MXPA96005184A - Drink of malta which has stabilized flavor and my production methods - Google Patents

Abstract

The present invention relates to a method for stabilizing the flavor of a fermented malt beverage, more particularly a beer, by the addition of an oxoaldehyde reductase enzyme, the fermented malt beverage prepared by this method, and to use during the fermentation process of oxoaldehyde reductase enzymes from natural sources, including those produced by yeast, to stabilize the flavor of the resulting beer product and to produce a beer having a stable flavor. The invention also relates to microorganisms, particularly yeasts, that have been specifically modified, selected, or genetically engineered to express or secrete an oxoaldehyde reductase enzyme that can be used during the fermentation process to stabilize the taste of the yeast. resulting beer product, and to produce a beer that has a stable flavor. The present invention also provides extracts from a natural source (eg, yeast), or a modified yeast, or extracts thereof, which will provide a sufficient amount of the enzymes necessary to block, inhibit or reduce the intermediate compounds of the invention. the Maillard reaction (e.g., 3-deoxyglucosone, which results in the formation of rancid flavor in fermented malt beverages.In addition, the present invention provides fermented malt beverage having improved flavor stability produced by these methods.

Description

DRINK OF MALTA WHICH HAS STABILIZED FLAVOR AND PRODUCTION METHODS OF THE SAME t FIELD OF THE INVENTION The present invention relates to the fields of biotechnology and food / beverage manufacturing. The invention relates to the production of malt beverages, and more particularly to the production of malt beverages having improved flavor stability. In particular, the invention relates to methods and compositions for improving the flavor stability of fermented malt beverages such as beer and malt beverages produced by these methods.

BACKGROUND OF THE INVENTION Related technique The process of brewing Summary. In the production of malt beverages, such as beer, a hot water extract of barley malt, with or without other grains not transformed into malt such as rice or corn, is boiled with hops, cooled and then subjected to fermentative action of the yeast. Water REF: 23440 hot used to extract the malt allows the action of several enzymes in the malt to hydrolyze the starch in the barley (and in the corn or rice) to fermentable sugar, which is triggered by the yeast to produce the alcohol in the drinks of fermented malt.

Conversion in Malta. The barley malt is soaked with water to produce soaked malt that germinates at a moderately low temperature. Germination is carried out with daily mixing and addition of water as needed, to maintain the moisture content at approximately 43%. The resulting green malt contains a high content of beer flavor precursors, beer flavor components, and coloring agents. After the germination is finished, the green malt is heated to a high moisture content to generate the precursors of the beer flavor, the flavor components of the beer and also to reduce the activity of the amylolitic enzymes. After heating, the malt is dried to a moisture content of 3.5-5.5% and a soluble protein content of 6.5-8%. The dried malt can then be matched (produce or re-pulped), to produce a wort that is boiled with hops, cooled, placed with brewer's yeast, and processed by conventional fermentation processes and fermentation equipment conventional Filling. The malt, which can really be a mixture of malts (ie, normal beer malt, low amylase malt, high color, etc.), rests and mixes with 2.5 to 4 times its weight of hot water in large vats and is pasted at 35-40 ° C for 5 to 15 minutes until a thick, sticky malt is formed. The pasted malt is then allowed to stand for 45-90 minutes without stirring, then heated in steps to 70-73 ° C while stirring, with a time allowed at each step for the various strains to turn the starches into sugars. fermentable Following heating, the matting is maintained for 15-30 minutes, and the temperature rises to 75 ° C, and the pasted malt is transferred to a filtration unit (lauter). If the rice and corn adjuncts are to be used, they are cooked separately and a sticky malt is obtained from the cooker. The production of the cooked malt from the cooker comprises the use of adjuncts together with a 10% -30% portion of malt, (or the addition of commercial enzymes) to convert the starch in the material into fermentable sugars. The adjuncts and the malt portion are gradually boiled and kept there until the products are completely gelatinized. During the final stages of the filling (at the highest temperatures), the mashed malt of the cooker and the pasted malt are combined. The filling serves a three-purpose purpose. First, it brings to the solution those malt substances (and adjuncts) that are easily soluble in hot water. Second, it allows malt enzymes to act on the insoluble substances and make them soluble. i Third, it provides a long-range enzymatic degradation of starches, proteins and gums in products of smaller size and lower molecular weight.

Filtration in Cuba and dew. The filtration in Cuba consists of the removal of the liquid, now called the "must", of the insoluble husks or "spent grains". The filtration in tank starts at the completion of the filling process, whereby the finished malt is transferred to a filtration tank. There it is allowed to rest for approximately ten to thirty minutes during which the spent grains settle to the bottom. The filtration tank is equipped with a false bottom that contains numerous perforations and an outlet that leads to the bottom of the tank. The pasted malt is then allowed to settle for 10-20 minutes and the runoff begins. The must is recycled until it is reasonably clear. The clear must is then pumped into a t fermentation kettle. Hot water is introduced through the spent grains to rinse, or spray, any remaining wort. The filtration temperature is approximately 72-77 ° C for both the bath and the spray water. The amount of spray water used is approximately 50-75% of the amount of the fermentation water.

Boiling and mixing of wort hops: Primary fermentation The must is boiled vigorously for one or two and a half hours in the fermentation kettle. You can add hops (or extracts thereof) in several stages of the boiling process, depending on the nature of the final product sought. The boiling of the must serves a number of purposes, including (1) concentration of the sprayed must (2) complete inactivation of the enzymes that may have survived the final filling process, (3) coagulation and precipitation of the high-weight proteins molecular and solid (called "break in the kettle" or "thermal break") (4) extraction of the desirable constituents of the hops, and (5) sterilization of the must.

Cooling, fermentation and storage: Maturation. After boiling, the must is strained to remove the solids or "turbidity", and the must is then cooled to a temperature of approximately 12-16 ° C. Fermentation begins when the must is placed with the appropriate amount of a pure brewer's yeast culture (typically around 0.32-0.68 Kg / bbl (0.7-1.5 lb / bbl)). After 24 hours, the fermentation is established and it is continued, at an accelerated speed. The fermentation typically continues for about 7 to 10 days. During this period, the must temperature must be controlled, since the fermentation process causes the must temperature to rise. Once the yeast has metabolized all the fermentable ingredients in the must, it settles to the bottom and is subsequently recovered and recycled for use in the placement of other beer stews. As the fermentation process reaches a conclusion, the temperature of the must begins to fall. The fermented must (called "green beer") is extracted during storage in a quarter, cold, or "ruh" tank, where its temperature is lowered to approximately 0-5 ° C.

