Enzymatic softening of beverages

Abstract

Methods of producing drinkable alcohol products involve the use of one or more enzymes to reduce or eliminate oral pain reactions that may be induced when drinking the product. The method comprises mixing at least one oxidase with a fermentation broth, and optionally distilling the fermentation broth to produce a drinkable alcohol product, such as a distilled liquor. The method specifically comprises mixing at least one oxidase comprising an aldehyde dehydrogenase.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 386,431, filed on December 7, 2022, entitled "Enzymatic Softening of Beverages", the entire content of which is incorporated herein by reference. Field of the Invention

[0003] The present disclosure relates to the field of potable beverages and their production systems and methods. Specific embodiments include methods for producing improved potable beverages by adding at least one dehydrogenase to the beverage during or after the beverage production process.

[0004] Sequence Listing

[0005] The present disclosure also incorporates herein by reference the Sequence Listing filed herewith. The.xml file of the Sequence Listing is identified as P305897WO02, has a size of 24,440 bytes, and was created on November 23, 2023. The Sequence Listing submitted in electronic form does not exceed the scope of the specification and does not contain new matter. Background of the Invention

[0006] Many potable beverages have an unpleasant pungent taste. Although often accompanied by an appealing flavor, this pungency is easily detectable and intolerable to consumers. As a result, cocktails may be mixed or salt, lime, etc. may be added to mask this pungency. For example, distilled alcoholic beverages typically have a pronounced pungency or "bite" that causes a mild pain response in the consumer's mouth / throat / oral cavity when consumed. Alcohol producers are keenly aware of this problem and have tried to remove the pungent components in distilled alcoholic beverages by using one or more aging or softening techniques during or after the distillation process. Examples of the latter include aging the distilled product, percolating the distilled product through a carbon filter, and / or adding flavoring substances to the distilled product to mask its pungency. Although these techniques can reduce the pungency to some extent, they do not eliminate it or reduce it to an undetectable level. These methods also consume a large amount of energy and take a long time.

[0007] The pungent taste is not limited to alcoholic beverages and is commonly felt when consuming a variety of beverages, including various non-distilled alcoholic beverages, coffee, and tea.

[0008] Therefore, there is a need to develop new technologies that can minimize or even completely eliminate the unpleasant pungency in various potable beverages and beverage components. Summary of the Invention

[0009] The present invention discloses an apparatus, system, and related method for producing an improved potable beverage by adding at least one dehydrogenase to a beverage or beverage component during production. According to the embodiments described herein, a method for preparing a distilled alcoholic beverage may include: forming a mash mixture—a substrate providing fermentable sugars for yeast; fermenting the mash mixture to form a fermented liquid; mixing at least one dehydrogenase with the fermented liquid; and distilling the fermented liquid to form distilled ethanol contained in or formulated to be contained in a distilled alcoholic beverage.

[0010] In some examples, the method further includes mixing at least one cofactor with the fermented liquid. In some examples, the at least one cofactor includes a dinucleotide cofactor, which may include NAD+ and / or NADP. In some examples, the cofactor is added in an amount from about 1 mg / L of the fermented liquid volume to about 2 g / L of the fermented liquid volume. In some examples, the dehydrogenase includes aldehyde dehydrogenase, which may be a native aldehyde dehydrogenase or an engineered dehydrogenase. The aldehyde dehydrogenase selectively oxidizes fatty aldehydes, which may include C2-C10 fatty aldehydes. In some examples, the distilled alcoholic beverage has a lower fatty aldehyde content compared to a distilled alcoholic beverage produced without mixing dehydrogenase with the fermented liquid. In some examples, the concentration range of fatty aldehydes in a distilled alcoholic beverage produced without mixing at least one dehydrogenase with the fermented liquid is from ppm to ppb. In some examples, the aldehyde dehydrogenase selectively oxidizes saturated and unsaturated aldehydes whose electrophilic index matches the cysteine nucleophilic index in the TRPA1 receptor.

[0011] In some examples, the method further includes adjusting the pH of the fermented liquid to about 5.0 to about 7.0. In some examples, the pH of the fermented liquid may not be adjusted. In some examples, the dehydrogenase is added in an amount from about 1 mg / L of the fermented liquid volume to about 2 g / L of the fermented liquid volume. In some examples, the method further includes aging the distilled ethanol in a wooden barrel. In some examples, the fermentation mash mixture may last up to about 5 days. In some examples, the mash mixture includes one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, or one or more other yeast organic raw materials. In some examples, the ethanol concentration of the distilled ethanol is from about 20% to about 95%. In some examples, the ethanol concentration of the fermented liquid is from about 1% to about 20%.

[0012] According to an embodiment described herein, a system for producing a distilled alcoholic beverage includes: a saccharification tank configured to stir and heat a mash mixture; a fermenter configured to ferment the mash mixture and form a fermented liquid; at least one dehydrogenase configured to be mixed with the fermented liquid; a distillation device configured to heat the fermented liquid and form a fermented liquid vapor; a condensation device configured to cool the fermented liquid vapor and form distilled ethanol; and a collection device for collecting the distilled ethanol.

[0013] In some examples, the system further includes a wooden barrel configured to receive and store the distilled ethanol that has undergone an aging process; in some examples, the distillate is bottled directly without aging. In some examples, the system further includes at least one cofactor configured to be mixed with the fermented liquid. In some examples, the at least one cofactor includes a dinucleotide cofactor, such as NAD+ or NADP. The dehydrogenase may include aldehyde dehydrogenase, which can be natural or engineered and can selectively oxidize fatty aldehydes, including C2-C10 fatty aldehydes. The system may further include at least one base configured to increase the pH value of the fermented liquid. In some examples, the components of the mash mixture include one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzyme, or one or more other yeast organic raw materials. In some examples, the system further includes one or both of a grinding device or a pressing device configured to grind or press one or more components of the mash mixture.

[0014] According to an embodiment of the present disclosure, a fermented liquid formed by a method for producing a distilled alcoholic beverage may include: a mash mixture composed of one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzyme, a sugar source, or sucrose. The fermented liquid may further include at least one aldehyde dehydrogenase and ethanol at a concentration of about 1% to about 20%.

