one aspect, ascorbic acid can be added in the form of a natural antioxidant, such as an antioxidant extract from a fruit or plant, etc. Any natural extract containing ascorbic acid (vitamin C), such as a fruit or plant extract, can be used in accordance with the present invention.

[0160] In one aspect, the extract may be selected from one or more of acerola extract, pomegranate extract, rosemary extract, lemon extract, green tea extract, a blend of rosemary and lemon extract, rosemary extract, and a blend of green tea and rosemary extract, preferably said natural antioxidant is acerola extract.

[0161] Additional enzymes In one aspect of the invention described herein, additional enzymes may be used during the brewing process.

[0162] For example, proline specific proteases can be used, for example, for reducing or removing haze in beer. Suitable proline specific proteases are known to those skilled in the art. In one aspect, the proline specific protease is an enzyme having at least 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity with any of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 or active fragments thereof.

[0163] The method may also include adding one or more of an ALDC (alpha acetolactate decarboxylase) enzyme, a glucoamylase, a maltogenic alpha-amylase, a pullulanase, or a transglucosidase. Optionally, the ALDC enzyme is an acetolactate decarboxylase as described in EC 4.1.1.5. Optionally, the glucoamylase is a 1,4-alpha-glucosidase as described in EC 3.2.1.3. Optionally, the maltogenic alpha amylase is a glucan 1,4-alpha-maltohydrolase as described in EC 3.3.1.133. Optionally, the pullulanase is an alpha-dextrin endo-1,6-alpha-glucosidase, limit dextrinase, amylopectin 6-glucanohydrolase, a debranching enzyme as described in EC 3.2.1.41. Optionally, the transglucosidase is a 1,4-α-glucan branching enzyme, oligoglucan branching glucosyl transferase, as described in EC 2.4.1.24.

[0164] The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA which are within the capabilities of one of ordinary skill in the art and are explained in the literature. For example, J. Sambrook, EFFritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FMet al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New, York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and DMJ Lilley and JEDahlberg, 1992, Methods of See Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is incorporated herein by reference.

[0165] The following examples further illustrate the present disclosure, but do not limit the scope of the claims of the present disclosure in any way.The accompanying drawings should be considered as part of the specification and description of the present disclosure.The following examples are presented to illustrate the disclosure described in the claims, but do not limit the disclosure.

[0166] Strecker aldehyde The present invention also encompasses a reduction in the level of Strecker aldehyde in the resulting beer. Flavor is a major quality attribute of beer and is important for brand recognition and identification. One well-established observation during beer roasting is the increase in aldehydes or so-called Strecker aldehydes, which have been shown to be associated with poor beer flavor. In a further aspect of the present invention, the addition of catalase compositions after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of beer or to the final beer itself reduces the production of Strecker aldehyde. Strecker aldehydes may include, but are not limited to, 2-butenal, hexanal, trans-3-penten-2-one, 4-hexen-3-one, nonanal, furfural, decanal, trans-2-nonenal, benzaldehyde, phenylacetaldehyde, and ethyl nicotinate.

[0167] In preferred aspects of the invention, Strecker aldehyde can be reduced by adding a catalase composition to beer, including a reduction in the production of trans-2-nonenal and / or benzaldehyde of at least 10%, at least 20%, at least 30%, at least 40% or at least 50%. EXAMPLES

[0168] Example 1 – Enzymes Highly active catalase preparation from Aspergillus niger (available from IFF, Food Enzymes, Denmark) having the amino acid sequence shown in SEQ ID NO: 1. The catalase preparation possesses 1,000 (Baker) units / g, where 1 Baker unit is defined as the amount of catalase that will decompose 264 mg of hydrogen peroxide in a "double vent" analysis based on the simultaneous inactivation of catalase by hydrogen peroxide and the decomposition of hydrogen peroxide by catalase after incubation at pH 7.0 and 25.0°C for 60 minutes.

[0169] Additionally, another highly active catalase preparation from Trichoderma reesei was utilized at 9,250 units / g, which also has the amino acid sequence shown in SEQ ID NO:1 (available from IFF, Food Enzymes, Denmark). In the following examples, selectively specific cases are identified where a catalase from Trichoderma reesei (catalase-2) was utilized, while in all other examples a catalase preparation from Aspergillus niger (catalase-1) was used.

[0170] Example 2 – Measurement of catalase activity Catalase activity was determined using the catalase assay procedure "K-CATAL 07 / 19" supplied by Megazymes (Ireland). This assay evaluates the activity of catalase based on the reduction of H2O2. Two separate reactions are required. In the first reaction, the catalase sample is mixed with a known concentration of H2O2 (approximately 65 mM). After a set time, the reaction is stopped with 15 mM sodium azide, which inhibits catalase. In the second reaction, the remaining H2O2 is measured using 3,5-dichloro-2-hydroxy-benzenesulfonic acid (DHBS), 4-aminoantipyrine (AAP) and peroxidase, which gives an enzyme-linked colorimetric detection at 520 nm. As a control, a blank reaction is performed simultaneously. Thus, 50 μl of substrate solution (165 mM H2O2, prepared according to Megazyme) was mixed with 50 μl of catalase solution diluted in reaction assay buffer (150 mM potassium phosphate buffer pH 7.0, supplied by Megazyme) and incubated for 5 min at 25° C. in an iEMS incubator / shaker (Thermo Scientific, Rockford, IL, USA). To stop the reaction, 20 μl of the reaction solution was mixed with 180 μl of 15 mM sodium azide, and then 3 μl of the stopped reaction solution was mixed with 225 μl of colorimetric reagent solution (peroxidase, 4-aminoantipyrine and 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS) solution supplied by Megazyme). The colorimetric reactions were incubated for 15 min at 25° C. in an iEMS incubator / shaker and the absorbance at 520 nm was recorded for blank and catalase reaction samples using a SpectraMAX MTP reader (Molecular Devices, Sunnyvale, Calif., USA).

