Enzymatic improvement of beverage mellowness

Abstract

A method for producing a consumable alcoholic product comprises using one or more enzymes to reduce or eliminate an oral pain response that would otherwise be experienced upon consumption of the product. The method comprises combining at least one oxidase with a fermentate and, optionally, distilling the fermentate to produce a consumable alcoholic product, such as distilled alcohol. The method comprises combining at least one oxidase, including an aldehyde dehydrogenase.

Description

[Technical Field]

[0001] The present disclosure is directed to the field of consumable beverages and systems and methods for their production. Implementations include methods for producing improved consumable beverages by adding at least one dehydrogenase enzyme to the beverage during or after production. [Background technology]

[0002] Many consumer beverages, while typically accompanied by an appealing flavor, have an unpleasant harshness quality that is easily detectable and overwhelming to the consumer, who may consequently cocktail or consume the beverage with salt, lime, and the like to mask the harshness. For example, distilled alcoholic beverages have a pronounced harsh taste or "bite" that, upon ingestion, evokes a mild painful reaction in the consumer's mouth / throat / oral cavity. Keenly aware of this problem, alcohol manufacturers have attempted to remove the harsh note from distilled alcoholic beverages by implementing one or more mellowing or smoothing techniques during or after the distillation process; some examples of the latter include aging the distilled product, dripping the distilled product through a carbon filter, and / or adding flavors to the distilled product to mask the harshness. While these techniques may mitigate the harshness level to some extent, they do not eliminate or reduce the harshness level to an undetectable level. These methods are also energy intensive and time consuming.

[0003] Astringency is not limited to alcohol, but is commonly detected during the consumption of a wide range of beverages, including various non-distilled alcohols, coffee, and tea.

[0004] Therefore, new technologies are needed to minimize and even eliminate the unpleasant astringent taste of a wide range of consumed beverages and beverage ingredients. Summary of the Invention

[0005] Cross-reference to related applications This application claims priority from U.S. Provisional Patent Application No. 63 / 386,431, filed December 7, 2022, entitled "Enzymatic Improvement of Beverage Mellowness," the contents of which are incorporated by reference in their entirety into this disclosure.

[0006] Sequence Listing This disclosure further incorporates by reference the Sequence Listing submitted herewith. The Sequence Listing xml file identified as P305897WO02 is 24,440 bytes in size and was created on November 23, 2023. The Sequence Listing submitted electronically herewith does not transcend the scope of the specification and does not contain any new matter.

[0007] The present disclosure describes devices, systems, and related methods for producing improved consumable beverages by adding at least one dehydrogenase enzyme to a beverage or beverage ingredient during production. According to embodiments described herein, a method for producing a distilled alcoholic beverage may include creating a mash mix (a substrate that provides fermentable sugars for yeast), fermenting the mash mix to produce a fermentate, combining the fermentate with at least one dehydrogenase, and distilling the fermentate to produce distilled ethanol, which is adapted to form or be included in a distilled alcoholic beverage.

[0008] In some examples, the method further includes mixing at least one cofactor with the fermentate. In some examples, the at least one cofactor may include a dinucleotide cofactor, and the dinucleotide cofactor may include NAD+ and / or NADP. In some examples, the cofactor is added at a loading level of about 1 mg / L of fermentation volume to about 2 g / L of fermentation volume. In some examples, the dehydrogenase includes an aldehyde dehydrogenase, which may be a native aldehyde dehydrogenase or an engineered dehydrogenase. The aldehyde dehydrogenase may selectively oxidize aliphatic aldehydes, and the aliphatic aldehydes may include C2 to C10 aliphatic aldehydes. In some examples, the distilled alcoholic beverage may have a lower aliphatic aldehyde concentration than the level of aliphatic aldehydes present in a distilled alcoholic beverage produced without mixing a dehydrogenase with the fermentate. In some examples, the level of aliphatic aldehydes present in a distilled alcoholic beverage produced without incorporating at least one dehydrogenase into the fermentate is in a range of ppm to ppb. In some examples, the aldehyde dehydrogenase may selectively oxidize saturated and unsaturated aldehydes with an electrophilic index that matches the nucleophilic index of cysteine ​​in the TRPA1 receptor.

[0009] In some examples, the method further includes adjusting the pH of the fermentate to about 5.0 to about 7.0. In some examples, the pH of the fermentate does not need to be adjusted. In some examples, the dehydrogenase is added at a loading level of about 1 mg / L to about 2 g / L of the fermentation volume. In some examples, the method also includes aging the distilled ethanol in a barrel. In some examples, the mash mix is ​​fermented for about to about 5 days. In some examples, the mash mix includes one or more additional organic ingredients: corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, or one or more additional organic ingredients for yeast. In some examples, the ethanol concentration of the distilled ethanol is about 20% to about 95%. In some examples, the ethanol concentration of the fermentate is about 1% to about 20%.

[0010] According to embodiments described herein, a system for producing distilled alcoholic beverages includes a mash tun configured to agitate and heat a mash mix, a fermentation tank configured to ferment the mash mix to produce a fermentate, at least one dehydrogenase configured for mixing with the fermentate, a still apparatus configured to heat the fermentate to produce a fermentate vapor, a condenser configured to cool the fermentate vapor to produce distilled ethanol, and a recovery apparatus for recovering the distilled ethanol.

[0011] In some examples, the system further includes a barrel configured to receive and store the distilled ethanol following an aging process, although in some examples, the distillate is not aged and is therefore immediately bottled. In some examples, the system also includes at least one cofactor configured for mixing with the fermentate. In some examples, the at least one cofactor includes a dinucleotide cofactor, such as NAD+ or NADP. The dehydrogenase may include an aldehyde dehydrogenase, which may be native or engineered and may selectively oxidize aliphatic aldehydes, such as C2 to C10 aliphatic aldehydes. The system may also include at least one base configured to increase the pH of the fermentate. In some examples, the mash mix ingredients include one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, or one or more additional organic ingredients for yeast. In some examples, the system further includes one or both of a grinder device or a press device configured to grind or press one or more of the ingredients of the mash mix.

[0012] According to embodiments of the present disclosure, the fermentate produced according to the method for producing a distilled alcoholic beverage may include a mash mix including one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, a sugar source, and sucrose. The fermentate may also include at least one aldehyde dehydrogenase and ethanol at a concentration of about 1% to about 20%.

[0013] In some examples, the fermentate further comprises a dinucleotide cofactor. The aldehyde dehydrogenase may be a native or engineered aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase is present at a concentration of about 1 mg / L to about 2 g / L of the fermentate. In some examples, the dinucleotide cofactor may be present at a concentration of about 1 mg / L of the fermentation volume to about 2 g / L of the fermentate.

[0014] According to embodiments of the present disclosure, a method of producing a distilled alcoholic beverage includes creating a mash mix, fermenting the mash mix to produce a fermentate, distilling the fermentate to produce distilled ethanol, and combining the distilled ethanol with at least one dehydrogenase to create the distilled alcoholic beverage.

[0015] In some examples, the method further includes mixing at least one cofactor with the distilled ethanol. The cofactor may include a dinucleotide cofactor such as NAD+. In some examples, the dehydrogenase includes an aldehyde dehydrogenase, which may be native or engineered. The aldehyde dehydrogenase may selectively oxidize aliphatic aldehydes, such as C2-C10 aliphatic aldehydes. In some examples, the distilled alcoholic beverage has a lower level of aliphatic aldehydes compared to the level of aliphatic aldehydes present in a distilled alcoholic beverage made without mixing at least one dehydrogenase with the distilled ethanol. In some examples, the level of aliphatic aldehydes present in a distilled alcoholic beverage made without mixing at least one dehydrogenase with the distilled ethanol is within a range ranging from ppm to ppb. In some examples, mixing the dehydrogenase with the distilled ethanol occurs in the barrel or bottle after distillation. In some examples, the mash mix includes one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, or organic ingredients for yeast. In some examples, the distilled ethanol has an ethanol concentration of about 20% to about 95%.

[0016] According to embodiments described herein, a method of producing a consumable alcoholic product may include creating a mash mix, fermenting the mash mix to produce a fermentate, combining the fermentate with at least one oxidase, and recovering the fermentate for inclusion in a consumable alcoholic product.

[0017] In some examples, the at least one oxidase comprises or consists of an aldehyde dehydrogenase. In some embodiments, the aldehyde dehydrogenase may be a native aldehyde dehydrogenase or may comprise a native aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase may be an engineered or modified aldehyde dehydrogenase or may comprise an engineered or altered aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase preferentially oxidizes aliphatic aldehydes. In some examples, the aliphatic aldehydes may comprise C2 to C10 aliphatic aldehydes. In some examples, the consumed alcoholic product may have a reduced level of aliphatic aldehydes compared to the level of aliphatic aldehydes present in a consumed alcoholic product made without incorporating at least one dehydrogenase into the fermentate. In some instances, the level of aliphatic aldehydes present in consumable alcoholic products made without incorporating at least one oxidase into the fermentate is in a range ranging from ppm to ppb.