Processing and packaging The "ruh" beer can be left to remain in the ruh tank for the completion of the maturation process, or it can be transferred to a separate maturing tank in the additional settlement of any remaining yeast and other solids: depending on the particular brewery, the Beer is allowed to mature from about 14 days to about 3 months. During this period, beer clarifies and develops its flavor. In maturation, beer is filtered in general to remove yeasts and other solids.

The beer can be subjected to a single pass or double pass filtration process. Double pass filtration consists of two steps. A primary (coarse) filtration, and a secondary (fine) filtration. The filtered beer is subsequently stored in a certain tank. To prepare beer for consumption, it is carbonated at a specified level. Then, depending on the shape of the packaging, the beer can be pasteurized. (In the case of beers "served directly from the barrel", filtered in cold, a microfiltration system is used to remove the contaminants, thus avoiding the passage of pasteurization). The beer packaged in cans and bottles is usually pasteurized, while the beer packaged in t casks or barrels (and sometimes bottles) remains unpasteurized. After the final processing of the packaged product (eg, labeling, etc.), the beer is ready for shipment to the consumer. Other conventional processing steps well known to those skilled in the art can be used in place of, or in addition to, the general fermentation methods described above. For example, the fermented must may be diluted with water to produce low-calorie, low-alcoholic malt beverages (40 or fewer calories per 354 mi (12 ounces)), (less than 0.5% by volume of alcohol) that simulates closely the taste, taste and mouthfeel of conventional beer.

Flavor Flavor is a key factor in the quality of a malt beverage such as beer. It is important that a beer maintains its original, fresh flavor and character during distribution and storage. In this way, the wrong flavors are a big problem for beer manufacturers and distributors. The taste modified by light is a well-known wrong taste formed during the storage of bottled beer, as is the wrong taste caused by contamination with microorganisms. Other flavors that are mistaken during storage are expressed as paper flavor, similar to cardboard, oxidized, or in general, rancid. At room temperature, the rancid flavor in bottle or pot begins to develop shortly after packaging, and increases gradually and continuously to the extent that most American beer makers return their product to the market if it is more than 4 years old. months from the date of packaging. Although oxygen in a beer bottle or canister is typically consumed by beer within 24 hours of packaging, the perceptible presence of a rancid taste does not appear in general for several weeks. In the past, the rancid taste of oxidized malt beverages, such as beer, has been generally attributed to the combined effects of oxidation, light and heat. Principally ^ the researchers (representing over 80% of the prior art) have focused on methods to reduce oxidation in the finished product. For example, the present practice of delaying the formation of rancid beer taste includes keeping a low level of air (or oxygen) in the bottled beer by minimizing the top space or space from the surface to the top of the beer. container. The modern beer filling machines are designed to achieve very low air levels in the product. Typically, the bottle is evacuated before it is filled with beer, or the air in the evacuated bottle is replaced with carbon dioxide before filling, or the bottle's over-foaming is used to replace the gases in the top space with the foam of beer. All of these practices can produce air levels of less than 0.5 ml per 358 ml (12 oz) bottles. But even these low levels of air still allow the beer to oxidize in 2-3 months. Wrong factors become more obvious when the malt beverage has been stored at high temperature (thermal reactions). The negative influence of isohumulones and melanoidins on the oxidation of alcohols at elevated temperatures has been known for several years. See, for example, Hashimoto, Rept. Res. Lab. Kirin Brewery Co. Ltd. 19: 1 (1979). However, although beer is ideally stored at cold temperatures, maintaining a uniformly cold temperature is not always possible during transport. This is a particular problem in warm and humid countries, where the temperature varies from 28-38 ° C, even more so in those countries where modern refrigeration is not always available. Therefore, there is clearly a need for a reliable method to stabilize the taste of the beer, which does not depend on the environmental conditions, specifically controlled, after the packaged product has left the brewery. A wide range of carbonyl compounds are known which are reduced during fermentation, particularly of malt and must, and produce the wrong flavors. See, Meilgaard and collaborators, Tech Q. Master »Brew. Assoc. Am. 12: 151-168 (1975). Two biological routes control the level of carbonyl compounds in the final product, the formation of aldehydes from the deposits of oxoacids and the enzymatic removal of the carbonyls of the must by the yeast of the beer. Higher alcohols and the corresponding aldehydes are formed, partially by the anabolic processes from the main source of carbon and partially through the catabolic pathway of exogenous amino acids. In addition, the aldehydes produced during fermentation, filling and boiling are known to be potential substrates for aldehyde dehydrogenases or reductases. Peppard et al., J. Inst. Brew. 87: 386-390 (1981). However, recent studies have indicated that aldehyde-reducing systems are more complex than previously assumed. See, Collins and collaborators, Proc. Congr. Eur. Brew. Conv. 23: 409-416 (1991) Kronlof et al., Proc. Cong. Eur. brew Conv. 22 355 -362 (1989). It is now recognized that many enzyme systems are involved in the reduction of carbonyl compounds in higher alcohols, and that each system probably operates with variable activities during the course of the fermentation process (DeJbourgr et al., J. Am. Soc. Brew. Chem. 52 (3): 100-106 (1994) For example, carbonyl compounds, particularly unsaturated carbonyls, are unstable.These compounds decompose to shorter chains, which are subjected to the condensation of aldol. , notably trans-2-nonenal, and related compounds comprised in the oxidation of long-chain fatty acids have long been associated with rancid taste in beer See, for example, Debourg et al. supra, and the US Patent No. 4,110,480 It is well known that the medium oxidation by enzymes of unsaturated fatty acids, such as linoleic acid, followed by oxidative cleavage. whether or not oxidative, subsequent to the carbon chain, will produce flavor-active compounds that have carbon lengths of 6 to 12. Therefore, those attempts to stabilize the flavor of the fermented malt beverage have focused in some cases, in the modification of the lipids included in the fermentation process. However, in beer, lipids are derived from malt in various forms including simple lipids (fatty acids, triglycerides and other neutral lipids), complex lipids (glycolipids and phospholipids) and bound lipids such as those bound to the starch grains. Numerous methods have been made to remove the lipids from the raw materials, including (1) removal of the germ from the grain, which contains a significant portion of lipids found in the cereals of the raw material (clarification), (2) removal of the lipids from cereals of the raw material by extraction with ethanol, (3) pretreatment of the grains of the cereals of the raw material with a lipid decomposition enzyme (Japanese Patent Examined Publication No. 2248/1973, Japanese Patent Not Examined Publication No. 55069/1987 and (4) Removal of lipids by separation with special filtration (US Patent No. 5, 460, 836) However, not all lipids have an adverse effect, ie the equilibrium of these forms of lipids imperceptibly affects the quality of the beer and the efficiency of the fermentation process of the beer.Thus, even after years of study, remains unknown balance is appropriate, or how altering the total lipid content will affect the flavor stability of the finished product, stored. Another recognized technique for stabilizing beer against oxidation is to add an oxygen scavenger, such as sulfur dioxide, usually in the bisulfite form, to beer. Sulfur dioxide is produced by the yeast during fermentation and will be combined with carbonyls to form bisulfite addition components that are hydrophilic, and thus less volatile. However, although effective, the increase in the concentration of S02, naturally or artificially, may be commercially unacceptable. In the United States of America, for example, S02 is limited by law to less than 10 ppm, and even those low levels produce undesirable and sulfur flavors in some beers, while in other countries, such as Germany, any addition of exogenous S02 is prohibited. . Even if allowed, the addition of bisulfite, which works by binding to the aldehydes, is not a panacea. Beer is a complex product that comprises many very different aldehydes (notably acetaldehyde, a normal product of fermentation), therefore, the action of a sulfite additive is often diminished. The addition of other oxygen scavengers has also been attempted, but with little effect on the long-term stability of the sabar in the fermented malt beverage. In view of the prior art and years of searching, however, the taste of the beer still becomes rancid. In this way, it is clear that until the present invention, a need long perceived in the art remained for a reliable method to stabilize the flavor of fermented malt beverages, having the following characteristics. (1) Do not significantly alter the desirable fresh taste of the finished product, (2) does not significantly decrease the efficiency of the fermentation process, (3) does not violate the law or regulation with respect to the addition of additives or preservatives and (4) does not depend on the maintenance of specific environmental conditions for transport and storage of the product packing. The present inventors have developed a completely new method for stabilizing the taste of fermented malt by focusing on one aspect of the fermentation reaction not previously considered in the prior art. The present invention demonstrates that the flavors of fermented malt can be stabilized by the use of inhibitors, blockers or reducing agents of the intermediates of the Maillard reaction, such as the NADPH-dependent enzyme, 3-deoxyglucosone reductase, or aminoguanidine.