[0171] Example 3 – Determination of catalase activity in beer after pasteurization To evaluate the effect of pasteurization on catalase activity after beer fermentation, 660 μl (1000 U / g) of the catalase preparation shown in SEQ ID NO: 1 was added to 330 ml of regular Pilsner type beer (Carlsberg, Denmark, 4.6% (v / v) alcohol) to obtain 2 U / mL of beer. The beer was incubated in a water bath at 62.1° C. Samples were drawn at 0, 10, 20, 30, 40, 45, 50 and 55 minutes during pasteurization of Carlsberg beer.

[0172] To determine the enzyme thermostability under the conditions used in this experiment, catalase activity was measured before and after pasteurization of beer according to Example 2. Beer without added catalase was used as a control. Accumulated energy input was converted to energy equivalent index pasteurization units (PU and accumulated PU) as calculated by the following formula:

[0173] Pasteurization Units or PU refers to a quantitative measure of pasteurization. One pasteurization unit (1 PU) of beer is defined as 1 minute at 60 degrees Celsius. It is calculated as follows: PU=t×1.393^(T), in the formula, t = time at pasteurization temperature (min) T = pasteurization temperature (in degrees Celsius) - here 60°C [^(T) represents the exponent of (T)]

[0174] Therefore, the thermostability of catalase in regular Carlsberg Pilsner (Denmark, 4.6% (v / v) alcohol) was determined. Data was calculated as % relative activity compared to the activity measured at pasteurization time 0 and the results are shown in Table 4. The catalase preparation shown in SEQ ID NO: 1 was found to be sufficiently thermostable to withstand 24 PU pasteurization (98.7% residual activity) performed at temperatures up to 62°C.

[0175] [Table 4]

[0176] Example 4 - Electron spin resonance (ESR) measurements: endogenous antioxidant capacity and T600 determination The functional principle of EAP determination is based on the application of the specific spin-trapping reagent α(4-pyridyl-1-oxide)- N -tert-butylnitrone (POBN), which, due to the pH effect on radical generation, reduces the distortion of the so-called lag time values ​​in beer caused by the former spin-trapping reagent α-pyridyl- N -tert-butylnitrone (PBN) ( 16 , 38 ).

[0177] For the lag time measurements with PBN and EAP measurements with POBN, the following ESR spectrometers were used: X-band spectrometer; Bruker (ESP-300); cavity type: Bruker 4108 TMH number: 8603 and X-band spectrometer; Magnettech Miniscope MS 100. The irradiation experiments for the artificial generation of radicals were performed by X-ray irradiation (X-ray tube manufactured by Seifert Debyflex). The term "X-ray system" for generating radicals, as used herein, describes the artificial generation of radicals via ionizing radiation (X-ray radiation), as known in the prior art.

[0178] reagent N-tert-Butyl-α-(4-pyridyl)nitrone N-oxide (POBN) 99%, C10H14N2O2, 194.23 g / mol (Sigma Aldrich, 1 g, at least 99%, No. 215430, CAS No. 66893-81-0, EC No. 266-512-3). Ethanol pro-analysis 99.8%, C2H5OH, 46.07 g / mol, density: 1 liter = 0.79 kg (Merck Darmstadt).

[0179] The determination of the endogenous antioxidant potential (EAP value) using ESR spectroscopy is based on the indirect detection of radical generation during accelerated beer aging (60°C). For a certain period of time, radical generation can be delayed or prevented by the endogenous antioxidant activity of the beer. After consumption of antioxidants, the ESR signal increases as spin-trap adducts, mainly hydroxyethyl radicals, are formed. The intersection of the two linear slopes gives a relative measure of stability, and the time at the intersection is defined as the EAP value (Deutsches Patent und Markenamt DE 10 2005 043 113 A1, 2005. US 20080248580, Method for determining the Endogenous Antioxidative Potential of beverages by means of ESR spectroscopy), 2008). The T600 value is defined as the ESR signal intensity after 600 minutes of assay and qualitatively indicates the content of radicals generated under forced aging conditions (60°C) (Deutsches Patent und Markenamt DE 10 2005 043 113 A1, 2005). US Patent No. 20080248580, Method for determining the Endogenous Antioxidative Potential of beverages by means of ESR spectroscopy, 2008).

[0180] Beer samples were degassed in an ultrasonic bath for 15 min (max. 20 °C). For each sample, 8.4 mg of POBN was diluted in 50 μl of double distilled water (final 3.5-3.6 mM POBN). For the preparation of ESR beer samples, amber vials containing 156 μl of ethanol (99%) and 12 ml of beer sample were used. Directly at the start of the measurement, 52 μl of POBN solution was added and 0.5 ml of enzyme sample was placed in the autosampler in a total volume of 12.708 ml at 60 °C. For the preparation of ESR wort samples, amber vials containing 10 ml of malt-based wort extract and 700 μl of ethanol were used. Directly at the start of the measurement, 105 μl of POBN solution was added and 0.5 ml of enzyme sample was placed in the autosampler in a total volume of 11.305 ml at 60 °C.

[0181] Example 5 – ESR analysis of catalase in malt extract To evaluate the influence of catalase during beer fermentation, different catalases shown in SEQ ID NO: 1 were prepared in double distilled water in different dilutions, added to wort samples and analyzed by ESR. For the preparation of ESR wort samples, amber vials containing 10 ml of malt-based wort extract, 700 μl of ethanol were used, and before starting the measurement, 105 μl of POBN solution and 0.5 ml of diluted enzyme sample were added and the solution was placed in the autosampler, total volume 11.305 ml, 60 °C. The results of the addition of different catalases to the wort are shown in both Figure 1 (from 0.0 to 44.2 U / ml) and Figure 2 (from 0.0 to 6.2 U / ml) at different dosages. Surprisingly, all dosages from a very low level of catalase (0.9 U / ml wort) showed very good oxidation protection and a complete drop in the ESR signal. The T600 measured 7.6 × 10 6 ~0.35×10 6 (Figure 1) and 64.43 × 10 5 ~34.17×10 4(Figure 2), clearly suggesting that catalase may act on pro-oxidant radical production by reducing hydrogen peroxide formed in the wort, but not on increasing the endogenous antioxidant capacity (EAP). Thus, the addition of catalase during beer fermentation in wort may significantly improve the oxidative stability of the wort and the final beer.