[0018] In some examples, the method may further include mixing the fermentate with at least one dinucleotide cofactor. In some examples, the dinucleotide cofactor may be NAD+, NADP+, or both, or may include NAD+, NADP+, or both. In some examples, the method may further include distilling the fermentate to produce distilled ethanol, which is prepared to form or be included in a consumable alcoholic product. In some examples, the method may further include aging the distilled ethanol in a barrel. In some examples, the distilled ethanol may have an ethanol concentration of about 20% to about 95%. In some examples, the consumable alcoholic product may be or include beer. In some examples, the method may further include adjusting the pH of the fermentate to about 5.0 to about 7.0. In some examples, the pH of the fermentate may not be adjusted. In some examples, the at least one oxidase may be added at a loading level of about 5 mg / L to about 2 g / L of fermentation volume. In some examples, fermenting the mash mix may include fermenting the mash mix for about 5 days. In some examples, the mash mix includes one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, a sugar source, and sucrose. In some embodiments, the ethanol concentration of the fermentate may be about 1% to about 20%. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a graph showing the activity levels of five different aldehyde dehydrogenases in the presence of acetaldehyde, according to certain embodiments of the present disclosure. [Figure 2] FIG. 2 is a graph showing the activity levels of the aldehyde dehydrogenase shown in FIG. 1 in the presence of nonanal, according to an embodiment of the present disclosure. [Figure 3] FIG. 3 is a graph showing the activity levels of aldehyde dehydrogenase in the presence of acetaldehyde and nonanal at various ethanol concentrations, according to certain embodiments of the present disclosure. [Figure 4] FIG. 4 is a graph showing aldehyde dehydrogenase activity levels determined based on the amount of octanoic acid produced at various ethanol concentrations, according to certain embodiments of the present disclosure. [Figure 5] FIG. 5 is a graph showing aldehyde dehydrogenase activity levels determined based on the amount of nonanoic acid produced at various ethanol concentrations, according to certain embodiments of the present disclosure. [Figure 6] FIG. 6 is a graph showing the activity level of aldehyde dehydrogenase determined based on the amount of decanoic acid produced at various ethanol concentrations, according to certain embodiments of the present disclosure. [Figure 7] FIG. 7 is a Western blot showing soluble and insoluble levels of four of the aldehyde dehydrogenases, according to an embodiment of the present disclosure. [Figure 8] FIG. 8 is a graph showing activity levels of the four aldehyde dehydrogenases depicted in FIG. 7 at a range of pH values ​​in 15% ethanol, according to an embodiment of the present disclosure. [Figure 9] FIG. 9 is a graph showing the relative activity levels of two of the aldehyde dehydrogenases depicted in FIG. 8 at a range of pH values ​​in 15% ethanol, according to an embodiment of the present disclosure. [Figure 10] FIG. 10 is a graph showing activity levels of two aldehyde dehydrogenases with associated cofactors in the presence of n-nonanal, according to an embodiment of the present disclosure. [Figure 11] FIG. 11 is a set of three graphs jointly showing the activity levels of two aldehyde dehydrogenases in the presence of octanal, nonanal, and dodecanal at 30° C. and 37° C. after 4 hours, according to an embodiment of the present disclosure. [Figure 12]FIG. 12 is a set of three graphs jointly showing the activity levels of two aldehyde dehydrogenases in the presence of octanal, nonanal, and dodecanal at 30° C. and 37° C. after 21 hours, according to an embodiment of the present disclosure. [Figure 13] FIG. 13 is a mass spectrometry graph showing analyte abundance in 40% ethanol after treatment with one of the aldehyde dehydrogenases, both with and without cofactors, according to an embodiment of the present disclosure. [Figure 14] FIG. 14 is a mass spectrometry graph showing analyte abundance in head samples after treatment with one of the aldehyde dehydrogenases, both with and without cofactors, according to an embodiment of the present disclosure. [Figure 15] FIG. 15 is a mass spectrometry graph showing analyte abundance in a fermentation sample after treatment with one of the aldehyde dehydrogenases, both with and without cofactors, according to an embodiment of the present disclosure. [Figure 16] FIG. 16 is a graph showing the substrate catalytic activity of various modified and native aldehyde dehydrogenases. DETAILED DESCRIPTION OF THE INVENTION

[0020] <Definition> As used in this disclosure, the term "beverage" encompasses a variety of consumer products, non-limiting examples of which may include distilled alcoholic beverages (e.g., vodka, whiskey, rum, brandy, gin, tequila, white liquor, spirits, cocktails, etc.), non-distilled alcoholic beverages (e.g., beer, wine, cider, sake, hard seltzer, etc.), coffee, and tea. Ethanol, which is not consumed alone but is used as a flavoring solvent, may also be processed according to the principles described in this disclosure. This disclosure primarily describes distilled alcoholic beverages and related production methods, but this is for ease of explanation only.

[0021] In the context, the term "beverage" may also be used to refer to a final, ready-to-consume beverage product (e.g., a distilled alcoholic beverage), a beverage component (e.g., ethanol), or an intermediate composition formed during beverage production (e.g., a ferment, which itself may be defined as a product resulting from the fermentation of organic ingredients).

[0022] As used in this disclosure, the term "dehydrogenase" may refer to one or more dehydrogenases, which may be or include aldehyde dehydrogenases. The term may also refer to the broader classes of dehydrogenases, oxidases, and oxidoreductases, which may include various enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism, and detoxification. Substrates for these enzymes may include glucose, steroids, advanced glycation end products, lipid peroxidation products, and / or environmental pollutants. In some instances, the term "enzyme" may be synonymous with dehydrogenases, such as aldehyde dehydrogenases, and may refer to enzyme compositions, which may include two or more enzymes and / or non-enzymatic components. In some instances, the term "enzyme" may include oxidases that selectively act on specific compounds produced by lipid peroxidation resulting from oxidative stress on yeast during the alcohol production process.

[0023] As used in this disclosure, "harshness" may refer to an unpleasant, strong bite of a beverage that evokes a mildly painful reaction in a person's mouth and throat upon ingestion of the beverage. Harshness, in some embodiments, may not refer to the burn / warmth of ethanol or to the negative flavor itself, such as aromas, compositions, or substances sometimes referred to as congeners or fusel oils.

[0024] As used in this disclosure, "user" may refer to a recipient, manufacturer, or handler of the enzyme compositions disclosed in this disclosure. User may also refer to a person who performs or manages a beverage production process involving the methods described in this disclosure.

[0025] The singular forms of various terms include the plural forms of the same terms. For example, the term "enzyme" should be understood to also include the terms "enzymes" and "enzyme systems."

[0026] In the alcoholic beverage industry, conventional wisdom teaches that improving distillation technology provides the optimal approach to enhancing beverage smoothness. For example, distillation, barrel aging, filtration, carbon filtration, and / or supplementation with additional masking flavors cover many of the existing approaches to combating harshness. The approach described in this disclosure defies conventional wisdom and embodies substantial improvements over existing technologies. Conventional technologies lack the sensitivity and specificity necessary to eliminate previously unrecognized harshness-causing compounds present in consumed beverages, often at very low concentrations.

[0027] The appeal of alcoholic beverages, especially distilled varieties, has been diminished by the harsh taste and harsh "bite" commonly associated with their consumption. From the lowest quality products to the finest, most expensive bottles, this negative sensory experience is not completely eliminated even by the most meticulous manufacturing techniques. The present disclosure is based on the previously undiscovered discovery of the exact compounds that produce the harsh bite sensation in the consumer, and a new method used to address those compounds and produce a fully smooth spirit without the harsh taste typically found in spirits or other consumable beverages that have not been treated according to the methods disclosed in this disclosure. To date, the only methods used to attempt to reduce harshness have been distillation, filtration, or aging, which have been largely ineffective. Additionally, in the limited cases where enzymes have been used in the production of certain alcoholic beverages, such cases have been limited to unique compositions and methods that can only increase the content of fermentable sugars or reduce the liquid viscosity. Such a narrow application would not result in a reduction in beverage harshness as contemplated herein. The harsh bite sensory experience associated with alcohol consumption, the pain receptors involved in that experience, and the unpleasant compounds produced during fermentation that are agonists for these specific pain receptors have never been linked. Consequently, the use of one or more enzymes described herein, e.g., dehydrogenase enzymes, to selectively target these unpleasant compounds without affecting other flavors or aromas in the consumed beverage has never been attempted.