In order to assess flavor stability, the inventors found that it is essential to have a sensitive, rapid and reproducible method with which changes in the taste of beer can be analyzed. The sensitive test has been the traditional means available to assess the organoleptic quality of beer. The taste test, although sensitive, suffers from human limitations, such as staff inclination and the tendency to make comparative (subjective) rather than objective evaluations (Mathews et al., Trends in Food Science &Technol., 4: 89-91). (1990)) . The Beer Making Technology Institute begins using high performance liquid chromatography (HPLC) analysis according to for example Greenhoff and Wheeler, J. Inst. Brew 86:35 (1981); Strat and Drost, Dev, in Food Sci. 17: 109-121 (1988). Improved methods were applied using purge and trap techniques, gas chromatography, and selective mass detection using the SIM technique, to establish high capacity and better separation, determination and identification. See, for example, Narzi e) MBAA Tech. Q. 30: 48-53 (1993). However, objective measurements of a particular quality parameter are meaningless unless they correlate to the human response to the drink as a whole, when purchased and consumed under normal conditions. In this way, the present inventors developed a system by which the organoleptic deterioration of beer could be evaluated, by analytical indices that provide a series of compounds (see Figure 1) that represent a reproducible continuum of fresh but deteriorated forms (rancid) . These analytical indices are then related to organoleptic evaluations, as demonstrated in Figures 2a and 2b, to provide a correlation between objective and organoleptic measures of flavor freshness. Bravo et al., IBTC Technical and Consortium Meeting # 35, Salzburg, Austria, September 1993; Bravo and collaborators IBTC Technical Consortium Meeting # 36, Caracas, Venezuela, November 1994. These compounds participate in the reactions comprised in beer rantering processing (substrates, and intermediate or final products), but do not necessarily produce the rancid taste. These analytical indices are relatively easy to detect and show a significant change in their relative maximum areas during the aging process (see Figures 2a and 2b). The concentration of furfural, 5-methylfurfuryl, 2-acetylfuran and 5-hydroxymethylfurfuran are useful index for measuring thermal damage in beer. For example, in an effort to establish a "quality deterioration test", methods have been developed to detect furfural and 5-methylfurfuril in fruit juices during storage. Harayama and collaborators Agrie. Biol. Chem. 53: 393-398 (1991) found by multivariable t analysis of the wrong taste in volatile compounds in the top space formed during beer storage, that certain furfural compounds were a valuable index for measuring a flavor of cardboard, particular in beer. Grongvist et al., EBC Cong. 421-428 (1993), using gas chromatography to measure the carbonyl compounds present during the production and aging of the beer, found that the concentration of furfural significantly increased during aging.

BRIEF DESCRIPTION OF THE INVENTION The present inventors, deduced that the products formed during the Maillard reaction could be used as indices of aging or maturation of the beer, developed a method, (using the indices measured t by a combination of capillary electrophoresis and HPLC) to reliably inspect the flavor stability and the organoleptic effect of aging in beer (Bravo et al., IBTC Technical Consortium Meeting # 35, Salzburg, Austria, September 1993; Bravo and collaborators IBTC Technical Consortium Meetin # 36, Caracas, Venezuela, November 1994). By using the method for the detection of the relevant chemical indices, the present inventors developed a new system, significantly and significantly advanced over those described and used to date, to reliably and efficiently assess the freshness degree of the beer, and to determine the storage conditions (time and temperature) of a beer exposed to a previously unknown environment. In addition, the present inventors have used these analytical systems to develop methods to improve the flavor stability of malt beverages such as beer, and to produce malt beverages by these methods. In the initial investigations designed to solve the problems described above, it was discovered that by enzymatically regulating the production of certain intermediates of the Maillard reaction, formed during the fermentation process, a fermented malt beverage could be produced reliably. refreshingly pure taste and improved flavor stability. The present inventors made additional investigations based on this finding and developed the present invention. The present invention relates to the production of malt beverages that have improved flavor stability.