[0182] Example 6 – ESR analysis of catalase in dark pilsners with low EAP It is well known that the temperature during the withering and kilning steps in malt production increases the production of stable organic radicals in the finished malt, resulting in a decrease in the intrinsic antioxidant capacity value and sulfur dioxide content of the final beer. To evaluate the effect of catalases on beers with low intrinsic antioxidant capacity values, different catalases shown in SEQ ID NO: 1 were prepared in different dilutions in double distilled water, spiked with a black pilsner sample (Koestritzer pilsner, 4.6% (v / v) alcohol, Germany) and analyzed by ESR. For the preparation of ESR beer samples, amber vials containing 156 μl ethanol (99%) and 12 mL degassed beer sample were used.

[0183] Directly at the start of the measurement, 52 μl of POBN solution was added and 0.5 ml of the diluted enzyme sample was placed in the autosampler at 60 °C, bringing the total volume to 12.708 ml. The results of various catalase additions to low EAP black pilsna gel are shown in Figure 3 (0.0-3.9 U / ml). A very clear effect of catalase was observed, showing very good oxidation protection and a reduction in the ESR signal. Beer samples without catalase showed an increase in the ESR signal over time without any assignable EAP. Even the addition of 0.2 U / ml of catalase resulted in a very significant decrease in the ESR signal (17.96 × 10 5 From 46.80 x 10 4 to 0.8 U / ml), and the addition of more than 0.8 U / ml catalase resulted in a complete reduction in the ESR signal (17.96 × 10 5 From 26.35 x 10 4These results clearly demonstrate that the addition of catalase to beer with low oxidative stability can significantly improve its taste and shelf life.

[0184] Example 7 – ESR analysis of catalase in beer using EAP To evaluate the effect of catalase on beers with moderate endogenous antioxidant capacity, different catalase dilutions shown in SEQ ID NO: 1 were prepared in double distilled water in different dilutions and added to Pilsner beer (Heineken Pilsner, 4.6% (v / v) alcohol, Germany and Becks, 4.6% (v / v) alcohol, Germany) samples and analyzed by ESR.

[0185] For the preparation of ESR beer samples, amber vials containing 182 μl ethanol (99%) and 14 ml degassed beer sample were used. Directly at the start of the measurement, 61 μl of POBN solution was added and 0.5 ml of diluted enzyme sample was placed in the autosampler at 60 °C, resulting in a total volume of 14.743 ml. The results of different catalase additions to low EAP black Pilsna gel are shown in Figure 4 (0.0 to 1.97 U / ml). A clear effect of catalase was observed, showing very good oxidation protection and reduction of the ESR signal. Further addition of 0.2 U / ml of catalase showed a complete reduction of the ESR signal (9.5 × 10 5 From 1.7 × 10 5 These results clearly show that the addition of catalase to beers with moderate oxidative stability (EAP of 72 and 116 min) can significantly improve the stability and thus the taste and shelf life of the beer.

[0186] Example 8 – ESR analysis of catalase in low SO2 beer during storage stability testing To evaluate the effect of catalase addition to beer with unknown intrinsic antioxidant capacity but low SO2, catalase of SEQ ID NO: 1 was prepared in double distilled water at a range of dilutions and two pilsner beer samples (Odense pilsner, 4.6% (v / v) alcohol, Denmark and Tuborg pilsner, 4.5% (v / v) alcohol, Denmark) were spiked and analyzed by ESR after storage. After catalase addition, the samples were gassed with CO2 and then stored at 28°C for one month. After one month, ESR spectroscopy and roast aldehyde measurements were performed. The following concentrations and samples were utilized: 1. Odense without additives (unused, not stored) 2. Tuborg without additives (unused, not stored) 3. 650 μl catalase of Odense + SEQ ID NO: 1, corresponding to 0.19 U / ml (1 month at 28° C.) 4. Tuborg + 650 μl H2O (1 month at 28°C) 5. Odense + 650μl H2O (1 month at 28℃) 6. 650 μl of Tuborg+ Catalase of SEQ ID NO: 1, equivalent to 0.19 U / ml (1 month at 28° C.)

[0187] Standard beer characteristics of the Tuborg and Odense samples were measured according to the "Mitteleuropaeischen Brautechnischen Analyse Kommission" (MEBAK; Brautechnische Analysenmethoden. Wuerze, Bier, Biermischgetraenke / herausgegeben vom Vorsitzenden Prof. Fritz Jacob, MEBAK, Freising-Weihenstephan, 2012) for the fresh beers as shown in Figure 5. These were: fresh Tuborg Pilsner had a gravity of 10°P, a real extract of 3.2% w / w, an alcohol content by volume of 4.5% v / v, a degree of fermentation of 85%, a color of 2.5 EBC, a pH of 4.2, a bitterness unit of 17 BU, an EAP value of not determinable, and a β-aminobutyric acid value of 1.1 x 10 6The fresh Odense had a gravity of 10.6, a fruit extract of 3.5, an alcohol content by volume of 4.6% v / v, a degree of fermentation of 83%, a color of 2.6 EBC, a pH value of 4.2, a bitterness unit of 20 BU, an EAP value of 68, and an undeterminable SO2 value. 6 The beer samples had a Tmax value of 1.1×10 and a SO2 content of 2.8 mg / l. ESR analysis of the beer samples was performed as described in Example 7 and the results are shown in FIG. 6. Here, the Tmax was 1.1×10 6 A fresh Odense with an EAP value of 68 minutes was then followed by a Tmax of 0.89×10 6 Fresh Tuborg had the highest signal intensity. Odense stored for one month had 0.88×10 6 The Tmax value of the Odense with catalase added at 0.8×10 6 The second lowest Tmax value was 0.57×10 6 was observed in Tuborg stored for 1 month with a Tmax of 0.40×10 6 Even with this very low dose of catalase (0.19 U / ml) and storage at 28°C for one month, a clear improvement in oxidative stability was observed, given the significant difference in T800 values ​​when comparing beer samples with and without added catalase.