[0028] As humans, we have three categories of sensory experiences: the experience of flavor (taste), the olfactory experience of smell perception, and touch, which is primarily responsible for sensations such as pain. The food and beverage industry has focused heavily on the first two, but not the last. The compositions, methods, and system embodiments described in this disclosure address this last one. In the production of alcoholic or distilled alcoholic beverages, a mash (a recipe of organic matter that yeast will use as a sugar source for fermentation) is created to be fermented by yeast. As the fermentation progresses and the ethanol concentration increases, yeast tends to experience oxidative stress. This stress leads to lipid peroxidation, ultimately resulting in the unpleasant compounds that are discovered and addressed in accordance with embodiments disclosed in this disclosure. Because the source of these irritating compounds is lipid peroxidation of yeast cell membranes, they are present independently of the type of alcoholic beverage being produced. In response to these compounds, humans will experience a mild pain response in the form of the strong, pungent taste discussed in this disclosure; however, these compounds are unpleasant, electrophilic, and often aldehydes. The present disclosure provides compositions, methods, and systems that effectively target and eliminate these irritants that contribute to the unpleasant harsh taste associated with consumed alcohol, thereby creating truly smoother alcoholic beverages, including, among other things, beverages typically categorized as premium beverages, as demonstrated by analytical and sensory testing disclosed in this disclosure.

[0029] One embodiment involves the use of aldehyde dehydrogenase to highly specifically target products of lipid peroxidation in yeast cell membranes, which occurs when yeast undergoes oxidative stress toward the end of alcoholic fermentation. This lipid peroxidation produces a series of compounds, many of which are aliphatic aldehydes, such as malondialdehyde or nonenal, that are well-known triggers of the TRPA1 receptor (transient receptor potential cation channel, subfamily A, member 1, or ankyrin 1). TRPA1 is not a flavor receptor, but a pain receptor responsible for the intense "bite" sensation, as further explained below. Astringency is not a savored flavor, but rather a mild pain sensation in response to the presence of unpleasant compounds, such as aliphatic saturated or unsaturated aldehydes. These compounds are eliminated by the enzymatic oxidation of carbonyl groups to carboxylic acids, weak organic acids, described in the present disclosure, which are often precursors for the formation of esters desirable in distilled alcoholic beverages. The approach described in this disclosure does not alter the flavor profile of an alcoholic beverage, but rather removes the harsh taste of the beverage, thereby creating a truly mellow beverage.

[0030] Given the vast variety of aldehydes that exist, not all aldehydes cause the pain sensation experienced. Many contribute to desirable flavors (e.g., cinnamaldehyde), some contribute to off-flavors (e.g., certain aldoses), and some are toxic. Toxicity depends on the electrophilicity and steric hindrance of the aldehyde. The TRPA1 receptor is designed to detect and respond only to aldehydes with specific toxic electrophilicities that adversely affect living cells. The embodiments disclosed in the present disclosure may act specifically on these toxic, unpleasant aldehydes, leaving desirable aldehyde species intact.

[0031] Disclosed embodiments The present disclosure provides methods, systems, and related reagents, ingredients, and devices for eliminating, minimizing, or reducing the level of harshness detected by consumers when consuming various consumable beverages. Non-limiting examples of such consumable beverages include distilled alcoholic beverages. Some embodiments may include adding at least one natural or modified enzyme to a beverage or its components during or after its production. The enzyme may include one or more dehydrogenases, such as at least one aldehyde dehydrogenase. In some embodiments featuring aldehyde dehydrogenases, the dehydrogenase may be added before, during, and / or after the initiation of a fermentation process and / or after the distillation process in embodiments related to the production of distilled alcoholic beverages. The dehydrogenase may oxidize to reduce the total content of one or more aldehydes present in the beverage, including aldehydes present at very low concentrations. Specifically targeted aldehydes may include aliphatic electrophilic aldehydes, such as C2-C10 aliphatic aldehydes. The dehydrogenase enzyme may not act on aldehydes that are responsible for flavor perceived by humans; the dehydrogenase enzyme may act selectively only on aldehydes identified in this disclosure as irritants that cause a harsh bite and pain response in consumers. Thus, by reducing the amount of one or more of these specific aldehydes, the harsh taste of the final beverage product may be eliminated or reduced to a level that is undetectable or substantially undetectable to most consumers, while maintaining the intended flavor of the product.

[0032] Enzyme composition The enzyme(s) used to reduce or eliminate the harsh taste typical of many consumed beverages may include at least one dehydrogenase, such as at least one aldehyde dehydrogenase. The enzyme(s) may selectively catalyze the oxidation of one or more electrophilic compounds identified by the present inventors as causative or contributing factors to the harsh taste of beverages, without also targeting compounds that contribute to the desirable flavor or quality of the final beverage product, even if both types of compounds belong to the same chemical class. For example, the methods disclosed herein may not affect aldehydes, which are positive flavors, due to steric hindrance. Some embodiments may selectively target one or more carbonyl groups, such as aldehydes, for oxidation, thereby converting the targeted compounds to carboxylic acids. The compounds targeted by the enzyme(s) may be present in very low concentrations in the beverage or beverage components. Thus, the enzyme(s) described herein may exhibit high levels of specificity and sensitivity. For ease of explanation, the singular term "enzyme" will be primarily used hereinafter, but it should be understood that "enzyme" can refer to one or more enzymes or enzyme systems.

[0033] In some embodiments, the enzyme may be substantially native (or wild-type) in amino acid composition, protein conformation, and activity. A native enzyme exhibiting the substrate specificity and sensitivity necessary to remove astringency from distilled alcoholic beverages was discovered after extensive testing of 15 different aldehyde dehydrogenases for their activity under conditions typical of commercial alcoholic fermentation and distillation. While one native enzyme demonstrated the most significant overall astringency reduction, additional dehydrogenases selected from the original 15 tested enzymes were also effective, including all 15 enzymes used individually or in combination in the alcohol production process. This indicates that aldehyde dehydrogenases, as a class of enzymes, can effectively reduce the astringency associated with the consumption of alcoholic beverages when used according to the methods described in this disclosure. The amino acid sequences of the enzymes evaluated for this effect correspond to SEQ ID NOs: 1 through 17. In some embodiments, the enzyme used in accordance with the methods of effectively reducing the harsh taste of beverages described herein may be approximately 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NO:1 through SEQ ID NO:17.

[0034] Additionally or alternatively, some embodiments may involve artificially modified or engineered enzymes that may differ from naturally occurring enzymes in amino acid composition, protein conformation, and / or activity. Some embodiments may involve enzyme compositions and systems that include a mixture of one or more naturally occurring and / or engineered enzymes. Depending on the specific fermentation conditions and the relative concentrations of the specific compounds being targeted, the naturally occurring and engineered enzyme species may be included in separate compositions that are used simultaneously during the beverage production process. In some embodiments, the engineered enzymes used in accordance with the methods disclosed herein for reducing harshness in beverages may be about 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOS: 1-17, or may be about 80%, 85%, 90%, 95%, 99%, or 100% identical to other naturally occurring dehydrogenases.

[0035] The form of the native enzyme that exhibited the most significant astringency reduction in the experiments described below (identified as 54.1 kDa Enzyme 2) has an amino acid sequence corresponding to SEQ ID NO: 6. One or more additional enzymes effective in significantly reducing the astringency of beverages using the techniques described herein may be about 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to SEQ ID NO: 6. The additional native enzyme that exhibited significant astringency reduction in the experiments described below (identified as 54.0 kDa Enzyme 3) has an amino acid sequence corresponding to SEQ ID NO: 8. One or more additional native enzymes that exhibit effective astringency reduction when used according to the methods described herein may be about 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to SEQ ID NO: 8. Although the native enzyme identified as Enzyme 2 in the experiments described below may exhibit greater pH tolerance than the native enzyme identified as Enzyme 3, both enzymes are effective astringency reducers responsible for a significant reduction in the harsh bite of potable alcohol and alcoholic components. The remaining enzymes corresponding to SEQ ID NOS: 1-5, 7, and 9-15 (which include the enzymes identified as Enzymes 1 and 4-15) subjected to the test conditions described below were also substantially or equally effective, due at least in part to their substantial homology with each other and with Enzymes 2 and 3, as well as with other aldehyde dehydrogenases. This will be understood by those skilled in the art to be due, at least in part, to the enzymatic activity common to all aldehyde dehydrogenases, which is characterized by the oxidation of an aldehyde to a carboxylic acid. Thus, while the experiments detailed in this disclosure evaluated a subset of enzymes, enzymes effective for driving astringency reduction according to embodiments described in this disclosure may also include others, such as all or substantially all dehydrogenases, including aldehyde dehydrogenases, and the approach described in this disclosure is therefore not limited to the specific enzymes tested in the experiments described below.