The invention has particular utility in the production of fermented malt beverages such as beer, although the invention can also be advantageously used in the production of other flavored malt beverages. The invention further relates to methods of fermentation or brewing, for producing fermented malt beverages, such as beer, beverages prepared by this method, and beverages having a substantially stabilized flavor. In particular, the present invention relates to a method for stabilizing the flavor of a fermented malt beverage, more particularly a beer, by the addition of an oxoaldehyde reductase enzyme, the fermented malt beverage prepared by this method, and a fermented malt beverage that has improved flavor stability. The invention further relates to the use, during the fermentation process, of oxoaldehyde reductase enzymes from natural sources, including those produced by yeasts, to stabilize the flavor of the resulting beer product and to produce a beer having a flavor stable. It also refers to microorganisms, particularly yeasts, that have been specifically modified, genetically engineered or engineered to express or secrete an oxoaldehyde reductase enzyme that can be used during the fermentation process to stabilize the taste of the resulting beer product and to produce a beer that has a stable flavor. The present invention also provides a method for improving the flavor stability of a malt beverage. According to the present invention, this method is suitable for improving the flavor stability of a fermented malt beverage, in particular, beer. Thus, it is an object of the present invention to provide a method of fermentation or brewing, wherein the flavor stability of the beer is improved. The present invention further provides the malt beverages prepared by these methods. According to the present invention, the malt beverage provided in this manner is a fermented malt beverage, particularly a beer. Thus, it is an object of the present invention to provide a beer in which flavor stability has been improved.

The present invention also provides extracts from a natural source (eg, yeast) or a modified yeast or extract thereof, which will provide a sufficient amount of the enzymes necessary to block, inhibit or reduce the intermediate compounds of the reaction of Maillard (for example, 3-deoxyglucosone), which results in the formation of rancid flavor in fermented malt beverages. Still other objects and advantages of the present invention will be apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Chromatogram of a fresh beer showing the chemical indices of beer aging: LCI, LC11, LC18 and 5-HMF.

Figure 2. a: Graph showing the changes in the intensity of the maximum LC18 height during storage at 5 ° C and its correlation with flavor evaluation. The LC18 is consumed at low temperatures and tends to disappear with time. b: Graph showing the changes in the concentration of 5-HMF during the storage of the beer at 28 ° C, and its inverted correlation with the degree of oxidation. t Figure 3. Diagram of the proposed mechanism of formation of LC18 in beer, and its possible complication in the deterioration of taste. LC18 is a precursor of the dicarbonyl compound of 5-HMF that can also be condensed with amino acids through the degradation of Strecker to produce aldehydes and pyrroles or pyrazines (CF).

Figure 4. Chromatogram of a model system of glucose-glycine treated with heat, consisting of glucose 1M + glycine 0.5 M, after 3 hours of reaction at 90 ° C, demonstrating the acquisition of the analytical indices of aging beer (LC8, LC11 and LC18) in a model system.

Figure 5. Composite chromatogram demonstrating the effect of the addition of 1,2-phenylenediamine to the must. A: must; B. must + 1,2-phenylenediamine. The addition of 1,2-phenylenediamine causes a specific reduction in the maximum value of LC18.

Figure 6. a: Bar graph showing the changes in the area of the hydrophobic quinoxalines that accompany the storage of beer at 5 ° C and 28 ° C for 15 days and at 60 ° C for 3 days. b: Bar graph showing the changes in the area of the hydrophilic quinoxalines that accompany the storage of beer at 5 ° C and 28 ° C for 15 days and 60 ° C for days. Figure 7. Scheme of the purification process of the enzyme reductase. Shock absorber A: 25 mM potassium phosphate, pH 7.5. Shock absorber B: 5 mM potassium phosphate, pH 6.5. Shock absorber C: 25 mM potassium phosphate, pH 7.0.

Figure 8. Elution profile of reductase 1 in Sephacryl S-200 chromatography. Start: SDS-polyacrylamide reductase gel electrophoresis 1. The gel was stained with Comassie's bright sugar.

Figure 9. Elution profile of reductase 2 in. chromatography of Sephacryl S-200. Start: SDS-polyacrylamide reductase gel electrophoresis 2. The gel was stained with Coomassie brilliant blue.

Figure 10. The substrate specificities of the enzymes reductase 1 and reductase 2. i Figure 11. Compound chromatogram showing the decrease in the maximum value of LC18 in the beer after the addition of reductase 1 and 2 isolated from the beer yeast.

Figure 12. Bar graphs showing organoleptically determined degrees of freshness of beers treated with reductase 1. Beers were incubated with a mixture of buffer C, NADPH (control beers) and reductase 1 (experimental beers) for 15 days at 28 ° C (panel a) or 3 days at 60 ° C (panel b). i DETAILED DESCRIPTION OF THE INVENTION Definitions Throughout this description, various terms are used that are generally understood by those skilled in the applicable art. Several terms with specific meaning are used, however, and are proposed to mean what is defined by the following: As used herein, the term "malt" is proposed to refer to any cereal grain, particularly barley, soaked in water until it is germinated and used in fermentation and distillation. The term "sticky malt" as used herein is defined as malt or crushed grain, soaked in hot water to make must. The term "wort" as used herein is defined as liquor runoff after the extraction of a prepared solid material, such as grain or ceneal malt, with hot water. As used herein, the term "fermented malt beverage" is proposed as any malt flavored beverage produced by fermentation, such as beer or sake. As used herein, the term "beer" is defined as an alcoholic beverage fermented from malt and hops. The term as used herein is intended to include thick and bitter beers, strong malt beers, aged beers, bitter and strong beers, malt liquors, low-calorie, low-alcohol and light-weight mixed liquors, and similar.

The reaction of Maillard More than 80 years ago, Luis Maillard first investigated the reaction to reduce sugars with free amino groups of amino acids and proteins. This complex reaction, called the Maillard reaction, or brown, non-enzymatic color, is responsible for the aroma and taste in cooked or preserved foods. Specifically, it is known to be comprised in the color and aroma resulting from fermented malt beverages, such as beer or sake. As shown in Figure 3, the Maillard reaction is initiated by the reaction of the primary amines (from amino acids, proteins and nucleic acids) with sugars to form amines (Schiff's bases) that are additionally subjected to rearrangement to form the Amadori products, which are responsible for the brown color and the fluorescent process, which subsequently results in the formation of numerous advanced glycosylation end products. Broadly, the advanced glycosylation end products are called a-carbonyl intermediates, which include, for example, 1-deoxydicetosas and 3-deoxyaldocetoses. When the reduced sugar is glucose, as in the fermentation of the malt, one of the intermediate compounds of a-carbonyl is 3-deoxyglucosone.