[0015] In some examples, the fermented liquid further includes a dinucleotide cofactor. The aldehyde dehydrogenase may be a natural aldehyde dehydrogenase or an engineered aldehyde dehydrogenase. In some examples, the concentration of aldehyde dehydrogenase in the fermented liquid is about 1 mg / L to about 2 g / L. In some examples, the concentration of the dinucleotide cofactor is about 1 mg / L to about 2 g / L of the volume of the fermented liquid.

[0016] According to an embodiment of the present disclosure, a method for producing a distilled alcoholic beverage may include: forming a mash mixture; fermenting the mash mixture to form a fermented liquid; distilling the fermented liquid to form distilled ethanol; and mixing at least one dehydrogenase with the distilled ethanol to form the distilled alcoholic beverage.

[0017] 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 can be natural or engineered. The aldehyde dehydrogenase can selectively oxidize fatty aldehydes, such as C2-C10 fatty aldehydes. In some examples, the distilled alcoholic beverage has a lower fatty aldehyde content compared to a distilled alcoholic beverage produced without mixing at least one dehydrogenase with the distilled ethanol. In some examples, the concentration range of fatty aldehydes in the distilled alcoholic beverage produced without mixing at least one dehydrogenase with the distilled ethanol is from ppm to ppb. In some examples, the dehydrogenase is mixed with the distilled ethanol in a wooden barrel or wine bottle after distillation. In some examples, the mash mixture includes one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzymes, or yeast organic raw materials. In some examples, the ethanol concentration of the distilled ethanol is about 20% to 95%.

[0018] According to the embodiments described herein, a method of producing a potable alcohol product may include: forming a mash mixture; fermenting the mash mixture to form a fermentation broth; mixing at least one oxidase with the fermentation broth; and collecting the fermentation broth for inclusion in the potable alcohol product.

[0019] In some examples, the at least one oxidase may comprise or consist of an aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase can be or include a natural aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase can be or include an engineered or modified aldehyde dehydrogenase. In some examples, the aldehyde dehydrogenase selectively oxidizes fatty aldehydes. In some examples, the fatty aldehydes may include C2-C10 fatty aldehydes. In some examples, the potable alcohol product has a lower fatty aldehyde content compared to a potable alcohol product produced without mixing at least one oxidase with the fermentation broth. In some examples, the concentration range of fatty aldehydes in the potable alcohol product produced without mixing at least one oxidase with the fermentation broth is from ppm to ppb.

[0020] In some examples, the method may further include mixing at least one dinucleotide cofactor with the fermentation broth. In some examples, the dinucleotide cofactor may be or include NAD+, NADP+, or both. In some examples, the method may further include distilling the fermentation broth to form distilled ethanol, which is included in or formulated to be included in the potable alcohol product. In some examples, the method may further include aging the distilled ethanol in a wooden barrel. In some examples, the ethanol concentration of the distilled ethanol may be about 20% to about 95%. In some examples, the potable alcohol product may be or include beer. In some examples, the method may further include adjusting the pH of the fermentation broth to about 5.0 to about 7.0. In some examples, the pH value of the fermentation broth may not be adjusted. In some examples, the addition amount of the at least one oxidase may be about 5 mg / L to about 2 g / L of the fermentation broth volume. In some examples, the fermentation mash mixture may last up to about 5 days. In some examples, the mash mixture may include one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzyme, sugar source, or sucrose. In some examples, the ethanol concentration of the fermentation broth may be about 1% to about 20%.

[0021] Brief Description of the Drawings

[0022] Figure 1 is a graph showing the activity levels of five different aldehyde dehydrogenases in the presence of acetaldehyde according to an embodiment of the present disclosure.

[0023] Figure 2 is a graph showing the activity levels of the aldehyde dehydrogenase according to an embodiment of the present disclosure Figure 1 in the presence of nonanal.

[0024] Figure 3 is a graph showing the activity levels of aldehyde dehydrogenase in the presence of acetaldehyde and nonanal at different ethanol concentrations according to an embodiment of the present disclosure.

[0025] Figure 4 is a graph showing the activity levels of aldehyde dehydrogenase determined based on the production of octanoic acid at different ethanol concentrations according to an embodiment of the present disclosure.

[0026] Figure 5 is a graph showing the activity levels of aldehyde dehydrogenase determined based on the production of nonanoic acid at different ethanol concentrations according to an embodiment of the present disclosure.

[0027] Figure 6 is a graph showing the activity levels of aldehyde dehydrogenase determined based on the production of dodecanoic acid at different ethanol concentrations according to an embodiment of the present disclosure.

[0028] Figure 7is a Western blot showing the soluble and insoluble levels of four aldehyde dehydrogenases according to an embodiment of the present disclosure.

[0029] Figure 8 is a graph showing the activity levels of the four aldehyde dehydrogenases shown at different pH values in 15% ethanol according to an embodiment of the present disclosure Figure 7 as shown.

[0030] Figure 9 is a graph showing the relative activity levels of two aldehyde dehydrogenases shown at different pH values in 15% ethanol according to an embodiment of the present disclosure Figure 8 as shown.

[0031] Figure 10 is a graph showing the activity levels of two aldehyde dehydrogenases and related cofactors in the presence of nonanal according to an embodiment of the present disclosure.

[0032] Figure 11 Provides three graphs showing the activity levels of two aldehyde dehydrogenases after 4 hours of the presence of octanal, nonanal, and dodecanal at 30 °C and 37 °C according to an embodiment of the present disclosure.

[0033] Figure 12 Provides three graphs showing the activity levels of two aldehyde dehydrogenases after 21 hours of the presence of octanal, nonanal, and dodecanal at 30 °C and 37 °C according to an embodiment of the present disclosure.

[0034] Figure 13 is a mass spectrum showing the analyte abundances after treatment with an aldehyde dehydrogenase (with and without cofactor) in 40% ethanol according to an embodiment of the present disclosure.

[0035] Figure 14 is a mass spectrum showing the analyte abundances after treatment with an aldehyde dehydrogenase (with and without cofactor) in a head sample according to an embodiment of the present disclosure.