[0036] Naturally occurring enzymes used in accordance with embodiments described herein may have a mass in the range of about 40 kDa to about 60 kDa, including masses of up to about 41 kDa, up to about 42 kDa, up to about 43 kDa, up to about 44 kDa, up to about 45 kDa, up to about 46 kDa, up to about 47 kDa, up to about 48 kDa, up to about 49 kDa, up to about 50 kDa, up to about 51 kDa, up to about 52 kDa, up to about 53 kDa, up to about 54 kDa, up to about 55 kDa, up to about 56 kDa, up to about 57 kDa, up to about 58 kDa, up to about 59 kDa, or more. Embodiments of the native enzyme may also have a mass in the range of about 53.5 kDa to about 57 kDa, including masses up to about 54.0 kDa, about 54.5 kDa, about 55.0 kDa, about 55.5 kDa, about 56.0 kDa, about 56.5 kDa, or greater. Specific embodiments of dehydrogenases may have a mass of about 54.1 kDa, about 57.5 kDa, or about 56.7 kDa.

[0037] The different properties exhibited by native and modified enzymes may vary. For example, a modified enzyme may exhibit normal activity levels under, for example, reduced pH and / or elevated temperature and / or elevated ethanol concentrations, while a native enzyme may exhibit reduced activity under the same conditions. In some embodiments, a native enzyme, including one having an amino acid sequence substantially similar or identical to one or more of SEQ ID NOS: 1-17, may exhibit substantially the same effective activity level as a modified enzyme. Different enzymes may also exhibit greater or less enzymatic activity under various conditions. Therefore, beverage production parameters may be adjusted depending on which enzyme is used.

[0038] The modified enzyme may maintain its activity under typical fermentation conditions, such as at a pH level of about 3.0 to 6.0, at a temperature of about 15°C to about 37°C, and at about 0 to 20% ethanol, where the ethanol concentration is, for example, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or more, for example, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, or up to about 95% ethanol, or any level therebetween, for enzyme addition after distillation. In some examples, the native enzyme may maintain a generally stable level of activity at elevated ethanol concentrations, including up to about 10% ethanol, up to about 11% ethanol, up to about 12% ethanol, up to about 13% ethanol, up to about 14% ethanol, up to about 15% ethanol, up to about 16% ethanol, up to about 17% ethanol, up to about 18% ethanol, up to about 19% ethanol, or greater.

[0039] The activity of a native enzyme at lower pH levels may, in some instances, be lower than the activity of the modified enzyme. For example, a native enzyme may exhibit detectable activity at a pH level of 3.5 to 7.0, but optimal activity may be achieved at a pH level of about 6.0 to about 7.0. Thus, while a native enzyme may exhibit sufficient activity at a pH level below about 6.0, a fermentation method utilizing a native enzyme may include increasing the pH level of the fermentate to 6.0 or above 6.0, e.g., to 6.7. In some embodiments, the enzymatic activity of one or more native enzymes may be substantially the same as the enzymatic activity of one or more modified enzymes at a lower pH level of about 3.5 to about 7.0, where non-limiting examples of such native enzymes include enzymes having any one of SEQ ID NOS: 1-17.

[0040] Enzyme compositions can be dry or liquid. Enzyme composition embodiments include, for example, spray-dried or freeze-dried powders, or liquid solutions consisting of powdered enzyme suspended in water and / or one or more buffers. In some instances, the enzyme composition, with or without added cofactors, may be immobilized on a solid substrate or structure. The pure enzyme content of the enzyme composition can vary greatly depending on the final form of the composition. For enzyme compositions in powder form, the enzymatic activity may be greater than about 2 units / mg protein, less than about 5 units / mg protein, less than about 10 units / mg protein, less than about 15 units / mg protein, less than about 20 units / mg protein, less than about 25 units / mg protein, less than about 30 units / mg protein, less than about 35 units / mg protein, less than about 40 units / mg protein, less than about 45 units / mg protein, less than about 50 units / mg protein, or any level greater or therebetween.

[0041] The enzymes may be provided to the user in a variety of forms. In some examples, the enzymes may be provided in one or more vials, containers, or tubs. Embodiments also include single-use add-packs containing the amount of enzyme needed for one round of a batch beverage production process. Similar add-packs may also be provided for one-time addition to a continuous beverage production process. Thus, multiple packs may be used for a continuous production process in which individual packs are added at regular intervals throughout production, including, for example, during or after fermentation, including after distillation.

[0042] As noted above, enzymes may exhibit high levels of substrate specificity and sensitivity. In addition to ethanol, distilled alcoholic beverages typically contain trace amounts of hundreds of organic molecules, including, for example, organic acids, other alcohols, ketones, esters, aldehydes, organic fats, and proteins, the large number and diversity of which significantly complicates any attempt to identify and effectively target only those compounds responsible for producing the harsh taste of a beverage. In some instances, enzymes may effectively reduce or even eliminate one or more harsh-causing aldehyde substrates present in a beverage or beverage component (e.g., a fermented product) at concentrations of only 500 ppm or less, e.g., about 5 ppm to about 10 ppm, or even less, e.g., on the order of ppb.

[0043] Without being bound by any particular theory, the pain response induced in a person upon consuming certain astringent beverages may be due to activation of the trigeminal nerve, which is triggered by activation of specific molecular receptors, such as TRPA1. TRPA1 is reactive to more than one compound and, due to the nature of its mechanism of action, cannot be blocked using masking agents or taste blockers. This makes it difficult to neutralize. As provided herein, electrophilic aliphatic compounds present in astringent beverages have been found to be the primary drivers of TRPA1 activation. Aliphatic molecules with reactive carbonyl groups, such as aldehydes, can form reversible covalent bonds with one or more cysteine ​​residues present in the active site of TRPA1, thereby inducing the pain response caused by TRPA1 activation. In particular, aliphatic molecules with α,β unsaturated bonds may drive the most pronounced pain response. These compounds can cause significant trigeminal activation even at very low levels, e.g., on the order of ppm to ppb, which cannot be eliminated even by the most sophisticated distillation techniques. The present discovery that such low substrate levels can be effectively targeted and enzymatically eliminated is surprising, especially in the context of distilled alcohol production, which requires implementing various process parameters not traditionally conductive to enzyme function.

[0044] In the context of alcohol production, increased ethanol production (e.g., during fermentation) creates environmental stresses that affect the yeast lipid bilayer. Oxidative stress leads to an increase in reactive oxygen species, which drives lipid peroxidation and the corresponding production of harmful aldehydes, including hydroxynonenal, malondialdehyde, and acrolein, each of which, when inoculated into humans, triggers the TRPA1 pain receptor.

[0045] The use of the enzymes described herein in the methods and systems described herein can prevent covalent bonding between cysteine ​​residues and reactive carbonyl groups in TRPA1 by catalyzing the conversion of aliphatic compounds (single or multiple) to carboxylic acids. Through this mechanism, the methods described herein can effectively reduce or eliminate the concentration of pain-inducing compounds present in various beverage products at very small but noticeable concentrations. In some examples, the enzyme(s) can catalyze the oxidation of multiple different substrates, such as multiple types of carbonyl electrophiles, including, but not limited to, crotonaldehyde, octanal (e.g., n-octanal), nonanal (e.g., n-nonanal), dodecanal (e.g., n-dodecanal), and / or acetaldehyde. In some instances, the oxidation of acetaldehyde may be catalyzed by the enzymes described herein, although its reduction, unlike the aforementioned aliphatic aldehydes, particularly the C2-C10 species thereof, may have little to no effect on the perceived astringency level.

[0046] Notably, the targeted enzyme-driven conversion of aldehydes to carboxylic acids can also result in the formation of ester precursors that may improve the taste and / or smoothness of the final beverage product in a manner previously achieved only through aging.

[0047] How to reduce the bitter taste of beverages The method described herein for reducing beverage astringency includes adding at least one enzyme described herein to a consumed beverage or a component thereof during the beverage production process. As described above, the enzymes described herein are configured to catalyze the oxidation-reduction reaction of aldehydes that contribute to the astringency of various beverages within the temperature, pH, and concentration ranges typical of alcoholic beverages, e.g., distilled alcoholic beverages. To reduce the concentration of aliphatic aldehydes and thereby reduce the overall astringency of the final distilled product, one or more enzymes may be added to one or more intermediate compositions formed during the alcohol production process, such as fermentation or distillation, or to a composition formed after distillation. Embodiments directed to the production of alcoholic beverages may generally include mash formation, fermentation, optional distillation, and optional aging. The specific subprocesses performed may depend on the final alcoholic product. For example, embodiments directed to beer production do not include distillation, unlike embodiments directed to whiskey or vodka production.