Thousands of compounds, including dextrin, polypeptides, alcohols, polyphenols, pyrroles; isohu ulonas, melanoides, fatty acids and aldehydes, as well as precursors and related intermediates, are included during the fermentation process in the Maillard reaction. For example, there are about 140 reductases and dehydrogenases in the reductase superfamilies comprised in the Maillard reaction. However, prior to the present invention, there is no suggestion in prior art t that by enzymatically regulating the production of an intermediate compound specific to the Maillard reaction, the taste of the beer could be effectively stabilized. In addition, the use of a specific reductase as a processing, enzymatic, regulatory aid in the production of beer has not been suggested until now. Historically, most tests to test beer flavor stability have been purely subjective (for example, classic beer tasters panels) and have not been conducive to quantification. Therefore, it was necessary for the present inventors to first develop a reliable, objective, analytical test to determine the flavor stability of the sample, which could be used in addition to the organoleptic evaluations, before new procedures can be implemented or characterized additives. in terms of its effects on flavor stability.

The attributes of fermented malt drinks.

Malt drinks, especially beer, have attributes easily discernible by the consumer. These attributes include foam, flavor and clarity. Of these, taste is finally the most important characteristic for the consumer. The taste (purity) and the after taste (refreshing sensation) are typically measured within the industry as having one of the following five degrees: 1: Not very clear taste and later taste has no cooling sensation. 2: (Bust is not clear and after taste has almost no refreshing sensation) 3: Usual 4: Taste is clear and after taste has refreshing sensation 5: Taste is very clear and after taste has very refreshing sensation. it typically evaluates in the packaged, stored product (usually at a storage temperature of 40 ° C) as having one of the following five grades: 1: Significantly rancid 2: Rancid 3: Usual. 4: Fresh. 5: Very cool. In addition, a growing number of consumers want a completely natural beer product that demonstrates the above qualities and is still completely free of artificial additives or supplements. It is known in the art that malted barley can be replaced in whole or in part by a so-called "fermentation attachment". The fermentation attachments, suitable include corn, rice, sugar and various syrups. A fermentation adjunct used in the production of a wort, such as corn, is usually crushed and a mat malta formed separately from the matting malt when adding enzymes. The prehydrolyzed products can be mixed with the matted malt, and syrups can be added to the wort at the time the must is boiling as described above. The use of fermentation adjuncts needs to be carefully controlled in order to produce a beer of acceptable taste and color. The use of adjuncts made from corn, rice and other grains extend the fermentation ingredients beyond the traditional ones listed above. However, an approach is not possible in certain countries, for example, in Germany, the law of purity of beer promulgated in 1516 (the "Reinheitsgebót") that limits fermentation ingredients to barley malt, water is still followed , hops and yeast.

The compounds added to the wort mixture before the primary fermentation step are called "processing aids". On the other hand, the compounds added to the wort mixture after the primary fermentation step are called "additives". The difference between the two is significant because the use of additives is regulated, while the use of processing aids is not regulated. By the methods of the present invention, a flavor stabilizer amount of at least one oxoaldehyde reductase enzyme is used as an additive for the fermented malt beverage. This enzyme additive provides improved flavor stabilization of the finished fermented malt beverage. The redacted oxoaldehyde enzyme (s) can be added at any stage of the fermentation process, which includes the grain malt, the must before the fermentation, the fermented must, the fermented malt beverage before processing, or to the fermented malt beverage, processed before packaging. Most preferably, the oxoaldehyde reductase enzyme is added to the must before fermentation to the fermented malt beverage before processing, to the fermented malt beverage, processed before packaging. Preferably, the oxoaldehyde reductase enzymes are of natural origin. Enzymes can be isolated using known methods of protein extraction from a number of sources, and can be purified as described below and then added to the fermentation beverage as a processing aid and / or as an additive. This scheme, Sta reductase enzymes can add to the fermentation process continuously or as an individual injection. The methods of the present invention can be carried out using either full-length enzymes, or biologically active fragments thereof. As an alternative form of the enzyme, certain synthetically formulated full-length or attenuated oxoaldehyde reductases can be used in place of the natural enzymes to stabilize the taste of the fermented malt product, while the alternative enzyme form possesses the activity biological of oxoaldehyde reductase enzyme, natural. Preferably, the oxoaldehyde reductase enzyme isolated and purified by, or used in, the methods of the present invention is an NADPH-dependent enzyme. Most preferably, the oxoaldehyde reductase enzyme is 3-deoxyglucosone reductase. Natural oxoaldehyde reductase enzymes are preferably isolated from yeast cells using routine protein extraction procedures as set forth in Example 1 below, or from animal or plant sources. Preferred as natural oxoaldehyde reductase sources are yeast cells, including beer or barley yeasts, for example, of the genus Saccharomyces, most preferably Saccharomyces cerevisiae species. The oxoaldehyde reductase enzyme isolated from natural sources can be purified by protein purification techniques that are routine for those skilled in the art. Preferably, the enzymes are purified by a combination of "salt displacement" and chromatographic purification such as liquid chromatography.