[0188] Example 9 - Use of catalase during the brewery mashing process and ESR analysis of wort samples To investigate the effect of catalase during mashing, various amounts of catalase having SEQ ID NO: 1 were tested in a laboratory scale malt-based mashing setup and the resulting wort was analyzed by ESR to assess oxidative stability.

[0189] During the mashing tests, mixtures resulting in dark beer worts were used. For these mixtures, 3.75 g of Caraaroma malt and 71.25 g of Pilsner malt were mixed with 375 ml of double distilled water. They were then milled according to FIG. 7.

[0190] The catalase spiked samples were mixed with catalase and then mashed. The following mash samples were produced for testing:

[0191] Dark beer mash (total volume 375ml): 1.95% Pilsner Malt + 5% Caraaroma 2.95% Pilsner malt + 5% Caraaroma malt + 0.63U / ml catalase (A. niger) 3.95% Pilsner malt + 5% Caraaroma malt + 1.26U / ml catalase (A. niger) 4.95% Pilsner malt + 5% Caraaroma malt + 0.63U / ml catalase (T. reesei) 5.95% Pilsner malt + 5% Caraaroma malt + 1.26U / ml catalase (T. reesei)

[0192] Samples were taken after wort filtration and whirlpool rest and investigated according to MEBAK (real extract: Vol. II 2.9.6.3, color: Vol. 2.12.2 and pH: Vol. 2.13) by ESR spectroscopy and beer analysis using a precision hydrometer DMA4500M (Alcoholizer ME, colour-&pH-module, Anton Paar GmbH, AT-8054 Graz). The results of the wort analysis are shown in Figure 8. Here, the wort without added catalase had an extract of 13.2 °P, a colour of 37.4 EBC and a pH of 5.6. The wort with the addition of 0.63 U / ml of catalase (A. niger) gave an extract with a P of 12.6, a color of 37.1 EBC and a pH of 5.5, whereas the wort with the addition of 1.23 U / ml of catalase (A. niger) had an extract with a P of 15.5, a color of 40.2 EBC and a pH of 5.5. Furthermore, the wort with the addition of 0.63 U / mL of catalase (T. reesei) gave an extract with a P of 13.6, a color of 36 EBC and a pH of 5.5, whereas the wort with the addition of 1.23 U / ml of catalase (T. reesei) had an extract with a P of 13.4, a color of 36.7 EBC and a pH of 5.5.

[0193] As controls, either 0.63 U / mL catalase (T. reesei) or 0.63 U / ml catalase (A. niger) was added to dark beer wort after wort boiling.

[0194] FIG. 9 shows the determination of T600 values ​​by ESR spectroscopy of mash test dark beer worts, including a control with catalase added to the dark beer wort after wort boiling.

[0195] Wort supplemented with 1.26 U / ml catalase (T. reesei) had a 2.6 × 10 6 The wort subsequently supplemented with 1.26 U / ml catalase (A. niger) gave the highest signal intensity of 2.4 × 10 6The signal intensity of the wort spiked with 0.63 U / ml catalase (T. reesei) was 2.3 × 10 6 and the signal intensity of the wort spiked with 0.63 U / ml catalase (A. niger) was 2.1 × 10 6 For comparison, the reference wort had a 1.6 x 10 6 The second lowest signal intensity was 2.4 × 10 5 A signal intensity of 2.0 × 10 was obtained from wort to which 0.63 U / ml catalase (T. reesei) was added after boiling, whereas 5 The lowest signal intensity of was obtained from the wort to which 0.63 U / ml of catalase (A. niger) was added after boiling. It is therefore very surprising that the addition of catalase during mashing had a negative effect on the oxidative stability, resulting in high T600 values ​​in the ESR analysis. This clearly indicates that catalase added during mashing may result in beer with significantly lower oxidative stability and thus improve taste evolution while shortening the beer's shelf life. If catalase is added to the final wort after mashing, as for example in the case of its addition during the fermentation of the beer, the results instead show that the addition of catalase significantly improves the stability of the beer / wort and therefore the shelf life of the resulting beer. The conditions and timing under which catalase is added to the brewing process are crucial for the function of the enzyme.

[0196] Example 10 - Analysis of accelerated oxidative off-flavor formation in beer by GC-MS Flavor is a major quality attribute of beer and is important for brand recognition and identification. One well-established observation during beer roasting is the increase of aldehydes or so-called Strecker aldehydes, which have been shown to be associated with off-flavors in beer. Oxygen and oxidation have been shown to significantly promote Strecker aldehyde formation in beer. In this example, conditions for accelerated off-flavor development were tested in a regular Pilsner type beer (Carlsberg, Denmark, 4.6% (v / v) alcohol).

[0197] Freshly produced 4.6% (v / v alcohol Carlsberg, Denmark) (within one month of production) gently opened beer bottles with 33 cl headspace were air flushed to exchange headspace CO2 with atmospheric air. The bottles were left open for 20 seconds and then tightly closed with a crown cork. To ensure uniform oxidation, the bottles were inverted repeatedly for 1 minute to ensure mixing of the liquid with headspace oxygen. An unopened control was kept at room temperature (RT) for 14 days. After headspace oxidation, beer samples were incubated at either 30°C or 60°C for up to 14 days to accelerate oxidation. The following six treatments were then performed and analyzed for off-flavor formation by GC-MS-SPME: FL.Blank - Unopened Carlsberg Pilsner stored at room temperature FL.1 - Carlsberg Pilsner, flash at 30℃, 1 day FL.2 - Carlsberg Pilsner, flash 30℃, 3 days FL.3 - Carlsberg Pilsner, flash 30℃, 7 days FL.4 - Carlsberg Pilsner, flash 30℃, 14 days FL.5 - Carlsberg Pilsner, flash 60℃, 5 days

[0198] Volatile off-flavors were quantified by SPME-GCMS, which was performed in full scan and SIM modes using a carbogen / PDMS fiber as described below.