[0048] Making mash The mash used for fermentation may contain various ingredients in various amounts and concentrations. The ingredients of the mash can vary based on the type of beverage being produced, such as wine, beer, vodka, gin, whiskey, bourbon, baijiu, rum, or any other consumable alcoholic beverage. Non-limiting examples of mash ingredients include grains (e.g., corn, rye, rice, barley, wheat), agave, dextrin, potato, fruit (e.g., grapes), molasses, water, one or more enzymes, one or more organic feedstocks for yeast, and / or additional sugars (e.g., sucrose), sugar equivalents, or sugar sources having a sugar concentration of about 0% to 100% by weight. source), where the sugar concentration ranges from less than 1% to 5%, up to about 10%, up to about 15%, up to about 20%, up to about 25%, up to about 30%, up to about 35%, up to about 40%, up to about 45%, up to about 50%, up to about 55%, up to about 60%, up to about 65%, up to about 70%, up to about 75%, up to about 80%, up to about 85%, up to about 90%, up to about 95% or more by weight, or any concentration therebetween.

[0049] The amount of each mash ingredient can vary. For example, corn-based embodiments may include from about 60% corn by weight to about 65% corn by weight, to about 70% corn by weight, to about 75% corn by weight, to about 80% corn by weight, to about 85% corn by weight, to about 90% corn by weight, or any amount therebetween. Rye or rye malt-based embodiments may include from about 10% rye malt by weight to about 12% rye malt by weight, to about 14% rye malt by weight, to about 16% rye malt by weight, to about 18% rye malt by weight, to about 20% rye malt by weight, to about 22% rye malt by weight, to about 24% rye malt by weight, to about 26% rye malt by weight, to about 28% rye malt by weight, or any amount therebetween. Embodiments based on barley or barley malt may include from about 2% by weight barley malt to about 4% by weight barley malt, to about 6% by weight barley malt, to about 8% by weight barley malt, to about 10% by weight barley malt, to about 12% by weight barley malt, to about 14% by weight barley malt, to about 16% by weight barley malt, to about 18% by weight barley malt, to about 20% by weight barley malt, or any amount therebetween.

[0050] One or more components of the mash may be pressed and / or milled before or after mixing with one or more other mash ingredients. Roller and / or hammer mills may be used to grind mash ingredients, such as grains. After one or more pre-treatment steps, the mash ingredients may be added to a vessel, such as a mash tun, where the ingredients may be agitated and heated.

[0051] ·fermentation The processed mash mix may be fermented through yeast-driven conversion of sugars present in the mash to alcohol. Fermentation may occur in large fermentation tanks or vessels into which the mash mix may be deposited. In certain embodiments, yeast may be mixed with the mash mix (filtered or unfiltered), and fermentation may occur for up to about 15 days, or until about 1% to about 20% ethanol is produced. The final ethanol concentration of the fermentate may be from about 1% to about 20%, or any percentage therebetween, such as up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, up to about 15%, up to about 16%, up to about 17%, up to about 18%, up to about 19%, or more.

[0052] The production of compounds that cause or contribute to the harsh taste of beverages, such as C2-C10 aliphatic aldehydes, can occur regardless of the fermentation organism or sugar source used to produce ethanol. The enzyme(s) described in this disclosure may be added at various loading levels before, during, or after the initiation of fermentation to minimize or eliminate such compounds. For example, aldehyde dehydrogenase may be added at a concentration of about 1 mg / L to about 2 g / L relative to the fermentation volume, or any amount therebetween, and examples of this amount include about 5 mg / L to about 2 g / L, about 50 mg / L, about 100 mg / L, about 150 mg / L, about 200 mg / L, about 250 mg / L, about 300 mg / L, about 350 mg / L, about 400 mg / L, about 450 mg / L, about 500 mg / L, about 550 mg / L, about 600 mg / L, about 650 mg / L, about 700 mg / L, about 750 mg / L, about 800 mg / L, about 850 mg / L, about 900 mg / L, about 950 mg / L, or more.

[0053] The timing of enzyme addition can be any time between before or during fermentation, or a shorter time thereafter, to just prior to distillation, such as during mash creation, during yeast addition, or after partial fermentation, such as to about 5 to about 10% completion, to about 20% completion, to about 30% completion, to about 40% completion, to about 50% completion, to about 60% completion, to about 70% completion, to about 80% completion, to about 90% completion, to about 95% completion, or to about 100% completion of a target total alcohol production described herein (e.g., up to about 20% ethanol). Alternatively, the enzymes described herein may be added during fermentation with residence times ranging from about 5 minutes to several days, including residence times of, for example, less than about 6 hours to about 6 hours, to about 12 hours, to about 36 hours, to about 48 hours, to about 60 hours, to about 72 hours, or longer. Enzyme addition may also occur after distillation and before or after bottling. Embodiments may also include adding the enzyme(s) before fermentation. Additional embodiments may include adding the enzyme(s) to the distilled ethanol in the aging barrel along with any components desired for the production of a particular beverage.

[0054] In some instances, one or more dinucleotide cofactors, such as NAD+ and / or NADP+, may also be added during the fermentation process at any of the aforementioned points or production stages. The cofactor(s) may be added at concentrations ranging from about 0 mg / L to about 1 mg / L of fermentation volume, or up to about 2 g / L of fermentation volume, or any amount therebetween, including about 25 mg / L, 50 mg / L, about 100 mg / L, about 150 mg / L, about 200 mg / L, about 250 mg / L, about 300 mg / L, about 350 mg / L, about 400 mg / L, about 450 mg / L, about 500 mg / L, about 550 mg / L, about 600 mg / L, about 650 mg / L, about 700 mg / L, about 750 mg / L, about 800 mg / L, about 850 mg / L, about 900 mg / L, about 950 mg / L, or more. Embodiments featuring the use of both aldehyde dehydrogenase and a cofactor may enhance or optimize the reduction of astringency. The combination of aldehyde dehydrogenase with, for example, NAD+ may enhance or maximize the reduction of astringency-causing compounds. In some embodiments, no exogenous cofactors may be added, and the astringency reduction / elimination effect may still be achieved equally. Thus, the methods described herein may be performed by adding the enzyme with or without cofactors. In some examples, the combination of aldehyde dehydrogenase (e.g., Enzyme 2) with a cofactor containing NAD+ may achieve optimal astringency reduction compared to other naturally occurring enzymes when added to the fermentation with or without NAD+. The combination of aldehyde dehydrogenase (e.g., Enzyme 3) with a cofactor containing NADP+ may also exhibit significant astringency reduction compared to other naturally occurring enzymes. Cofactor addition may not be necessary in certain embodiments, such as those involving the use of modified enzymes or those involving the use of enzymes having amino acid sequences that are about 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to at least one naturally occurring enzyme, such as enzymes 2 and / or 3 having amino acid sequences substantially similar or identical to SEQ ID NO:6 and SEQ ID NO:8, respectively.

[0055] In some examples, the pH of the fermentate may be adjusted. For example, a basic compound, such as calcium carbonate, may be added at one or more points during fermentation to increase the pH of the fermentate to a range of about 5.0 to about 7.0, particularly when native enzymes are used. Certain embodiments may include increasing the pH to about 6.0 to about 7.0, e.g., about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9. In some examples, pH adjustment may not be necessary, for example, when modified enzymes are used or when native enzymes have activity within the required range. According to these embodiments, the pH of the fermentate may be about 3.0 to 6.0.

[0056] The temperature of the fermentation may also vary, and in some instances may be from about 15°C (59°F) to about 37°C (98.6°F), or any temperature therebetween, such as about 17°C (62.6°F), 19°C (66.2°F), 21°C (69.8°F), 23°C (73.4°F), 25°C (77°F), 27°C (80.6°F), 29°C (84.2°F), 31°C (87.8°F), 33°C (91.4°F), 35°C (95°F), or higher. The temperature may be maintained at a constant or substantially constant level throughout or for most of the fermentation cycle. In some embodiments, the temperature may be changed one or more times throughout the course of the fermentation. In such embodiments, the temperature may be maintained within the range of about 15°C (59°F) to about 37°C (98.6°F) for at least about 50% up to about 100% of the fermentation time, or up to about 55% of the fermentation time, up to about 60% of the fermentation time, up to about 65% of the fermentation time, up to about 70% of the fermentation time, up to about 75% of the fermentation time, up to about 80% of the fermentation time, up to about 85% of the fermentation time, up to about 90% of the fermentation time, up to about 95% of the fermentation time, or longer.