[0036] Figure 15 is a mass spectrum showing the analyte abundances after treatment with an aldehyde dehydrogenase (with and without cofactor) in a fermentation sample according to an embodiment of the present disclosure.

[0037] Figure 16 is a graph showing the substrate catalytic activities of various engineered and native forms of aldehyde dehydrogenase.

[0038] Detailed Description

[0039] Definitions:

[0040] As used herein, the term "beverage" encompasses a variety of edible products, including but not limited to distilled alcoholic beverages (such as vodka, whiskey, rum, brandy, gin, tequila, baijiu, spirits, cocktails, etc.), non-distilled alcoholic beverages (such as beer, wine, cider, sake, hard seltzer, etc.), coffee, and tea. Ethanol, although not consumed alone but used as a flavor solvent, can also be processed in accordance with the principles of this disclosure. For ease of illustration, this disclosure is mainly described with reference to distilled alcoholic beverages and related production methods.

[0041] Accordingly, the term "beverage" can refer to a final beverage product for direct consumption (such as a distilled alcoholic beverage), a beverage component (such as ethanol), or an intermediate product formed during the beverage production process (such as a fermentation broth, which can itself be defined as a product obtained by fermenting organic raw materials).

[0042] As used herein, the term "dehydrogenase" can refer to one or more dehydrogenases, which can be or include aldehyde dehydrogenase. This term can also generically refer to a broader class of dehydrogenases, oxidases, and oxidoreductases, the latter of which includes a variety of enzymes involved in catalyzing redox reactions in biosynthesis, intermediary metabolism, and detoxification. The substrates of these enzymes can include glucose, steroids, advanced glycation end products, lipid peroxidation products, and / or environmental pollutants. In certain examples, the term "enzyme" can be synonymous with dehydrogenases (such as aldehyde dehydrogenase) and can refer to an enzyme composition (which may contain more than two enzymes and / or non-enzyme components). In certain examples, the term "enzyme" can include oxidases that selectively act on specific compounds produced by lipid peroxidation reactions caused by oxidative stress in yeast during the alcohol production process.

[0043] As used herein, "pungency" refers to the unpleasant, pungent biting sensation of a beverage that elicits a slight pain response in the mouth and throat when the beverage is consumed. In certain embodiments, this term does not refer to the burning / warming sensation of ethanol itself, nor does it include negative flavors themselves, such as flavors, components, or substances commonly referred to as liquor aromatics or fusel oils.

[0044] As used herein, "user" refers to the recipient, producer, or operator of the enzyme preparation described in this disclosure, and can also refer to a person who executes or manages the beverage production process in accordance with the methods of this disclosure.

[0045] The singular forms of various terms include their plural meanings. For example, the term "enzyme" should be understood to include both "multiple enzymes" and "enzyme systems" simultaneously.

[0046] In the alcoholic beverage industry, conventional wisdom holds that improving distillation technology is the best way to enhance the smoothness of beverages. Current methods for improving pungency mainly include, for example, distillation, oak barrel aging, filtration, activated carbon filtration, and / or the addition of additional flavor masking substances. The method of the present disclosure breaks through conventional wisdom and achieves significant improvements compared to the prior art, which lacks sufficient sensitivity and specificity to eliminate previously unrecognized pungency-causing compounds that are present in trace amounts in beverages.

[0047] The appeal of alcoholic beverages (especially distilled varieties) is often diminished by the pungency and painful "bite" they impart. From the lowest-end products to the highest-end, most expensive spirits, even the most rigorous production processes have not been able to completely eliminate this negative sensory experience. The present disclosure is based on an unprecedented discovery: the precise identification of the compounds that cause the pungent bite experienced by consumers, and a novel method for targeting these compounds and producing a completely smooth spirit or other conventional alcoholic beverage that is free of the typical pungent bite of spirits or other conventional alcoholic beverages that have not been treated by this method. To date, attempts to mitigate pungency have only included distillation, filtration, or aging, but these methods have had limited success. Additionally, in the limited cases where enzymes are used in the production of specific alcoholic beverages, they are limited to specific components and methods that can increase the fermentable sugar content or reduce the viscosity of the liquid. This narrow application does not reduce the pungency of the beverage as envisioned herein. The pungent bite experience associated with drinking, the nociceptors involved in this experience, and the harmful compounds produced during fermentation (which are agonists of specific nociceptors) have never been linked before. Therefore, the method of the present disclosure, which uses one or more dehydrogenases of the present disclosure, such as aldehyde dehydrogenase, to selectively target these harmful compounds without affecting other flavors or aromas of the beverage, has not been practiced.

[0048] Human sensory experiences can be classified into three categories: taste (gustation), olfaction for perceiving aroma, and somatosensation (primarily responsible for sensations such as pain). The food and beverage industry has long focused on the first two while neglecting the latter. Embodiments of the compositions, methods, and systems of the present disclosure are directed precisely at the latter. During the production of alcoholic or distilled alcoholic beverages, mash (a formulation composed of organic matter that yeast uses as a sugar source for fermentation) is fermented by yeast. As the ethanol concentration increases during the fermentation of the mash by yeast, oxidative stress occurs, which in turn triggers a lipid peroxidation reaction, ultimately generating the harmful compounds discovered by the present invention and targeted for elimination. Since these irritating compounds originate from the lipid peroxidation of the yeast cell membrane, their presence is independent of the type of alcoholic beverage produced. When humans are exposed to these electrophilic harmful compounds (usually aldehyde compounds), they experience a mild pain response in the form of the irritating bite sensation described herein. Through the analytical and sensory tests disclosed herein, it has been confirmed that the compositions, methods, and systems provided by the present invention can effectively target and eliminate these irritants that make alcoholic beverages unpleasant and irritating, thereby producing truly smooth alcoholic beverages, including those typically classified as premium beverages, etc.