[0199] Volatile substances analysis Each treated sample (1.0 g) was weighed into a 22 ml headspace vial. A standard solution (100 μl) containing 5-methyl-2-hexanone (3.3 μg / ml) and o-cresol (2.3 μg / ml) was added to each vial before vial crimping. SPME was performed using a Gerstel MultiPurpose Robotic PRO (Gerstel, Muelheim Germany) equipped with an SPME fiber (85 μm carbocene / PDMS, Supelco). The vials were stored in a cooling tray at 10° C. and then incubated at 50° C. for 1 min. Extraction was performed at 50° C. for 30 min with stirring (250 rpm). The fiber was desorbed in a GC inlet (Agilent, 7890) for 30 s. The inlet was equipped with a SPME liner operated at 300° C. with a split ratio of 5:1. Separation was performed on a CP-WAX 52B (50 m × 0.32 μm × 1.2 μm, Agilent CP77731). The oven program started at 70 °C (held for 2 min) and then increased at 12 °C / min to 240 °C (held for 10 min). Helium was used as the carrier gas at 2 ml / min (39.4 cm / sec). Mass selective detection (Agilent, 5977B) was used for detection. The ion source and quadrupole temperatures were 230 °C and 150 °C, respectively. The mass spectrometer was operated in both scan mode (m / z 29-250) and selected ion monitoring (SIM) mode with a dwell time of 100 ms for all ions. The monitored ions and onset times are shown in Table 5.

[0200] [Table 5]

[0201] Tentative identification of the analytes was achieved from deconvolution of the chromatograms. The characteristic ions of the analytes were used for peak integration in ChemStation.

[0202] Relative quantification of the following off-flavors was performed: 2-methylfuran, hexanal, 2-butenal, trans-3-penten-2-one, 4-hexen-3-one, furfural, trans-2-nonenal, benzaldehyde, phenylacetaldehyde and ethyl nicotinate as well as two unidentified substances (ions, RT: 10.21; ions: 95.00 / unidentified and RT: 9.25; ions: 81.00 / unidentified). The results are shown in Figure 10 - as it is clear, flushing the headspace of the beer bottles with air followed by incubation at 60°C for 5 days increased the oxidative off-flavor formation the most. Significant off-flavors were also formed after 14 days at 30°C, but below 60°C. A gradual decrease in off-flavors was observed as the incubation time at 30°C decreased.

[0203] Example 11 – Use of catalase to reduce off-flavor formation in beer To investigate the effect of catalase on accelerating Strecker aldehyde and off-flavor formation in beer, various concentrations of catalase were added to a regular Pilsner-type beer (Carlsberg, Denmark, 4.6% (v / v) alcohol). Off-flavors were quantified as described in Example 10 using an incubation of 14 days at 30°C. For comparison, sulfite, a very strong antioxidant commonly found in beer, was added in excess in the form of sodium metabisulfite (Na2S2O5). 10 g of sodium metabisulfite (Na2S2O5, Sigma Aldrich) was dissolved in 100 ml of doubly diluted H2O and added to a concentration of 10 ppm SO2 in the beer. Catalase of SEQ ID NO: 1 from Aspergillus niger or Trichoderma reesei was added according to the following concentrations: The following treatments were used: · O1GC_1_no; Control - unopened Carlsberg Pilsner stored at room temperature ·O1GC_4; Carlsberg Pilsner_Flushing, 14 days at 30°C · O1GC_5; Carlsberg Pilsner + 0.64U / ml catalase (Aspergillus niger) flushing, 14 days at 30°C · O1GC_10; Carlsberg Pilsner + 29.6U / ml catalase (Trichoderma reesei) flushing, 14 days at 30°C · O1GC_12; Carlsberg Pilsner + 3.7U / ml catalase (Trichoderma reesei) flushing, 14 days at 30°C · O1GC_16; Carlsberg Pilsner + 10 ppm sulfite_flushing, 14 days at 30°C

[0204] The relative amount of off-flavors produced in the beer at 30° C. after 14 days was analyzed as described in Example 10, and the results are shown in Table 6. The results show that the addition of catalase significantly reduced off-flavor production, especially for 2-butenal, 4-hexen-3-one, trans-3-penten-2-one, benzaldehyde, phenylacetaldehyde, and trans-2-nonenal. The effect of catalase also exceeded the effect of 10 ppm added sulfite, and this effect was most pronounced at increasing doses. For all off-flavors analyzed, the highest dose of catalase (29.6 U / ml) showed significantly lower levels of off-flavors compared to the unadded control. This clearly suggests that catalase can significantly improve the oxidative stability and reduce a broad set of off-flavors produced in beer, thus extending the shelf life of beer.

[0205] [Table 6]

[0206] Example 12 – Freshness analysis of beer with several potential additives to reduce free radicals The following procedure was used to test 330 ml bottles of Carlsberg Pilsner (still fresh and within its expiration date).

[0207] All beer bottles were opened except for the O1FA_1_no control treatment. The headspace was gently flushed with air for 10 seconds to replace the CO2 contained in the headspace from the bottling. This was to ensure oxidation of the beer. The flushing was done in an extraction hood to avoid contamination and was done gently to avoid loss of beer. The opened beer was then dosed with the additives listed in Table 7 below.

[0208] All beers were then tightly closed with crown corks and then inverted to ensure mixing with the headspace air within the bottle of liquid.All beers were then incubated at 30°C for 14 days.

[0209] After 14 days the beers were placed in a refrigerator until analysis - this provides a final freshness index for each beer sample. Samples were compared to each other using a Bruker automated micro ESR system. Samples were run in duplicate for preparation and analysis but no sensory testing was performed.