[0057] Fermentation may be carried out under a variety of pressures, in some embodiments, fermentation may be carried out at from about 5 psi to about 20 psi, or up to about 6 psi, up to about 7 psi, up to about 8 psi, up to about 9 psi, up to about 10 psi, up to about 11 psi, up to about 12 psi, up to about 13 psi, up to about 14 psi, up to about 15 psi, up to about 16 psi, up to about 17 psi, up to about 18 psi, up to about 19 psi, or more, or any pressure therebetween.

[0058] The resulting mixture may be fermented in the presence of baker's yeast (Saccharomyces cerevisiae) for up to about 10 days. In some embodiments, enzymes may be added to the mixture and remain active therein for at least about 5 minutes to at least about 5 days, or any time therebetween or longer, for example, up to about 1 day, up to about 2 days, up to about 3 days, up to about 4 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, or more. After fermentation, the fermentate may have an ethanol content of about 1% to about 20%, or any concentration therebetween, including, for example, an ethanol content of about 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or more.

[0059] ·distillation Once fermentation is complete, a distillation process may be carried out, for example, to obtain an ethanol concentration approximately equal to or exceeding 10% alcohol by volume (ABV). In some embodiments, the fermentate (filtered or unfiltered) may be heated in a batch or continuous manner along the desired distillation process (with the exception of non-distilled beverages (e.g., beer, wine, etc.), which may be subjected to one or more post-fermentation processes that do not involve distillation). Heating of the fermentate may be carried out in a distiller such as a pot still. Heating may evaporate ethanol present in the fermentate, thereby separating the ethanol from unwanted grain particles, liquid, etc., present in the fermentate. The ethanol vapor may then be condensed by cooling in a condenser and subsequently collected in a collector. In some instances, the distiller, condenser, and / or collector may be included in a single unitary apparatus, which in some instances may be referred to as a still.

[0060] The fermented liquid may be distilled to an ethanol concentration of about 20% to about 95%. The dehydrogenase may not pass through the distillation but remain in the portion of the distilled liquid that is not recovered as a consumable alcoholic beverage.

[0061] ·Bottling / Aging The distilled ethanol may be diluted to any desired concentration and then bottled or poured into wooden barrels for further aging. After the desired aging period is complete, the liquid in the barrel may be adjusted to reach the desired ethanol concentration and then bottled for distribution. In some embodiments, one or more dehydrogenases may be added to the distilled ethanol after bottling and / or kegging so that the astringent aldehyde content is reduced directly in the bottle / keg prior to consumption. In such embodiments, the dehydrogenase(s) may be added at various loading concentrations of about 5 mg / L to about 2 g / L of the distilled alcohol volume, or any amount therebetween, including about 50 mg / L, about 100 mg / L, about 150 mg / L, about 200 mg / L, about 250 mg / L, about 300 mg / L, about 350 mg / L, about 400 mg / L, about 450 mg / L, about 500 mg / L, about 550 mg / L, about 600 mg / L, about 650 mg / L, about 700 mg / L, about 750 mg / L, about 800 mg / L, about 850 mg / L, about 900 mg / L, about 950 mg / L, or more.

[0062] The final ethanol concentration of the distilled alcoholic beverage may be from about 10% to about 70%, or may be about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or higher. In some examples, the ethanol concentration may be diluted for later consumption to reduce the final concentration to less than 10%, such as about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or any concentration therebetween.

[0063] As described, the enzyme(s) described in this disclosure may be added at any time from before or after fermentation, or shortly thereafter, to just before distillation, with residence times within the ranges described herein, and / or may include adding the enzyme(s) to the distilled ethanol in the aging barrel along with any components desired to produce a particular beverage. Some embodiments may also include adding the enzyme(s) before fermentation. Moreover, the discovery of adding an enzyme to the fermentate or distillate during the beverage production process is surprising, given the traditional difficulties associated with pinpointing effective mechanisms for delivering the enzyme(s).

[0064] ●Beverage products Beverages produced by the foregoing methods may have reduced aldehyde content compared to beverages that have been similarly formulated and processed except for the enzymatic treatment described herein, including the type of enzyme(s), timing of enzyme addition, and concentration of enzyme addition, all of which may be important for the effective oxidation of carbonyl groups present on target compounds. Embodiments of the beverages may particularly have reduced amounts of aliphatic electrophilic aldehydes, which may include or comprise C2-C10 aliphatic aldehydes, compared to beverages that have not been treated with enzyme(s) according to the techniques described herein. [Example]

[0065] ●Experimental example A series of experiments were conducted to demonstrate the effectiveness of aldehyde dehydrogenases in effectively oxidizing astringent compounds under conditions commonly applied during the production of consumable alcoholic products. Additional experiments were conducted to confirm the effectiveness of aldehyde dehydrogenases in effectively oxidizing astringent compounds during the production of consumable alcoholic products. Taste tests were also conducted to confirm the reduction in astringency perceived by users upon consumption of consumable alcoholic products produced by practicing the methods disclosed in this disclosure. As described below, certain specific aldehyde dehydrogenases were selected for initial evaluation and subsequent testing for exemplary purposes only. Therefore, it should be understood that the astringency reduction effects disclosed in this disclosure are not limited to those achieved with the specific enzymes tested in the following experiments, which comprise a subset of enzymes tested to demonstrate the effective oxidation of astringent compounds under conditions commonly applied during distilled alcohol production, as well as the readily observable phenomenon of resulting beverage astringency. Enzymes at least similar to or identical to any one of SEQ ID NOs: 1-17, including all or substantially all aldehyde dehydrogenases, may be effective under conditions similar to those applied below.

[0066] ●Experiment 1 - Screening and activity evaluation of aldehyde dehydrogenase Experiment 1 was conducted to demonstrate that dehydrogenase enzymes from various sources can effectively target compounds responsible for harsh tastes. Specifically, spectrophotometric measurements were performed to determine the activity of 15 different aldehyde dehydrogenases (corresponding to SEQ ID NOS: 1-15) in the presence of various aldehyde substrates. The source organisms of the enzymes varied, indicating that different enzymes from different sources may be effective in achieving the results disclosed in this disclosure. For example, aldehyde dehydrogenase from humans was tested, as well as enzymes from Bacillus, E. coli, cynomolgus monkey (macaca fascicularis), garnet galago (otolemur garnettii), black flying fox (Pteropus alecto), Geobacillus, Parageobacillus caldoxylosilyticus, Citrobacter rodentium, Shigella boydii, Klebsiella pneumonia, and Acinetobacter.

[0067] Because aldehyde dehydrogenase activity is NAD(P)+ dependent, enzyme activity was measured by the conversion of NADP+ to NAD(P)H. Because NAD(P)H absorbs light at 340 nm, whereas NAD(P)+ does not, NAD(P)H production was monitored spectrophotometrically at 340 nm for 10 min in the presence of equimolar amounts of NAD(P)+ and five different aldehyde substrates, including acetaldehyde, crotonaldehyde, n-octanal, n-nonanal, and n-dodecanal, which were provided at 5 mM (220-292 ppm) and 0.5 mM (22-92 ppm), respectively, in separate samples.

[0068] Since all aldehyde dehydrogenases act on aldehydes, the experimental goal was to select the most active one under specific conditions. Of the 15 enzymes, five that showed the highest activity for all substrates at 500 μM (identified as enzymes 1–5, corresponding to SEQ ID NOS: 5, 6, 8, 14, and 15, respectively) were subjected to further evaluation. This evaluation began with assessing the enzymes' tolerance to ethanol concentrations typically present during fermentation and distillation processes. First, each enzyme was screened against 5 mM acetaldehyde and 5 mM n-nonanal at ethanol concentrations ranging from 5% v / v to 50% v / v ethanol. As shown in Figures 1 and 2, respectively, the activity of each enzyme remained relatively stable in the presence of acetaldehyde and n-nonanal up to approximately 20–30% v / v ethanol, but activity levels generally decreased at these ethanol concentrations. This demonstrates that numerous aldehyde dehydrogenases, even from distinct source organisms, can effectively act and catalyze the aldehydes responsible for the harsh bite associated with alcohol. Additional dehydrogenases, including but not limited to the 15 species tested in this experiment, may also effectively act and catalyze the aldehydes responsible for the harsh taste typically associated with consumed beverages and their components.

[0069] To further elucidate the ethanol tolerance of the enzymes and identify any background enzyme activity, the activity of each enzyme was also assessed in 5% v / v and 50% v / v ethanol with and without available substrates (acetaldehyde and nonanal). The results, shown graphically in Figure 3, indicate that in the absence of substrate, each enzyme exhibited activity in 50% v / v ethanol greater than or equal to 5% v / v. However, the results also demonstrate that in the presence of acetaldehyde and nonanal, each enzyme exhibited increased activity compared to the activity measured in the absence of the aldehyde substrate.