HPLC, FPLC, affinity chromatography, ion exchange chromatography, size exclusion chromatography, and immunoaffinity chromatography. Most preferably, the purified enzymes are purified by a combination of ammonium sulfate precipitation and HPLC purification or FPLC. These purified oxoaldehyde reductase enzymes can then be added to the product, in the flavor stabilizing amounts t as described above, to improve the flavor stability of the fermented malt beverage. In an alternative embodiment, the crude oxoaldehyde reductase preparations can be added to the product without purification. Crude preparations encompassed by this embodiment of the invention include extracts or digestion products of natural yeast, animal or vegetable sources. Preferable is an enzyme digestion product or extract from natural or genetically modified yeast cells (as described below), which include cells of the genus Saccharomyces, and most preferably Saccharomyces cerevisiae species. The methods for preparing these extracts and enzymatic digestion products are well described in the microbiological literature (see for example Difco Manual, Difco, Inc., Norwood, Massachusetts). In another alternative embodiment, sources (such as yeast) capable of producing oxoaldehyde reductase enzymes may be added per se in an amount sufficient to produce an effective amount of the oxoaldehyde reductase in situ to stabilize the taste of the finished product. These sources can also be used to prepare a crude preparation, preferably an enzymatic digest or extract, comprising improved amounts of an oxoaldehyde reductase enzyme, which is then used as described above to stabilize the taste of the beverage of fermented malt. Preferably, yeasts of the genus Saccharomyces and in a more preferential form of the species Saccharomyces cerevisiae, are used in this modality. In yet another embodiment, yeast cells, preferably of the genus Saccharomyces, and in the most preferred form of the species Saccharomyces cerevisiae, are genetically modified to produce improved quantities of oxoaldehyde reductase relative to their wild-type or parent strains. Methods for genetically modifying yeast cells and other microorganisms are well known and routine to those skilled in the art (see, for example, Watson, J. D., And collaborators in: Recombinant DNA, 2nd Ed., New York : Scientific American Books, pp. 235-253 (1992)). These genetically modified yeasts provide an easily adequate source of reductase (such as a crude or purified preparation) that will be added during the fermentation process. In the alternative, as in the previous embodiments, the genetically modified yeast having the improved expression of oxoaldehyde reductase may be added per se in an amount sufficient to provide in situ flavor stabilization in the finished malt product. If added per se, the yeast cells capable of producing oxoaldehyde reductase can be immobilized in a solid phase carrier, at a density sufficient to provide sufficient enzyme support to substantially stabilize the taste of the finished fermented malt beverage. The carrier is important in terms of providing an adequate environment for the growth of the yeast and contact with the aqueous substrate. The materials used to provide a carrier may include, for example, alginate beads (which provide a gel-like carrier), latex particles, or DEAE-cellulose granules. The yeast cells can be immobilized in a carrier and cultured according to any means known in the art (see for example, U.S. Patent No. 5,079,011). Optimal amounts of the oxoaldehyde reductase, necessary to stabilize the taste of the finished malt product were determined using the analytical methods set forth in the following examples. According to these methods, the optimum concentration varies for the enzymes of oxalyaldehyde rebluctase in the finished malt beverage are about 5-500 units / ml, preferably about 10-250 units / ml, more preferably about 25-100 units / ml and in the most preferred form of approximately 50 units / ml. As used herein an enzyme unit is defined as the amount of enzyme that catalyzes the oxidation of 1 micro-mol of NADPH per minute at 25 C. It should be noted that while these ranges are described in terms of an oxoaldehyde reductase-dependent of NADPHin particular, the methods of the present invention contemplate the addition of other flavor stabilizing proteins, simultaneously, sequentially, or by the individual injection of two or more premixed components. Having now described the present invention in detail, it will be more clearly understood by reference to the following examples, which are included therein for purposes of illustration only and are not intended to be limiting of the invention.

Examples Materials and Methods. The following materials and methods were generally used in the Examples.

Organoleptic Test. The organoleptic test was designed to give an indication of the stability of beer in the bottle, as determined by the subjective methods (for example, "taste test"). In this approach, filtered filtered beer, treated with enzymes, is packed in standard 275 ml bottles, and the samples are subjected to an alternating cooling cycle (0 ° C for 24 hours) and heating (40 ° C for 24 hours) ). The flavor of the beer is then evaluated organoleptically by experienced tasters. A control sample of the beer not treated with enzyme is cycled at the previous temperature at the same time to provide a standard. The taste indices of these treated and untreated beers are then compared to determine the improved t stability achieved by treating the beer with an oxoaldehyde reductase enzyme. The results of this organoleptic test are then compared to those obtained by the chromatographic measurements of the chemical flavor indices described below.

Analysis of CL18 and 5-HMF Analyzes of LC18 and 5-HMF chemical indices were performed by liquid chromatography, using a Waters system HPLC consisting of a 600 pump, a Wisp autosampler 717, a handler of Millennium Chromatography 2010 of 2.1.

The separation was carried out on an Aminex HPX-87H 300 x 7.8 mm, 9 μm column maintained at 55 ° C. The elution was inspected with a Waters photodiode array detector 991 (200 mm-300 mm) and the quantification of the maximum values of 5-HMF and LC18 was carried out at 283 mm. For the analysis, 50 μl of degassed beer was injected into duplicate samples and eluted with 0.05 M H2SO4 for 25 minutes at a flow rate of 1.0 ml / min. Quantification of 5-HMF was performed using an external calibration curve of the respective, pure compounds. { Sigma, San Luis, Missouri).

Analysis of CE3 All beer samples were degassed in an ultrasonic bath before injection and analyzed in duplicate. Analyzes were performed in a capillary electrophoresis system of applied biosystems 270A-HT. A fused, untreated silica capillary with an internal diameter of 50 μm and 72 cm in length (50 cm for the detector) was used in all separations. The samples were injected under vacuum for 3.5 seconds, and the electrophoretic separations were carried out in the 20 mM sodium citrate buffer, pH 2.5, at a voltage of 15 KV for 20 minutes. Detection was performed at 200 nm. The acquisition of a data processing was performed using the programming elements of the data analysis system model 600 (Applied Biosystems) for the Macintosh.

Derivation of dicarbonyl compounds with 1, 2-f enilendiamine and determination of quinoxalines by HPLC A fixed volume (2.2 ml) of 5% phenylenediamine (OPD) in methanol was added to a beer bottle (222 ml) which was then returned to cover and kept at 20 ° C for 12 hours. After 12 hours of reaction, 25 ml of sample were extracted with chloroform (3 x 8 ml). The organic phase of chloroform was removed by centrifugation at 3000 rpm for 10 minutes, collected, washed with 0.1 M HCl (3 x 8 ml), in order to remove the unreacted 1,2-phenylenediamine, and semi-dried with magnesium sulfate. The semi-dried organic phase, which contained the hydrophobic, quinoxaline derivatives, was then dried in a rotary evaporator to dryness, and the residue was redispersed in 250 μl of acetonitrile, and diluted 1/10 (50% of the solvent a and 50% of the solvent b) before the chromatographic analysis. The hydrophobic quinoxalines were analyzed using method II (see below). The aqueous phase containing the hydrophilic quinoxalines was directly injected and analyzed using a method II (see below). The chromatographic conditions were as follows: i a Nova-Pak C18 column (Waters) of 3.9 x 150 mm, 4 μm was used. The mobile phase was: solvent A-95% water (Milli-Q) and 5% acetonitrile; solvent B-90% acetonitrile and 10% water; flow rate 0.7 ml / min. The elution was inspected with a Water 991 photodiode array detector (200 nm-360 nm). Typical results are shown in Figures 5 and 6 Methods I and II are as follows.