[0210] [Table 7-1] [Table 7-2]

[0211] The following measurements were made for each beer sample and the results are shown in Table 8. Free radical generation results are shown in Figure 11a, while Figures 11b, 11c and 11d show the same data separated by common trends within grouped samples. Oxidation rates are shown in Figure 11e.

[0212] There was a very clear difference in freshness between the beer samples. Most samples had either poor freshness / no freshness protection versus acceptable protection. However, oxidation decreased across the sample range. This suggests that fewer free radicals are generated after protection is lost.

[0213] [Table 8-1] [Table 8-2]

[0214] Example 13 - Use of natural extracts to reduce oxidation, including acerola extract (natural ascorbic acid) The characteristics and effects of natural extracts containing acerola, a natural source of ascorbic acid, were investigated. Acerola extract, liquid rosemary extract, powdered rosemary extract, a blend of rosemary and lemon extracts, and a blend of green tea and rosemary extracts, all provided by International Flavors & Fragrances (IFF), were added to the beer at the concentrations listed below. The effects of these natural extracts were then evaluated by ESR measurements.

[0215] In a first test, the extracts were dissolved and diluted as necessary to obtain the following weight percentages in dark beer (Schwarzbier, Koestritzer, Germany, 4.8% (v / v) alcohol): 0.25% acerola extract, 0.02% powdered rosemary extract, 0.03% blend of rosemary & lemon extracts, liquid 0.12% rosemary extract, 0.009% blend of green tea & rosemary extracts and a reference dark beer without any added extracts.

[0216] The observed ESR measurements of this first trial are shown in Figure 12a. The acerola extract produced a significantly lower signal intensity compared to all other extracts, with a T600 value of 4.98 x 10 5The next lowest T600 value was 1.1 × 10 for a blend of green tea and rosemary extract. 6 The T600 value of the control beer was 1.4 x 10 6 It was.

[0217] In a second test, the extracts were dissolved and diluted as necessary to obtain the following weight percentages in Pilsner beer (Juebilaeums, Berliner Kindl, Germany, 5% (v / v) alcohol): 0.25% acerola extract, 0.04% powdered rosemary extract, 0.06% blend of rosemary & lemon extracts, 0.24% liquid rosemary extract, 0.018% blend of green tea & rosemary extracts and a reference Pilsner beer without any added extracts.

[0218] The observed ESR measurements of this first trial are shown in Figure 12b. The acerola extract produced a significantly lower signal intensity compared to all other extracts, with a T600 value of 0.13 x 10 6 The next lowest T600 value was 0.56 × 10 for the liquid rosemary extract. 6 The T600 value of the control beer was 0.7×10 6 It was.

[0219] Example 14 – ESR analysis of natural antioxidant extracts in beer using EAP To evaluate the impact of catalase on beers with low intrinsic antioxidant capacity, various natural antioxidant extracts (acerola extract, powdered rosemary extract, blend of rosemary and lemon extracts, liquid rosemary extract and blend of green tea and rosemary extracts, all kindly provided by IFF, Brabrand, Denmark) were prepared in double distilled water at various dilutions, added to black pilsner samples (Koestritzer, 4.6% (v / v) alcohol, Germany) and analyzed by ESR. For the preparation of ESR beer samples, amber vials containing 156 μl ethanol and 12 ml degassed beer sample were used. Directly at the start of the measurement, 52 μl of POBN solution was added and 0.5 ml of diluted natural antioxidant extract sample was placed in the autosampler at 60 °C, making a total volume of 12.708 ml. The results of the addition of various antioxidant extracts to black pilsner with low EAP are shown in Figure 5 (ranging from 0.02 to 0.25% (w / w)). The beer sample without catalase showed an increase in the ESR signal over time with no assignable EAP. A very clear effect of acerola was observed, with very good oxidation protection and a decrease in the ESR signal, an increase in the EAP value at 754 min, but a decrease of 1.25 × 10 5 compared to beer without extract, which had a T1000 value of 6.5 x 10 5 The acerola extract showed an increase in the T1000 value of EAP compared to beer without added extract. All other extracts tested did not show a clear increase in EAP and T1000 compared to beer without added extract. Therefore, acerola extract can significantly improve the oxidative stability of beer by improving the EAP.

[0220] Numbered paragraphs Aspects of the invention are now set forth in numbered paragraphs.

[0221] 1. A method for producing beer comprising adding a catalase composition after mash boiling and / or wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of the beer and / or comprising adding a catalase composition to the final beer, further comprising adding an antioxidant after mash boiling and / or wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of the beer and / or comprising adding an antioxidant composition to the final beer.

[0222] 2. The method of paragraph 1, wherein the catalase composition has a catalase activity of at least 0.1 U / ml in the mash boil preparation, and / or in the wort preparation, and / or in the fermentation preparation, and / or in the maturation preparation, and / or in the stabilization preparation, and / or in the pasteurization preparation, and / or in the final beer; and / or the catalase composition provides a catalase activity of at least 0.1 U / ml in the mash boil preparation, and / or in the wort preparation, and / or in the fermentation preparation, and / or in the maturation preparation, and / or in the stabilization preparation, and / or in the pasteurization preparation, and / or in the final beer.

[0223] 3. The method of paragraph 1 or 2, wherein the catalase is a microbial catalase.

[0224] 4. The method of any one of paragraphs 1 to 3, wherein the microbial catalase is of bacterial or fungal origin.

[0225] 5. The method according to any one of paragraphs 1 to 4, wherein the microbial catalase is derived from a fungus, preferably from an Aspergillus sp. or Trichoderma sp., preferably from A. niger or T. reesei.

[0226] 6. The method of any one of paragraphs 1 to 5, wherein the catalase has the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, or a sequence having at least 70% identity thereto.

[0227] 7. The method of paragraph 6, wherein the catalase has a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0228] 8. The method of any one of paragraphs 1 to 7, wherein the catalase is produced by recombinant techniques.

[0229] 9. The method of any one of paragraphs 1 to 8, wherein the catalase composition further comprises glucose oxidase.

[0230] 10. The method according to any one of paragraphs 1 to 9, wherein the catalase is added during maturation and / or stabilization and / or pasteurization of beer and / or to the final beer.