[0070] The activity of each enzyme in the presence of low substrate concentrations was further evaluated by gas chromatography. To learn more about the enzymes' ethanol tolerance, each enzyme was also tested in the presence of 10 ppm n-octanal, n-nonanal, and n-dodecanal in 20% v / v ethanol. Based on the amount of octanoic acid produced, shown graphically in Figure 4, all enzymes showed activity in the presence of 10 ppm or less of octanal. As shown in Figure 5, all enzymes also showed activity in the presence of 5 ppm and 10 ppm nonanal, with enzyme 2 again maintaining high activity levels at each substrate concentration in both 5% v / v and 20% v / v ethanol. The results, shown in Figure 6, indicate that all enzymes showed appreciable activity in both 5 ppm and 10 ppm dodecanal in both 5% v / v and 20% v / v ethanol. Enzyme 2 showed the highest activity at both 5 ppm in 5% v / v ethanol and 10 ppm in 20% v / v ethanol.

[0071] Spectrophotometric and gas chromatographic screening indicated that enzyme 2 exhibited strong ethanol tolerance, evidenced by the absence of a drop in activity at 20% v / v ethanol. Enzyme 2 also exhibited high activity levels in the presence of only 5-10 ppm of various aldehyde substrates. Collectively, these results indicate that one or more of the enzymes evaluated, including all of the enzymes, can function effectively under conditions relevant to alcoholic beverage production, e.g., distilled alcohol production, and enzyme 2 exhibited the highest activity in the particular experiments performed.

[0072] ● Experiment 2 - Characterization of aldehyde dehydrogenase Experiment 2 was conducted to select specific enzymes for further testing in craft distilleries to demonstrate a positive sensory impact in removing beverage astringency. Along with a negative control, enzyme powders were made for Enzymes 1-4 (corresponding to SEQ ID NOS: 5, 6, 8, and 14, respectively), and the relative amounts of soluble versus insoluble powder were determined. As shown in the Western blots in Figure 7, Enzyme 2 (54.1 kDa) and Enzyme 3 (54.0 kDa) produced the highest relative levels of soluble protein. Based on the higher relative amounts of soluble protein present in the powders, Enzymes 2 and 3 may function most effectively when added as powders to substantially liquid fermentation and / or distillate samples.

[0073] To evaluate the enzymes' function at elevated alcohol contents and reduced pH levels often encountered during fermentation, the activity levels of enzymes 1-4 were evaluated by the above-described NAD(P)H spectrophotometric measurements at pH levels ranging from 3.5 to 7.5 in 10% v / v ethanol and 15% v / v ethanol. Figure 8 shows that enzymes 1 and 4 exhibited lower overall activity across all pH values ​​in 15% v / v ethanol compared with enzymes 2 and 3, which were the most stable enzymes at lower pH values. Figure 9 shows that enzyme 2 was more stable than enzyme 3 at most pH levels in 15% v / v ethanol, especially at pH values ​​of 3.5 and 4.0.

[0074] Enzyme 2 and Enzyme 3 were evaluated for their cofactor preference for NAD+ and NADP+ using NAD(P)H spectrophotometric measurements. As shown in Figure 10, both enzymes exhibited activity with both cofactors, with Enzyme 2 exhibiting approximately 1.5-fold greater activity than Enzyme 3 with NAD+ and Enzyme 3 exhibiting approximately 1.2-fold greater activity with NADP+. Thus, although both Enzyme 2 and Enzyme 3 can be effective in the absence of cofactors such as NAD+ and NADP+, the activity of Enzyme 2 can be most significantly enhanced in the presence of NAD+, and the activity of Enzyme 3 can be most significantly enhanced in the presence of NADP+.

[0075] Enzymes 2 and 3 were further evaluated head-to-head, but this time in the presence of 100 ppm octanal, nonanal, or dodecanal in 15% ethanol at pH 5.5 at both 30°C and 37°C. Each enzyme was tested at concentrations ranging from 0.001 g / L to 11 g / L of enzyme powder (1:100,000 to 1:10 molar ratios of enzyme to aldehyde). Activity levels of each enzyme were measured at 1, 4, and 21 hours. NAD+ was used as a cofactor for Enzyme 2, and NADP+ was used as a cofactor for Enzyme 3. Reactions were quenched, extracted with ethyl acetate, and analyzed by gas chromatography. The percent substrate conversion was determined by dividing the product peak area by the sum of the product and substrate peak areas.

[0076] As shown in Figure 11, Enzyme 2 exhibited higher activity levels compared to Enzyme 3 under all test conditions at 4 hours. Activity levels generally increased with increasing enzyme loading up to approximately 0.1 g / L enzyme, after which enzyme activity remained relatively constant. The highest activity level for Enzyme 2 was measured at 15% v / v ethanol, pH 5.5. Figure 12 shows the activity levels of both enzymes at 21 hours. As shown, activity levels for the enzymes were similar at 21 hours compared to 4 hours. For both enzymes, activity levels were higher at 30°C than at 37°C.

[0077] Thus, aldehyde dehydrogenases can effectively reduce the harsh taste of consumed beverages and beverage components, including one or more enzymes having an amino acid sequence similar to or identical to any one of SEQ ID NOS: 1-17, such as enzyme 2 corresponding to SEQ ID NO: 6. The foregoing results further confirm that one or more of the evaluated enzymes, including all of the enzymes evaluated, can function effectively under conditions relevant to alcoholic beverage production, such as distilled alcohol production, and that enzyme 2 exhibited the highest activity in the particular experiments conducted.

[0078] ●Experiment 3 - Enzyme 2 Distillation Analysis When a pot still is used to produce distilled alcoholic beverages, the first portion of the distillate is generally discarded or otherwise unrecovered (the "heads"). After the heads are discarded, the "heart" of the distillate is recovered and used to make the beverage. Finally, a portion of the distilled product consisting of off-flavors, called the "tails," is also often discarded.

[0079] In Experiment 3, the distillation head, final 40% v / v ethanol product, and post-fermentation / pre-distillation samples obtained from the distillation alcohol production process disclosed in this disclosure, with and without Enzyme 2 treatment, were characterized using a gas chromatography-mass spectrometry protocol. Specifically, for each intermediate and final product in the fermentation / distillation process, a control sample, a sample obtained after treatment with Enzyme 2, and a sample obtained after treatment with Enzyme 2 and an NAD+ cofactor were obtained. Analyte peaks were identified for each sample to determine whether Enzyme 2 reduced the presence of compounds that may cause a harsh taste at one or more stages of the distillation alcohol production process.

[0080] Figure 13 shows the analyte peaks identified in the 40% ethanol sample. The number and height of the peaks were highest in the control sample (labeled with stars), indicating greater presence and amount of analyte in the sample not treated with aldehyde dehydrogenase. The sample treated with Enzyme 2 had fewer peaks than the control (labeled with circle dots), and the sample treated with Enzyme 2 and NAD+ cofactor had the fewest peaks (labeled with squares), indicating that Enzyme 2 is effective when used with the NAD+ cofactor.

[0081] Figure 14 shows the analyte peaks identified in the head sample. The control sample had the greatest number of peaks and peak heights (labeled with stars). The sample treated with Enzyme 2 had significantly smaller peaks (labeled with circles), and the sample treated with Enzyme 2 and NAD+ cofactor also had the fewest peaks (labeled with squares). These further demonstrate that while Enzyme 2 is most effective in some instances when used with an NAD+ cofactor, Enzyme 2 alone is also effective, and this is true for any one of the aldehyde dehydrogenases that are about 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to any one of SEQ ID NOS: 1-17, or other aldehyde dehydrogenases.

[0082] Figure 15 shows the analyte peaks identified in the fermentation samples. As shown, more than five times more peaks were identified overall, indicating that more distinct analytes were present in the fermentation compared to the 40% ethanol and head samples. Enzyme 2 reduced the number of peaks relative to the control (labeled with a star) both with and without NAD+ (peaks labeled with circles and squares, respectively), further indicating that Enzyme 2 can be effective with or without cofactors in the fermentation. This is also true for any one of the aldehyde dehydrogenases that are about 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% identical to any one of SEQ ID NOS: 1-17, or other aldehyde dehydrogenases, at least due to their shared activity and homology.

[0083] Experiment 4 - First Still Trial with Enzyme 2: Immature Whisky Test The ability of Enzyme 2 (with or without cofactors) to act on various aldehydes present at very low levels over a unique range of pH and temperature was further evaluated in distillation trials to produce sample whiskeys for tasting. The trials were conducted at a commercial craft distillery. The efficient oxidation of targeted aldehydes by aldehyde dehydrogenase was determined by collecting sensory data generated from test subjects after consuming distilled alcoholic beverages treated with the enzyme.