Method I Method II Example 1: Purification of NADPH-dependent 2-oxoaldehyde reductase from the beer evacuation Brewing yeasts (Polar, Caracas, Venezuela) for placement were washed twice with 25 mM potassium phosphate buffer pH 7.5, (buffer A), dispersed in the same buffer and broke with glass beads (diameter 0.5 mm) in a Disintegrator-S (IMA) at 3000 rpm for 10 minutes. The cell homogenates were centrifuged at 1000 xg for 40 minutes, and the supernatant (cytosolic fraction) was used for the purification of NADPH-dependent oxoaldehyde reductase activities. The enzymes were purified by column chromatographs, successive in an FPLC system (Pharmacia), as summarized in Figure 7. All procedures were carried out at 5 ° C. The cytospic fraction i was applied to a DEAE-Sepharose column previously equilibrated with buffer A. The column was first washed with the same buffer and then with buffer A containing 250 mM and 500 mM KCl, and the amount of the enzyme was eluted as two maximum values (peaks) The first maximum value (reductase 1) was eluted with the wash buffer, and the second (reductase 2) was eluted with the buffer containing 25 mM KCl. Both fractions were combined separately and precipitated by the addition of ammonium sulfate. Reductase 1 was precipitated with ammonium sulfate to give 50% saturation, the mixture was stirred for 30 minutes at 5 ° C and then centrifuged for 20 minutes at 4360 xg. The resulting supernatant was brought to a saturation of 90% ammonium sulfate, stirred for 30 minutes and centrifuged for 20 minutes at 4360 xg. The reductase 2 was precipitated with ammonium sulfate to give an 80% saturation and was continued as described above. The pellets obtained after this centrifugation were re-dispersed, separately, in a minimum amount of 5 mM potassium phosphate, pH 6.5 (buffer B) and dialysed overnight against the same buffer. The fractions of the dialysed enzymes were applied separately in identical CM-Sephadex columns previously equilibrated with buffer B. In both cases, the reductase activity did not interact with the resin and the proteins were eluted with the equilibrium buffer. The fractions with reductase activity were combined and concentrated by ultrafiltration with an Amico'n YM-10 membrane. The fractions of the combined enzymes were then separately absorbed onto identical Cibacron Blue columns previously equilibrated with 25 M potassium phosphate, pH 7.0 (shock absorber C). Reductase 1 was eluted with the buffer containing 400 mM KCl, while reductase 2 was eluted with a linear gradient of 0-1 M KCl in buffer C. Fractions showing reductase activity were combined separately and concentrated to a volume of 2 ml as previously described, and then applied to a Sephacryl S-300 column equilibrated with C-buffer. The reductase 1 was further purified by means of a blue Cibacron column equilibrated with C-buffer. The column was washed with the same buffer and proteins were eluted with buffer C containing 500 mM KCl. As a final purification step, both enzyme preparations (reductase 1 and reductase 2) were subjected to an inverted, preparative phase column (Pharmacia Biotech Resource RPC 1 ml) connected to an HPLC system plus module 1 Waters LC. The elution of the proteins was inspected by measuring the absorbance at 215 nm. The purified enzymes were freeze-dried and stored at -70 ° C. The activities of oxoaldehyde reductase enzymes, isolated and purified, were determined in a mixture containing 9 mM methylglyoxal, NADPH, 0.1 mM, 20 mM potassium phosphate buffer (pH 7.0), and the fraction enzyme (8 μg). approximately) in a total volume of 0.5 m The reaction was inspected at 340 nm. All tests were carried out at 25 ° C. One unit of the enzyme was defined as the amount of the enzyme that catalyzes the oxidation 1 μmol of NADPH per minute at 25 ° C.

Example 2: Characterization of reductase.

The chromatographic fractions of Example 1 that showed enzymatic activity were used for the estimation of the molequal weight by both gel filtration chromatography and gel electrophoresis with 12.5% polyacrylamide containing sodium dodecylsulfate (SDS-PAGE) as described by Weber and Osborn (J. Biol. Chem. 244: 4406-4412 (1969)). The protein was determined by the method of Lowy et al. (J. Biol. Chem. 193: 265-275 (1951)); using some of bovine serum as a norm. The gel filtration, analytical HPLC was performed on a Sephacryl S-200 column (Waters), which was equilibrated and eluted with C buffer. Both enzymes were eluted as individual maximum values; The molecular weight of inactive reductase 1, as determined by this method, was shown to be 8 kDa. However, the SDS-PAGE analysis of reductase 1 showed two major molecular weight bands of 44 and 47 kDa (Figure 8), while a single band of 39.5 kDa was seen for reductase 2 (Figure 9).

Example 3: Tests of substrate specificity Several carbonyl compounds were evaluated as substrates for oxoaldehyde reductase enzymes, isolated and purified. As shown in Figure 10, both reductase 1 and reductase 2 act on the 2-oxoaldehydes such as methylglyoxal and 3-deoxyglucosone. Reductase 1 showed a higher activity of reductase 2 in the compounds with an individual keto or aldo group such as acetaldehyde and pyridine-3-aldehyde. It was found that glucoronate is a better substrate for reductase 2 than for reductase 1, while metyrapone was an acceptable substrate for both enzymes. Both reductases i showed little or no effect on the aldosas valued (Glucose, galactose and xylose) It is notable that no enzyme showed any appreciable activity in pyruvate These results show that reductase 1 and reductase 2 are chemically and kinetically distinguishable.