[0231] 11. The method of any one of paragraphs 1 to 10, wherein the proline specific protease is added during the production of beer.

[0232] 12. The method of any one of paragraphs 1 to 11, wherein the oxidative stability of beer is improved.

[0233] 13. The method according to any one of paragraphs 1 to 12, wherein the production of Strecker aldehyde in the mash boil preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer is reduced.

[0234] 14. The method of any one of paragraphs 1 to 13, wherein the production of Strecker aldehyde in the final beer is reduced.

[0235] 15. The method according to any one of paragraphs 1 to 14, wherein trans-2-nonenal production is reduced in the mash boiling preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0236] 16. The method of any one of paragraphs 1 to 15, wherein trans-2-nonenal production in the final beer is reduced.

[0237] 17. The method of any one of paragraphs 15 to 16, wherein trans-2-nonenal production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably trans-2-nonenal production is reduced by at least 50%.

[0238] 18. The method according to any one of paragraphs 1 to 17, wherein trans-benzaldehyde production is reduced in the mash boiling preparation, and / or the wort preparation, and / or the fermentation preparation, and / or the maturation preparation, and / or the stabilization preparation, and / or the pasteurization preparation, and / or the final beer.

[0239] 19. The method of any one of paragraphs 1 to 18, wherein trans-benzaldehyde production in the final beer is reduced.

[0240] 20. The method of any one of paragraphs 18-19, wherein benzaldehyde production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably benzaldehyde production is reduced by at least 50%.

[0241] 21. The method of any one of paragraphs 1 to 20, wherein the shelf life of beer is extended.

[0242] 22. The method of any one of paragraphs 1 to 21, wherein the shelf life of the beer is extended by at least about 4 months and / or by at least about 4 months.

[0243] 23. The method of any one of paragraphs 1 to 22, wherein the flavor stability of beer is improved.

[0244] 24. The method of any one of paragraphs 1 to 23, wherein the antioxidant is one or more of ascorbic acid, an extract from a natural source of ascorbic acid, acerola extract, pomegranate extract, rosemary extract, lemon extract, green tea extract, a blend of rosemary extract and lemon extract, rosemary extract, and a blend of green tea extract and rosemary extract.

[0245] 25. The method of any one of paragraphs 1 to 24, wherein the antioxidant is ascorbic acid.

[0246] 26. The method of any one of paragraphs 1 to 25, wherein the antioxidant is ascorbic acid derived from a natural source.

[0247] 27. The method of any one of paragraphs 1 to 26, wherein the antioxidant is ascorbic acid derived from a natural source, and the natural source is an acerola extract.

[0248] 28. The method according to any one of paragraphs 1 to 27, wherein the amount of antioxidant in the mash boiled preparation, and / or the wort preparation, and / or the fermented preparation, and / or the matured preparation, and / or the stabilised preparation, and / or the pasteurised preparation, and / or the final beer is at least 0.01% by weight.

[0249] 29. The method according to any one of paragraphs 1 to 28, wherein the catalase and the antioxidant are added during maturation and / or stabilization and / or pasteurization of beer and / or to the final beer, respectively.

[0250] 30. The method of any one of paragraphs 1 to 29, wherein the catalase and the antioxidant are in a catalase composition.

[0251] 31. Use of catalase and an antioxidant in the production of beer, wherein the catalase is added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization and / or is added to the final beer; and the antioxidant is added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization and / or is added to the final beer.

[0252] 32. The use according to paragraph 31, wherein the catalase is a catalase composition as defined in any one of paragraphs 1 to 30.

[0253] 33. The use according to any one of paragraphs 31 to 32, in which the flavor stability of beer is improved.

[0254] 34. Beer obtained or obtainable by a method according to any one of paragraphs 1 to 30.

[0255] 35. Beer according to paragraph 34, comprising catalase and an antioxidant, preferably ascorbic acid.

[0256] 36. Beer containing catalase and an antioxidant, preferably ascorbic acid.

[0257] 37. Beer according to any one of paragraphs 34 to 36, comprising glucose oxidase.

[0258] 38. A beer according to any one of paragraphs 34 to 37, wherein the shelf life of the beer is extended to at least about 4 months or by at least about 4 months.

[0259] 39. A beer according to any one of paragraphs 34 to 38, wherein the flavor stability of the beer is improved.

[0260] All publications mentioned herein above are incorporated herein by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

[0261] Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry, molecular biology, or related fields are intended to be within the scope of the following embodiments.

[0262] array SEQ ID NO:1 (showing the mature amino acid sequence of catalase from Aspergillus niger): [ka]

[0263] SEQ ID NO:2 (showing the deduced amino acid sequence of catalase from Aspergillus niger with the potentially truncated N-terminal region underlined and in bold): [ka]

[0264] SEQ ID NO:3 (showing the deduced amino acid sequence of catalase expressed from Aspergillus niger): [ka]

[0265] SEQ ID NO:4 (showing the deduced amino acid sequence of catalase expressed from Trichoderma reesei): [ka]

[0266] SEQ ID NO:5: [ka]

[0267] SEQ ID NO:6 (Aspergillus niger): [ka]

[0268] SEQ ID NO:7 (Aspergillus transmontanensis): [ka]

[0269] SEQ ID NO:8 (Aspergillus homomorphus): [ka]

[0270] SEQ ID NO:9 (Rasamsonia emersonii): [ka]

[0271] SEQ ID NO:10 (Penicillium antarcticum): [ka]

[0272] SEQ ID NO:11 (Penicillium oxalicum): [ka]

[0273] SEQ ID NO:12 (Aspergillus awamori): [ka]

[0274] SEQ ID NO:13 (Aspergillus bertholletius): [ka]

Claims

1. 1. A method for producing beer, comprising adding a catalase composition after mash boiling and / or wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of the beer, and / or comprising adding a catalase composition to the final beer, and further comprising adding an antioxidant after mash boiling and / or wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization of the beer, and / or comprising adding an antioxidant composition to the final beer.