[0084] To produce the whiskey ("White Dog" Whiskey), a mash mix consisting of a blend of corn, rye malt, barley malt, and dextrin was prepared and added to a 5.5-gallon fermenter. No acid was added. The starting temperature was 75°F, and the starting pH was 5.7. The mash was then fermented with brewer's yeast. Approximately 2.75 pounds (lbs) of dextrose equivalent were added to the fermenter approximately 24 and 48 hours after the start of fermentation. The pH of the fermentate fluctuated throughout the course of fermentation, but was measured at approximately 3.5 after 48 hours. After sequential adjustments with the addition of teaspoons of calcium carbonate, a common ingredient used by distillers to adjust pH, a final pH of 6.7 was measured. Fermentation continued until the fermentate contained approximately 15.6% ethanol.

[0085] The fermentation was then divided into three 5 L samples, one of which served as a negative control and two of which served as test samples. The control sample was not treated with Enzyme 2, the first test sample was treated with Enzyme 2 at a 1:1000 dose at 90°F and pH 6.7, and the second test sample was treated with Enzyme 2 (at a 1:1000 dose) and dinucleotide cofactor (NAD+) at 91°F and pH 6.7. The control sample was 15.6% alcohol by volume (ABV), the first test sample was approximately 12% alcohol by volume (ABV), and the second test sample was approximately 13% alcohol by volume (ABV).

[0086] Each sample was distilled at an initial pressure of about 2.5 psi and a boiling temperature of about 130° F. For a heart of 125-135 proof, the same volume percentage was collected for each sample and the final distillate was diluted to 80 proof.

[0087] Sensory testing was then conducted to determine whether the effect on astringency was readily detectable. The control sample and each test sample were tasted by 30 test subjects. Compared to the control sample, the first and second test samples showed an 85% and 100% reduction in astringency, respectively. Thus, adding aldehyde dehydrogenase to the fermentate dramatically reduced the final astringency level of the unaged whiskey, with the combination of aldehyde dehydrogenase and cofactors having a particularly pronounced effect. This data demonstrates that by combining aldehyde dehydrogenase with the fermentate, as well as by combining a combination of aldehyde dehydrogenase and cofactors with the fermentate and producing a distilled alcoholic beverage from the enzyme-treated fermentate, compounds that cause astringency, among the hundreds of compounds produced during fermentation, were eliminated or reduced to concentrations below sensory detection.

[0088] The ability of the enzyme to act on various aldehydes present in very low concentrations at the specific range of pH and temperature typically found in these types of fermentations was further evaluated by tastings by additional subjects who independently tasted the samples and reported their results separately. The effective oxidation of target aldehydes by aldehyde dehydrogenase was determined by collecting sensory data generated from test subjects after consuming distilled alcoholic beverages that were either treated or not with the enzyme.

[0089] All 30 subjects were able to not only distinguish between the treated and untreated samples, but also clearly identify which samples were treated and which were untreated, based on the overall greater astringency of the untreated samples.

[0090] Experiment 5 - Second Still Trial with Enzyme 3: Vodka Test This experiment demonstrated the ability of Enzyme 3 (corresponding to SEQ ID NO:8) to dramatically reduce astringent compounds and thereby sensory perception in vodka using the cofactor NADP. A vodka mash was prepared using a white winter wheat malt vodka mash by adding water and calcium chloride and heating to 152°F for 1 hour. After cooling to 70°F and aeration, yeast starter and yeast nutrients were added to initiate fermentation. At the end of fermentation, the pH was adjusted to 6.5 using baking soda. A portion of this post-fermentation liquid was treated with Enzyme 3 (1:1000 addition) and cofactor (NADP+) for 24 hours at 86°F before distillation. At the end of fermentation, the fermentate had an alcohol by volume (ABV) of 9.4% and proceeded to distillation. The fermentate was distilled, and a 184-proof head was collected and subsequently diluted to 80 proof for tasting.

[0091] A sensory test was then conducted with 10 people tasting the control sample versus the enzyme-treated sample. All 10 test subjects were able to clearly identify the treated sample as being at least 80% smoother and characterized by the elimination of the harsh bite experienced when consuming the control sample.

[0092] ● Experiment 6 - Test using natural enzymes and genetically modified enzymes To demonstrate that native and engineered oxidase enzymes are effective alcohol astringency reducers, a series of tests were conducted to demonstrate the efficacy of the engineered enzymes in catalyzing highly specific reactions that convert compounds responsible for astringency. As shown in Figure 16, the activity of native dehydrogenases corresponding to SEQ ID NOS: 1-17 and dehydrogenases engineered from enzymes having SEQ ID NOS: 1-17 demonstrated efficient substrate catalysis, as evidenced by the robust rates of target substrate catalysis exhibited for the native enzymes (grouped within the dashed triangle) and engineered enzymes (grouped within the dashed parallelogram). In comparison, samples not treated with any form of enzyme demonstrated little or no substrate catalysis (grouped within the dashed circle). These data further demonstrate that various aldehyde dehydrogenases, either native or engineered, can be utilized in accordance with the methods disclosed herein to catalyze substances identified as causing the harsh bite typically associated with consumed alcoholic beverages and their components.

[0093] The above description of specific embodiments is merely exemplary in nature and is not intended to limit the implementation(s) described in the present disclosure or their application or uses. Reference is made to the accompanying drawings, which form a part of this disclosure, and these drawings are shown as illustrative depictions of specific embodiments in which the systems and methods described in the present disclosure may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the systems and methods described in the present disclosure, and it should be understood that other embodiments may be utilized and structural and logical changes may be made without departing from the spirit and scope of the present system. For example, it should be recognized that any one of the examples, embodiments, or processes described in the present disclosure may be combined with one or more other examples, embodiments, and / or processes, or may be separated and / or implemented within separate devices or device parts in accordance with the present systems, devices, and methods. Additionally, for purposes of clarity, detailed descriptions of certain features have not been provided where such details would be apparent to those skilled in the art so as not to obscure the description of the present system. This detailed description is therefore not to be taken in a limiting sense, and the scope of the present system is defined only by the appended claims.

Claims

1. Making a mash mix, fermenting the mash mix to produce a fermentate; mixing at least one oxidase with the fermentate; and recovering said fermentate for inclusion in a consumable beverage; 1. A method for producing a consumable alcoholic product, comprising:

2. 10. The method of claim 1, wherein the at least one oxidase comprises an aldehyde dehydrogenase.

3. The method of claim 2 , wherein the aldehyde dehydrogenase is a naturally occurring aldehyde dehydrogenase.

4. 3. The method of claim 2, wherein the aldehyde dehydrogenase is an engineered or modified aldehyde dehydrogenase.

5. 3. The method of claim 2, wherein the aldehyde dehydrogenase selectively oxidizes aliphatic aldehydes.

6. 6. The method of claim 5, wherein the aliphatic aldehyde comprises a C2 to C10 aliphatic aldehyde.

7. 7. The method of claim 6, wherein the consumed alcoholic product has a reduced concentration of fatty aldehydes compared to the concentration of fatty aldehydes present in a consumed alcoholic product made according to the method of claim 1, but without combining the at least one dehydrogenase with the fermentate.

8. 8. The method of claim 7, wherein the concentration of the aliphatic aldehydes present in a consumable alcoholic product made according to the method of claim 1, but without mixing the at least one oxidase with the fermentate, is a concentration within a range ranging from ppm to ppb.

9. 10. The method of claim 1, further comprising mixing at least one dinucleotide cofactor with the fermentate.

10. 10. The method of claim 9, wherein the dinucleotide cofactor comprises NAD+, NADP+, or both.

11. 10. The method of claim 1, further comprising distilling the fermentate to form distilled ethanol that constitutes or is prepared for inclusion in the consumable alcoholic product.

12. 12. The method of claim 11, further comprising aging the distilled ethanol in a barrel.

13. 13. The method of claim 12, wherein the distilled ethanol has an ethanol concentration of about 20% to about 95%.

14. The method of claim 1 , wherein the consumable alcoholic product comprises beer.

15. 10. The method of claim 1, further comprising adjusting the pH of the fermentate to about 5.0 to about 7.

0.

16. The method of claim 1 , wherein the pH of the fermentate is not adjusted.

17. 10. The method of claim 1, wherein the at least one oxidase is added at a dosage concentration of about 5 mg / L to about 2 g / L of fermentation volume.

18. 10. The method of claim 1, wherein fermenting the mash mix comprises fermenting the mash mix for up to about 5 days.

19. 10. The method of claim 1, wherein the mash mix comprises one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, a sugar source, and sucrose.

20. 2. The method of claim 1, wherein the ethanol concentration of the fermentate is from about 1% to about 20%.

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