Example 4: Effect of reductases in LC18 In order to determine the effect of both reductases on the intensity of the LC18 maximum value, at 1 ml of the flask beer mixture, a mixture of 1 ml of fresh beer, potassium phosphate buffer was incubated at 25 ° C for 30 minutes. 25 mM (pH 7.0), 0.1 mM NADPH and the required volume of enzyme to obtain 50 units / ml. After incubation, the treated beer was analyzed on an Aminex HPX-87H column connected to a Waters HPLC system under the conditions described above. As shown in Figure 11, the treatment of beer with Reductase 1 or Reductase 2 caused a significant decrease in the area of the LC18 maximum value (arrows), relative to that in an untreated beer. The treatment of beer with Reductase 1 induced a greater decrease in the maximum value of LC18 than did the treatment with Reductase 2, perhaps reflecting the higher specific activity of the former for several substrates of keto- and aldo-carbonyl, Individuals as shown in Figure 10. These results demonstrate that treatment with either Reductase 1 or Reductase 2, and preferably with »Reductase 2, can reduce the formation of rancid taste indices such as LC18 in fresh beer .

Example 5: Taste evaluation For sensitive taste evaluations of beer, a panel of six experienced tasters was used. Each participant was asked to compare the flavor profiles and determine the presence or absence of flavor components, associated with the freshness degree of the beer in the following samples: 1) fresh beer at 5 ° C; 2) control beer at 28 ° C; and 3) beer with reductases 1 added at 28 ° C. The scale used to report the freshness of the beer was from "1" to "5" (with "5" indicating the freshest taste). Beers were prepared as follows: 1) Beers of control: 10 ml of beer were taken from each of six bottles of 222 ml of fresh beer, pasteurized under a current of C02. This volume was replaced with 6 ml of buffer C and 4 ml of 3 mM NADPH, and then the bottles were recapped. Three bottles were stored at 5 ° C for 15 days and the others at 28 ° C for 15 days. 2) Experimental beers: 10 ml of beer were taken from each of three bottles of 222 ml of fresh beer, pasteurized under a stream of C02, and this volume was replaced with 5.4 ml of buffer A, 4 ml of NADPH 3 mM, and 0.6 ml of reductase 1. The bottles were recapped and stored at 28 ° C for 15 days. The fresh, control and experimental beers were then subjected to the evaluation by the panel of tasters. As shown in Figure 12, these taste evaluation tests showed a significant increase in the degree of freshness in the beers containing reductase 1, compared to the control beers at 28 ° C.

Together with those for the previous chromatographic test, these results indicate that the treatment of beer with reductase 1 stabilizes the taste of the beer. Having now fully described the present invention, it will be understood by those skilled in the art that it may be performed within a broad range and equivalent of conditions, formulations and other parameters without affecting the scope of the invention or any modality thereof. . All publications, patents and patent applications cited herein are indicative of the level of those skilled in the art to which the invention pertains, and are hereby incorporated by reference in their entirety.

It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (21)

1. A method for improving the flavor stability of a fermented malt beverage, characterized in that it comprises contacting the beverage with a flavor stabilizing amount of an oxoaldehyde reductase enzyme.

2. A genetically modified yeast cell, characterized in that it produces improved amounts of an oxoaldehyde reductase enzyme as compared to the cells of the wild-type yeast strain, parent.

3. A composition comprising an extract or enzymatic digestive product of the yeast cell of claim 2, the composition is characterized in that it comprises an oxoaldehyde reductase enzyme which affectively stabilizes the taste of a fermented malt beverage.

4. An oxoaldehyde reductase enzyme, purified, characterized in that the enzyme affectively stabilizes the taste of a fermented malt beverage.

5. A method for improving the taste of a fermented malt beverage, characterized in that it comprises contacting the beverage with a flavor stabilizing amount of the enzymatic digestion product or extract of claim 3.

6. A method for improving the taste of a fermented malt beverage, characterized in that it comprises contacting the beverage with a purified flavor stabilizing amount of the purified oxoaldehyde reductase enzyme of claim 4.

7. The method according to any of claims 1, 5 or 6, characterized in that the fermented malt beverage is beer.

8. The method according to claim 1 or claim 6, characterized in that the enzyme is an NADPH-dependent enzyme.

9. The method according to claim 8, characterized in that the enzyme is 3-deoxyglucosone reductase.

10. The method in accordance with the claim 9, characterized in that the enzyme is produced by yeast. i

11. The method according to claim 10, characterized in that the yeast is of the genus Saccharomyces.

12. The method according to claim 11, characterized in that the yeast is of the species Saccharomyces cerevisiae.

13. The method according to claim 10, characterized in that the yeast has been genetically modified to allow the improved production of an oxoaldehyde reductase enzyme which effectively stabilizes the taste of a fermented malt beverage.

14. A fermented malt beverage, characterized in that the flavor has been stabilized by the method of any of claims 1, 5 or 6.

15. The fermented malt beverage according to claim 14, characterized in that the beverage is beer.

16. A method for producing a fermented malt beverage, the method is characterized in that it comprises the steps of producing a grain malt, producing a wort from the grain malt, adding a stabilizing amount of the flavor of an oxoaldehyde reductase enzyme, fermenting the must to produce a fermented malt beverage, process the fermented malt beverage to produce a fermented malt beverage, processed, and package the high, fermented, processed beverage.

17. The method according to claim 16, characterized in that the enzyme is added to the must before the fermentation step.

18. The method in accordance with the claim 16, characterized in that the enzyme is added to the fermented malt beverage before the processing step.

19. The method according to claim 16, characterized in that the enzyme is added to the fermented malt beverage, processed before the packaging step.

20. A fermented malt beverage produced by the method of claim 16.

21. A fermented malt beverage according to claim 20, characterized in that the beverage is beer. SUMMARY OF THE INVENTION The present invention relates to a method for stabilizing the flavor of a fermented malt beverage, more particularly a beer, by the addition of an oxoaldehyde reductase enzyme, the fermented malt beverage prepared by this method, and the use during the fermentation process of oxoaldehyde reductase enzymes from natural sources, which include those produced by yeast, to stabilize the flavor of the resulting beer product and to produce a beer that has a stable flavor. The invention also relates to microorganisms, particularly yeasts, that have been specifically modified, selected, or genetically engineered to express or secrete an oxoaldehyde reductase enzyme that can be used during the fermentation process to stabilize the enzyme. flavor of the resulting beer product, and to produce a beer that has a stable flavor. The present invention also provides extracts from a natural source (eg, yeast), or a modified yeast, or extracts thereof, which will provide a sufficient amount of the enzymes necessary to block, inhibit or reduce the intermediate compounds of the invention. the Maillard reaction (e.g., 3-deoxyglucosone, which results in the formation of rancid flavor in fermented malt beverages.In addition, the present invention provides fermented malt beverage having improved flavor stability produced by these methods.