2. 2. The method of claim 1, wherein the catalase composition has a catalase activity of at least 0.1 U / ml in the mash boil preparation, and / or in the wort preparation, and / or in the fermentation preparation, and / or in the maturation preparation, and / or in the stabilization preparation, and / or in the pasteurization preparation, and / or in the final beer; and / or the catalase composition provides a catalase activity of at least 0.1 U / ml in the mash boil preparation, and / or in the wort preparation, and / or in the fermentation preparation, and / or in the maturation preparation, and / or in the stabilization preparation, and / or in the pasteurization preparation, and / or in the final beer.

3. The method of claim 1 or 2, wherein the catalase is a microbial catalase.

4. The method according to any one of claims 1 to 3, wherein the catalase is a microbial catalase, the microbial catalase being of bacterial or fungal origin.

5. The method according to any one of claims 1 to 4, wherein the catalase is a microbial catalase, the microbial catalase being derived from a fungus, preferably from Aspergillus sp. or Trichoderma sp., preferably from A. niger or T. reesei.

6. 6. The method of any one of claims 1 to 5, wherein the catalase has the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 or a sequence having at least 70% identity thereto.

7. The method of claim 6, wherein the catalase has a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

8. The method of any one of claims 1 to 7, wherein the catalase is produced by recombinant techniques.

9. The method of any one of claims 1 to 8, wherein the catalase composition further comprises glucose oxidase.

10. 10. The method according to any one of claims 1 to 9, wherein the catalase is added during maturation and / or stabilization and / or pasteurization of the beer and / or to the final beer.

11. The method according to any one of claims 1 to 10, wherein a proline specific protease is added during the production of the beer.

12. The method according to any one of claims 1 to 11, wherein the oxidative stability of the beer is improved.

13. 13. The method according to any one of claims 1 to 12, wherein the production of Strecker aldehyde in the mash boiled preparation, and / or the wort preparation, and / or the fermented preparation, and / or the matured preparation, and / or the stabilised preparation, and / or the pasteurised preparation, and / or the final beer is reduced.

14. The method according to any one of claims 1 to 13, wherein the formation of Strecker aldehyde in the final beer is reduced.

15. 15. The method according to any one of claims 1 to 14, wherein trans-2-nonenal production in the mash boiled preparation, and / or the wort preparation, and / or the fermented preparation, and / or the matured preparation, and / or the stabilised preparation, and / or the pasteurised preparation, and / or the final beer is reduced.

16. The method according to any one of claims 1 to 15, wherein trans-2-nonenal formation in the final beer is reduced.

17. 17. The method according to claim 15 or 16, wherein trans-2-nonenal production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably trans-2-nonenal production is reduced by at least 50%.

18. 18. The method according to any one of claims 1 to 17, wherein trans-benzaldehyde production in the mash boiled preparation, and / or the wort preparation, and / or the fermented preparation, and / or the matured preparation, and / or the stabilised preparation, and / or the pasteurised preparation, and / or the final beer is reduced.

19. The method according to any one of claims 1 to 18, wherein trans-benzaldehyde formation in the final beer is reduced.

20. 20. The method of claim 18 or 19, wherein benzaldehyde production is reduced by at least 10%, at least 20%, at least 30%, at least 40% or at least 50%, preferably benzaldehyde production is reduced by at least 50%.

21. The method according to any one of claims 1 to 20, wherein the shelf life of the beer is extended.

22. 22. The method according to any one of claims 1 to 21, wherein the shelf life of the beer is extended by at least about 4 months and / or by at least about 4 months.

23. The method according to any one of claims 1 to 22, wherein the flavor stability of the beer is improved.

24. 24. The method of any one of claims 1 to 23, wherein the antioxidant is one or more of ascorbic acid, an extract from a natural source of ascorbic acid, acerola extract, pomegranate extract, rosemary extract, lemon extract, green tea extract, a blend of rosemary extract and lemon extract, rosemary extract, and a blend of green tea extract and rosemary extract.

25. The method of any one of claims 1 to 24, wherein the antioxidant is ascorbic acid.

26. 26. The method of any one of claims 1 to 25, wherein the antioxidant is ascorbic acid from natural sources.

27. 27. The method of any one of claims 1 to 26, wherein the antioxidant is ascorbic acid from a natural source, the natural source being an acerola extract.

28. 28. The method according to any one of claims 1 to 27, wherein the amount of antioxidants in the mash boiled preparation, and / or the wort preparation, and / or the fermented preparation, and / or the matured preparation, and / or the stabilised preparation, and / or the pasteurised preparation, and / or the final beer is at least 0.01 wt.-%.

29. 29. The method according to any one of claims 1 to 28, wherein the catalase and the antioxidant are added during maturation and / or stabilization and / or pasteurization of the beer and / or to the final beer.

30. 30. The method of any one of claims 1 to 29, wherein the catalase and the antioxidant are in the catalase composition.

31. Use of catalase and an antioxidant in the production of beer, wherein the catalase is added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization and / or is added to the final beer; and the antioxidant is added after mash boiling and / or during or after wort preparation, and / or fermentation, and / or maturation, and / or stabilization, and / or pasteurization and / or is added to the final beer.

32. The use according to claim 31, wherein the catalase is a catalase composition according to any one of claims 1 to 30.

33. 33. The use according to claim 31 or 32, in which the flavor stability of the beer is improved.

34. Beer obtained or obtainable by the method according to any one of claims 1 to 30.

35. 35. Beer according to claim 34, comprising catalase and an antioxidant, preferably ascorbic acid.

36. Beer containing catalase and an antioxidant, preferably ascorbic acid.

37. The beer according to any one of claims 34 to 36, comprising glucose oxidase.

38. 38. The beer of any one of claims 34 to 37, wherein the shelf life of the beer is extended to at least about 4 months or by at least about 4 months.

39. The beer according to any one of claims 34 to 38, wherein the flavor stability of the beer is improved.

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