[0049] Embodiments of the present invention employ aldehyde dehydrogenase to specifically target the lipid peroxidation products of the yeast cell membrane, which are generated once the yeast undergoes an oxidative stress process at the end of alcoholic fermentation. This lipid peroxidation reaction generates a series of compounds, many of which are fatty aldehydes, such as malondialdehyde or nonenal, which have been confirmed to be triggers of the TRPA1 receptor (transient receptor potential cation channel subfamily A member 1 / ankyrin 1). TRPA1 is not a taste receptor but a pain receptor responsible for generating the irritating "bite sensation," as described below. This irritation is not a taste that is tasted but a mild pain response to harmful compounds such as aliphatic saturated / unsaturated aldehydes. These compounds are eliminated by the enzymatic oxidation of the carbonyl groups disclosed herein into carboxylic acids, which are weak organic acids and are usually precursors for forming esters, which are precisely what is required for distilled alcohol. The methods disclosed herein do not change the taste characteristics of alcoholic beverages but eliminate the irritation of such beverages, thereby creating truly smooth beverages.

[0050] Not all aldehydes trigger a pain response because there is a wide variety of existing aldehyde compounds. Many of these aldehydes can produce desirable flavors (such as cinnamaldehyde), some cause off-flavors (such as certain aldoses), and some are toxic. The toxicity of aldehyde compounds depends on their electrophilicity and steric hindrance. The TRPA1 receptor is designed to detect and respond only to those aldehydes with specific toxic electrophilicity that have a negative impact on living cells. The embodiments disclosed herein can specifically act on these toxic and harmful aldehydes while completely preserving the desirable varieties.

[0051] Embodiments of the Present Disclosure

[0052] The present disclosure relates to methods, systems, and related reagents, ingredients, and devices for eliminating, minimizing, or reducing the level of pungency perceived by consumers when consuming a variety of potable beverages, non-limiting examples of which include distilled alcoholic beverages. Embodiments may include adding at least one natural or engineered enzyme during or after the production of the beverage or its components. The enzyme may comprise one or more dehydrogenases, such as at least one aldehyde dehydrogenase. In embodiments employing aldehyde dehydrogenase, in embodiments related to the production of distilled alcoholic beverages, the dehydrogenase may be added before, during, and / or after the start of the fermentation process, and / or after the distillation process. The dehydrogenase can reduce the total content of one or more aldehydes, including trace amounts of aldehydes, in the beverage through oxidation. Specifically targeted aldehydes include aliphatic electrophilic aldehydes, such as C2-C10 fatty aldehydes. The dehydrogenase does not act on aldehydes related to human taste perception, but selectively acts on the pungent aldehydes identified herein that cause pungent biting sensations and nociceptive responses in consumers. Thus, by reducing the content of one or more specific aldehydes, the pungency of the final beverage product can be eliminated or reduced to a level that is imperceptible or substantially imperceptible to most consumers, while fully retaining the intended flavor profile of the product.

[0053] Enzyme Composition

[0054] Enzymes for reducing or eliminating the pungency typical of many potable beverages may include at least one dehydrogenase, such as at least one aldehyde dehydrogenase. The enzyme can selectively catalyze the oxidation of one or more electrophilic compounds that have been identified by the inventors as causative or contributing factors for beverage pungency, without targeting compounds that contribute to the desirable flavor or quality of the final beverage product, even if these two classes of compounds may belong to the same chemical class. For example, the methods disclosed herein may not affect aldehydes that produce a positive flavor due to steric hindrance. Embodiments may selectively target one or more carbonyl groups (such as aldehydes) for oxidation, thereby converting the target compound into a carboxylic acid. The compounds targeted by the enzyme may be present in low amounts in the beverage or beverage component. Thus, the enzymes of the present disclosure can exhibit a high degree of specificity and sensitivity. For ease of explanation, the singular term "enzyme" will be primarily used hereinafter, but it should be understood that "enzyme" may refer to one or more enzymes or enzyme systems.

[0055] In some embodiments, the enzyme may substantially retain its native (or wild-type) characteristics in terms of amino acid composition, protein conformation, and activity. Through extensive testing of the activities of 15 different aldehyde dehydrogenases under typical conditions of commercial alcohol fermentation and distillation, we discovered native enzymes with specific substrate selectivity and sensitivity that can effectively remove the pungency of distilled alcoholic beverages. Although one of the native enzymes exhibited the most significant reduction in pungency, other dehydrogenases among the 15 enzymes initially tested (including all 15 enzymes used alone or in combination during alcohol production) also showed effectiveness, indicating that aldehyde dehydrogenases, as a class of enzymes, can effectively reduce the pungency associated with the consumption of alcoholic beverages when used according to the methods described herein. The amino acid sequences of the enzymes evaluated to show this effect correspond to SEQ ID NOS: 1-17. In an embodiment, an enzyme that can effectively reduce the pungency of a beverage and is implemented according to the method of the present disclosure has about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with any one of SEQ ID NOS: 1-17.

[0056] Embodiments may also optionally include artificially modified or engineered enzymes that may differ from native enzymes in terms of amino acid composition, protein conformation, and / or activity. Embodiments may include enzyme compositions and systems containing a mixture of one or more native and / or engineered enzymes. Depending on the specific fermentation conditions and the relative concentrations of the target compounds, native and engineered variants may also be included in separate compositions used simultaneously during beverage production. In an embodiment, an engineered enzyme implemented according to the method of reducing beverage pungency of the present disclosure has about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with any one of SEQ ID NOS: 1-17, or about 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with other native dehydrogenases.

[0057] In the experiments described below, the native enzyme form (designated as Enzyme 2, 54.1 kDa) that showed the most significant pungency reduction effect has an amino acid sequence corresponding to SEQ ID NO: 6. According to the methods of the present disclosure, one or more other enzymes capable of significantly reducing the pungency of a beverage may have at least about 80%, 85%, 90%, 95%, 99% or 100% identity with SEQ ID NO: 6. Another native enzyme (designated as Enzyme 3, 54.0 kDa) that showed a significant pungency reduction effect in the experiments described below has an amino acid sequence corresponding to SEQ ID NO: 8. One or more other native enzymes that show effective pungency reduction when used according to the methods of the present disclosure may have at least about 80%, 85%, 90%, 95%, 99% or 100% identity with SEQ ID NO: 8. The native enzyme designated as Enzyme 2 in the experiments described below may exhibit greater pH tolerance than the native enzyme designated as Enzyme 3, but both enzymes are effective pungency reducers, responsible for significantly reducing the pungent bite of potable alcohol and alcohol components. The remaining enzymes that were subjected to the test conditions described below, corresponding to SEQ ID NO: 1 - 5, 7 and 9 - 15 (including the enzymes designated as Enzyme 1 and Enzymes 4 - 15) may also have substantially equivalent or comparable effectiveness, which is at least partially attributed to their high homology with each other and with Enzyme 2 and Enzyme 3 and other aldehyde dehydrogenases, as would be understood by those skilled in the art, which is at least partially due to the enzymatic activity of oxidizing aldehydes to carboxylic acids that is common to all aldehyde dehydrogenases. Thus, while the experiments detailed herein evaluated a subset of the enzymes, according to the disclosed embodiments, the enzymes capable of effectively reducing pungency may include more, such as all or substantially all dehydrogenases, including aldehyde dehydrogenases. Accordingly, the methods of the present disclosure are not limited to the specific enzymes tested in the experiments described below.

[0058] The native form enzymes used according to the embodiments of the present disclosure may have a molecular weight range from about 40 kDa to about 60 kDa, including up to about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa or greater. The molecular weight range of other embodiments of the native enzymes may also be from about 53.5 kDa to about 57 kDa, including 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 the dehydrogenase may have a molecular weight of about 54.1 kDa, about 57.5 kDa or about 56.7 kDa.

[0059] The different properties exhibited by natural and engineered forms of an enzyme may vary. For example, under conditions of decreasing pH, and / or increasing temperature, and / or increasing ethanol concentration, an engineered enzyme may exhibit normal activity levels, while a natural enzyme may exhibit decreased activity under the same conditions. In some embodiments, a natural form of the enzyme (including those having an amino acid sequence that is substantially similar or identical to one or more of the sequences in SEQ ID NO: 1-17) may exhibit an effective activity level that is substantially equal to that of the engineered form of the enzyme. Different enzymes may also exhibit more or less enzyme activity under a range of conditions. Thus, beverage production parameters can be adjusted based on the enzyme used.

[0060] Engineered enzymes can maintain their activity under typical fermentation conditions, for example, within a pH range of about 3.0 to 6.0, a temperature range of about 15°C to 37°C, and an ethanol concentration range of 0% to 20%, such as about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or higher, and for cases where the enzyme is added after distillation, such as up to 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% or about 95%, or any value in between. In some examples, natural form enzymes can maintain a generally stable activity level at higher ethanol concentrations (including up to about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or higher).

[0061] In some examples, the activity of natural form enzymes at lower pH values may be lower than that of engineered form enzymes. For example, natural form enzymes can be detected with activity between pH 3.5 and 7.0, but their optimal activity occurs within a pH range of about 6.0 to 7.0. Thus, while natural form enzymes can still exhibit sufficient activity levels at pH values below about 6.0, fermentation methods using natural enzymes may involve raising the pH of the fermentation broth to 6.0 or higher (such as 6.7). In some embodiments, the enzyme activity of one or more natural form enzymes (non-limiting examples include enzymes having any of the sequences in SEQ ID NO: 1-17) within a lower pH range of about 3.5 to 7.0 may be substantially equivalent to that of one or more engineered form enzymes.

[0062] The enzyme can be in a dry or liquid form. Embodiments of the enzyme composition can comprise a spray-dried or lyophilized powder, or a liquid solution consisting of a powdered enzyme suspended in water and / or one or more buffers. In some examples, the enzyme composition (which may or may not include added cofactors) can be immobilized on a solid matrix or structure. The content of pure enzyme in the enzyme composition can vary, mainly depending on the form of the final formulation. For an enzyme composition in powder form, the enzyme activity can range from about 2 units / mg protein to about 5 units / mg protein, to about 10 units / mg protein, to about 15 units / mg protein, to about 20 units / mg protein, to about 25 units / mg protein, to about 30 units / mg protein, to about 35 units / mg protein, to about 40 units / mg protein, to about 45 units / mg protein, to about 50 units / mg protein, or higher, or any value in between.

[0063] The enzyme can be provided to the user in a variety of forms. In some examples, the enzyme can be encapsulated in one or more vials, containers, or drums. Embodiments can also provide a single-use addition pack containing the amount of enzyme required for a single batch of the beverage production process. Similar addition packs can also be used for single-use addition in a continuous beverage production process. Thus, multiple addition packs can be used during continuous production and added at different stages of the production process at predetermined intervals (such as during or after fermentation, including after distillation).

[0064] As described above, the enzyme can exhibit a high degree of substrate specificity and sensitivity. In addition to ethanol, distilled alcoholic beverages typically also contain trace amounts of hundreds of organic molecules, such as organic acids, other alcohols, ketones, esters, aldehydes, organic fats, and proteins, the large number and variety of which make the task of identifying and effectively targeting those compounds that cause the beverage to be pungent very complex. In certain examples, the enzyme can effectively reduce or even eliminate one or more pungent aldehyde substrates present in the beverage or beverage component (such as the fermentation broth) at a concentration of only 500 ppm or lower, for example, about 5 ppm to about 10 ppm, or even lower, such as ppb.

[0065] Without being bound by any particular theory, the pain response triggered by consumers when consuming certain stimulating beverages can be attributed to the activation of the trigeminal nerve, which is caused by the activation of certain 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 and is thus difficult to neutralize. As described herein, it has been found that electrophilic aliphatic compounds present in stimulating beverages may be the main cause of activating TRPA1. Aliphatic molecules with reactive carbonyl groups (such as aldehydes) can form reversible covalent bonds with one or more cysteine residues within the active site of TRPA1, thereby inducing a pain response caused by TRPA1 activation. In particular, aliphatic molecules with α,β-unsaturated bonds may cause the most obvious pain response. These compounds may cause significant trigeminal nerve activation at very low levels (such as ppm to ppb), and even the most sophisticated distillation techniques cannot eliminate them. It is surprising that such low levels of substrates can currently be effectively targeted and eliminated by enzymes, especially in the context of distilled alcohol production, which requires the implementation of various processing parameters that are traditionally unfavorable to enzyme function.

[0066] During the alcohol production process, an increase in ethanol production (such as during fermentation) causes environmental stress, affecting the lipid bilayer structure of yeast. Oxidative stress leads to an increase in reactive oxygen species, thereby triggering lipid peroxidation reactions and correspondingly producing harmful aldehydes, including hydroxy nonenal, malondialdehyde, and acrolein, each of which will trigger the TRPA1 nociceptor when ingested by humans.

[0067] By using the enzymes, related methods, and systems described in the present disclosure, the covalent bond formation between the cysteine residues of TRPA1 and the reactive carbonyl group can be prevented by catalyzing the conversion of aliphatic compounds into carboxylic acids. Through this mechanism, the methods of the present disclosure can effectively reduce or eliminate the levels of pain-causing compounds that are present at extremely low but still perceivable levels in a variety of beverage products. In some examples, the enzyme can catalyze the oxidation reactions of a variety of different substrates, such as various types of carbonyl electrophiles, non-limiting examples of which can include crotonaldehyde, octanal (such as n-octanal), nonanal (such as n-nonanal), dodecanal (such as n-dodecanal), and / or acetaldehyde. In certain examples, although the oxidation reaction of acetaldehyde can be catalyzed by the enzymes of the present disclosure, unlike the aforementioned aliphatic aldehydes (especially C2-C10 aliphatic aldehydes), the reduction of acetaldehyde content has little effect on the perceived level of irritation.

[0068] Notably, the enzymatic conversion of the target aldehydes into carboxylic acids can also form ester precursors, and these precursors can improve the taste and / or smoothness of the final beverage product, and the effects could only be achieved through the aging process in the past.

[0069] Method for Reducing Beverage Irritancy

[0070] The present disclosure relates to a method of adding at least one enzyme of the present disclosure to a potable beverage or its components during the beverage production process to reduce the beverage pungency. As described above, the enzymes of the present disclosure can be configured to catalyze the redox reactions of aldehydes that cause various beverage pungencies within the temperature, pH, and concentration ranges characteristic of alcoholic beverages (such as distilled alcoholic beverages). To reduce the concentration of fatty aldehydes and thus reduce the overall pungency of the final distilled product, one or more enzymes can be added to one or more intermediate compositions (such as fermentation broth or distillate) formed during the alcohol production process or to the composition formed after distillation. Embodiments for the production of alcoholic beverages generally can include mash formation, fermentation, optional distillation, and optional aging. The specific sub-processes employed can depend on the final alcohol product. For example, embodiments related to the production of whiskey or vodka are different, and embodiments involving beer production do not include distillation.

[0071] Preparing the mash - The mash for fermentation can contain a variety of components with varying contents and concentration ranges. The components of the mash can vary depending on the type of beverage produced, such as wine, beer, vodka, gin, whiskey, bourbon, baijiu, rum, or any other potable alcoholic beverage. Non-limiting examples of mash components can include one or more of the following: grains (such as corn, rye, rice, barley, wheat), agave, dextrin, potato, fruit (such as grapes), molasses, water, one or more enzymes, one or more yeast organic raw materials, and / or other saccharides (such as sucrose), sugar equivalents, or sugar sources, with a sugar concentration ranging from about 0 wt% to 100 wt%, including less than 1 wt% to about 5 wt%, to about 10 wt%, to about 15 wt%, to about 20 wt%, to about 25 wt%, to about 30 wt%, to about 35 wt%, to about 40 wt%, to about 45 wt%, to about 50 wt%, to about 55 wt%, to about 60 wt%, to about 65 wt%, to about 70 wt%, to about 75 wt%, to about 80 wt%, to about 85 wt%, to about 90 wt%, to about 95 wt% or higher, or any concentration between the above.

[0072] The content of each mash component may vary. For example, an embodiment characterized by corn may contain from about 60 wt% corn to about 65 wt% corn, to about 70 wt% corn, to about 75 wt% corn, to about 80 wt% corn, to about 85 wt% corn, to about 90 wt% corn, or any content between any of the above values. An embodiment characterized by rye or rye malt may contain from about 10 wt% rye malt to about 12 wt% rye malt, to about 14 wt% rye malt, to about 16 wt% rye malt, to about 18 wt% rye malt, to about 20 wt% rye malt, to about 22 wt% rye malt, to about 24 wt% rye malt, to about 26 wt% rye malt, to about 28 wt% rye malt, or any content between any of the above values. An embodiment characterized by barley or barley malt may contain from about 2 wt% barley malt to about 4 wt% barley malt, to about 6 wt% barley malt, to about 8 wt% barley malt, to about 10 wt% barley malt, to about 12 wt% barley malt, to about 14 wt% barley malt, to about 16 wt% barley malt, to about 18 wt% barley malt, to about 20 wt% barley malt, or any content between any of the above values.

[0073] One or more components of the mash may be pressed and / or crushed before or after mixing with one or more other mash components. A roller mill and / or a hammer mill may be used to grind mash components such as grains. After one or more pretreatment steps, the mash components may be added to a container (such as a mash tun), where the components are stirred and heated.

[0074] Fermentation process - The treated mash mixture may be fermented by yeast to convert the sugars contained therein into alcohol. The fermentation process may be carried out in a large fermenter or container into which the mash mixture may settle. In a specific embodiment, the yeast may be mixed with the mash mixture (filtered or unfiltered), and the fermentation process may be carried out for up to about 15 days, or until about 1% to about 20% ethanol is produced. The final ethanol concentration of the fermentation broth may range from about 1% to about 20%, or any percentage therebetween, such as up to about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19% or higher.

[0075] Regardless of the fermentation microorganism or sugar source used to produce ethanol, compounds that cause or exacerbate beverage pungency may be produced, such as C2-C10 fatty aldehydes. The enzymes described in the present disclosure can be added in different amounts before the start of fermentation, during fermentation, or after fermentation has ended to minimize or eliminate such compounds. For example, the amount of aldehyde dehydrogenase added can range from about 1 mg / L to about 2 g / L of the fermentation broth volume, or any value in between, including 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 higher.

[0076] The enzyme can be added at any point before the start of fermentation or during fermentation, or at any point shortly after fermentation has ended until just before distillation begins. For example, it can be added when the mash is formed, when yeast is added, or when partial fermentation is complete, such as when about 5% to about 10% of the total target alcohol production of the present disclosure is complete, up to about 20% complete, up to about 30% complete, up to about 40% complete, up to about 50% complete, up to about 60% complete, up to about 70% complete, up to about 80% complete, up to about 90% complete, up to about 95% complete or up to about 100% complete (e.g., up to about 20%). Alternatively, the enzyme can be added during fermentation, with a residence time ranging from about 5 minutes to several days, including, for example, less than about 6 hours to about 6 hours, up to about 12 hours, up to about 36 hours, up to about 48 hours, up to about 60 hours, up to about 72 hours or longer. The enzyme can also be added after distillation, before bottling, or after bottling. Embodiments can also include adding the enzyme before the start of fermentation. Other embodiments can include adding the enzyme to the distilled ethanol in an aging barrel along with any other ingredients required to produce a particular beverage.

[0077] In some examples, one or more dinucleotide cofactors (such as NAD+ and / or NAP+) can be added at any of the above time points or production stages during the fermentation process. The addition concentration range of the cofactor can be from about 0 mg / L to about 1 mg / L of the fermentation broth volume, or up to about 2 g / L of the fermentation broth volume, or any concentration value in between, 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 higher. The implementation of using aldehyde dehydrogenase in combination with cofactors can enhance or optimize the reduction of irritation. For example, the combination of aldehyde dehydrogenase and NAD+ can enhance or maximize the reduction of irritating compounds. In some embodiments, without adding exogenous cofactors, the effect of reducing / eliminating irritation can still be achieved equivalently. Therefore, the methods of the present disclosure can be implemented by adding enzymes alone, or adding enzymes and cofactors. In some examples, when adding aldehyde dehydrogenase (such as enzyme 2) and a cofactor containing NAD+ to the fermentation broth, compared with other native enzymes (with or without adding NAD+), the best irritation reduction effect can be obtained. The combination of aldehyde dehydrogenase (such as enzyme 3) and a cofactor containing NADP+ also shows a significant irritation reduction effect compared with other native enzymes. In some embodiments, it may not be necessary to add cofactors, for example, when using engineered enzymes, or when using enzymes having at least about 80%, 85%, 90%, 95%, 99% or 100% amino acid sequence identity with at least one native enzyme form (such as enzyme 2 and / or enzyme 3), and the amino acid sequences of these enzymes are substantially similar or identical to SEQ ID NO:6 and SEQ ID NO:8 respectively.

[0078] In some examples, the pH value of the fermentation broth can be adjusted. For example, an alkaline compound (such as calcium carbonate) can be added at one or more time points during the fermentation process to raise the pH value of the fermentation broth to the range of about 5.0 to about 7.0, especially in the case of using native enzymes. Specific embodiments may involve raising the pH value to about 6.0 to about 7.0, such as 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, it may not be necessary to adjust the pH value, for example, when using engineered enzymes or when native enzymes are active within the required pH range. According to these embodiments, the pH value range of the fermentation broth can be about 3.0 to 6.0.

[0079] The temperature of the fermentation broth can also vary and in some examples can range from about 15 °C (59 °F) to about 37 °C (98.6 °F), or any temperature in between, 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. Throughout or most of the fermentation cycle, the temperature can be kept constant or substantially constant. In some embodiments, the temperature can vary one or more times during the fermentation process. According to such embodiments, for at least about 50% 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% or more of the fermentation time, the temperature can be maintained within the range of about 15 °C (59 °F) to about 37 °C (98.6 °F).

[0080] Fermentation can be carried out under a variety of pressure conditions. In some embodiments, the pressure range for carrying out the fermentation can be 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 higher, or any pressure value in between.

[0081] In the presence of Saccharomyces cerevisiae, the fermentation of the resulting mixture can last up to about 10 days. In some embodiments, the enzyme can be added to the mixture and remain active for at least about 5 minutes to at least about 5 days, or any duration in between or longer, such as 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 longer. After fermentation, the ethanol content of the fermentation broth can range from about 1% to about 20%, or any concentration in between, including about 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18% or higher.

[0082] Distillation - Once fermentation is complete, the distillation process can be carried out to obtain, for example, an ethanol concentration of about 10% alcohol by volume (ABV) or greater. In some embodiments, the fermented liquid (filtered or unfiltered) can be heated in a batch or continuous manner depending on the desired distillation process (except for non-distilled alcoholic beverages such as beer and wine, which can be subjected to one or more post-fermentation processes that do not involve distillation). The heating of the fermented liquid is typically carried out in a distillation apparatus such as a pot still. Heating causes the ethanol in the fermented liquid to vaporize, thereby separating it from unwanted impurities such as grain particles and liquids in the fermented liquid. The ethanol vapor is then condensed by cooling in a condenser and subsequently collected in a collection device. In some examples, the distillation apparatus, condenser, and collection device may be integrated into a single unit, which is referred to as a still in some instances.

[0083] The fermented liquid can be distilled to an ethanol concentration of about 20% to 95%. Dehydrogenase is not transferred through distillation and remains in the portion of the distillate that is not collected as a potable alcoholic beverage.

[0084] Bottling / Aging - The distilled ethanol can be diluted to the target concentration as needed and bottled directly, or transferred to a wooden barrel for further aging. When the required aging period is completed, the liquid in the barrel needs to be adjusted to the desired ethanol concentration and finally bottled for distribution. In some embodiments, one or more dehydrogenases can be added to the distilled ethanol after bottling and / or barreling to directly reduce the content of irritating aldehydes in the bottle / barrel before consumption. According to such embodiments, the amount of dehydrogenase added ranges from about 5 mg / L to about 2 g / L of the volume of distilled alcohol, or any amount in between, 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 higher.

[0085] The final ethanol concentration of distilled alcoholic beverages ranges from 10% to 95%, or to about 15%, to about 20%, to about 25%, to about 30%, to about 35%, to about 40%, to about 45%, to about 50%, to about 55%, to about 60%, to about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90% or higher. In some examples, the ethanol concentration can subsequently be diluted for consumption, reducing the final concentration to below 10%, such as about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or other intermediate concentration values.

[0086] According to what is provided, the enzymes of the present disclosure can be added at any time point before fermentation, during fermentation, or shortly after fermentation up to just before distillation, and have the residence time ranges of the present disclosure; and / or may involve adding the enzymes to distilled ethanol in an aging barrel together with any ingredients required for producing a specific beverage. Certain embodiments may also involve adding the enzymes before fermentation. Additionally, in view of the difficulties previously encountered in determining effective enzyme delivery mechanisms, the discovery of adding enzymes to the fermentation broth or distillate during the beverage production process is surprising.

[0087] Beverage Product

[0088] Compared to beverages with the same formulation and process but not treated with the enzymes disclosed herein (including enzyme types, enzyme addition timing, and enzyme addition concentration, all of which may be crucial for the effective oxidation of carbonyl groups present on target compounds), beverages produced by the above methods may have a lower aldehyde content. In embodiments of the beverage of the present invention, specifically, the content of aliphatic electrophilic aldehydes (including or consisting of C2–C10 fatty aldehydes) is reduced compared to conventional beverages not treated with enzymes according to the method disclosed herein.

[0089] Experimental Examples

[0090] To verify the efficacy of aldehyde dehydrogenase in effectively oxidizing pungency-causing compounds under the conventional conditions of producing drinking alcohol products, a series of experimental studies were conducted. Supplementary experiments further confirmed the efficacy of aldehyde dehydrogenase in effectively oxidizing pungency-causing substances during the production of drinking alcohol products. Taste tests were also conducted to verify the reduced pungency felt by users when drinking drinking alcohol products produced by implementing the methods disclosed herein. As described below, the selection of certain aldehyde dehydrogenases for preliminary evaluation and continuous testing is only for illustrative purposes. It should be understood that the pungency-reducing effect of the present disclosure is not limited to the effect achieved by using the specific enzymes tested in the following experiments, which include a subset of the enzymes tested to demonstrate the effective oxidation of pungency-causing compounds under the conditions commonly applied during the production of distilled alcohol, and the resulting significant reduction in beverage pungency. Each class (including all or substantially all aldehyde dehydrogenases) that is at least similar or identical to any of SEQ ID NOS: 1-17 may be effective under conditions similar to those applied below.

[0091] Experiment 1 - Aldehyde Dehydrogenase Screening and Activity Evaluation

[0092] Experiment 1 was conducted to verify that dehydrogenases from different sources can effectively target irritant compounds. Specifically, spectrophotometry was used to determine the activities of 15 different aldehyde dehydrogenases (corresponding to SEQ ID NOS: 1 - 15) in the presence of various aldehyde substrates. The source organisms of these enzymes were diverse, indicating that enzymes from multiple sources can achieve the technical effects of the present disclosure. For example, human aldehyde dehydrogenase was tested, as well as enzymes from Bacillus, Escherichia coli, Cynomolgus monkey, Otolemur gamettii, Pteropus alecto, Geobacillus, Parageobacillus caldoxylosilyticus, Citrobacter rodentium, Shigella boydii, Klebsiella pneumoniae, and Acinetobacter spp.

[0093] Enzyme activity was determined by the conversion of NADP+ to NAD(P)H, as aldehyde dehydrogenase activity depends on NAD(P)+. Since NAD(P)H absorbs light at 340 nm while NAD(P)+ does not, in the presence of equimolar amounts of NAD(P)+ and five aldehyde substrates (acetaldehyde, crotonaldehyde, n - octanal, n - nonanal, and n - dodecanal), the formation of NAD(P)H was monitored spectrophotometrically at 340 nm for 10 minutes. Each aldehyde substrate was provided at 5 mM (220 - 292 ppm) and 0.5 mM (22 - 92 ppm) in separate samples.

[0094] Although all aldehyde dehydrogenases can act on aldehydes, the purpose of this experiment was to screen for the enzyme with the highest activity under specific conditions. Among the 15 enzymes, 5 enzymes showed the highest activity towards all substrates at a concentration of 500 μM (identified as Enzymes 1 - 5, corresponding to SEQ ID NOS: 5, 6, 8, 14, and 15 respectively), and these 5 enzymes were further evaluated. First, their tolerance at ethanol concentrations commonly found in fermentation and distillation processes was evaluated. First, each enzyme was screened for 5 mM acetaldehyde and 5 mM n - nonanal in the ethanol concentration range of 5% v / v to 50% v / v. As shown respectively in Figure 1 and Figure 2 , in the presence of acetaldehyde and n - nonanal, the activity of each enzyme remained relatively stable until the ethanol concentration reached approximately 20 - 30% v / v, at which point the enzyme activity generally decreased. This indicates that many aldehyde dehydrogenases, even from different organisms, can effectively act on and catalyze alcohol - related irritant aldehydes. Other dehydrogenases (including but not limited to the 15 tested in this experiment) may also effectively act on and catalyze irritant aldehydes commonly associated with potable beverages and their components.

[0095] We also evaluated the activity of each enzyme at 5% v / v and 50% v / v ethanol concentrations, with and without substrates (acetaldehyde and nonanal), to further elucidate the ethanol tolerance of the enzymes and determine any background enzyme activity. Figure 3 The graphical results shown indicate that, in the absence of substrates, the activity of each enzyme in 50% v / v ethanol was indeed equal to or higher than that in 5% v / v ethanol. However, the results further showed that each enzyme exhibited an increase in activity in the presence of acetaldehyde and nonanal compared to the activity measured in the absence of aldehyde substrates.

[0096] We further evaluated the activity of various enzymes at low substrate concentrations by gas chromatography. To gain a deeper understanding of the ethanol tolerance of these enzymes, we tested various enzymes in 20% v / v ethanol in the presence of 10 ppm n-octanal, n-nonanal, and n-dodecanal, respectively. According to Figure 4 the amount of caprylic acid produced, all enzymes exhibited activity in the presence of octanal at a concentration of 10 ppm or lower. As Figure 5 shown, all enzymes also showed activity in the presence of 5 ppm and 10 ppm nonanal, and enzyme 2 maintained a high activity level at various substrate concentrations in both 5% v / v and 20% v / v ethanol. Figure 6 The results showed that all enzymes exhibited significant activity at 5 ppm and 10 ppm dodecanal in both 5% v / v and 20% v / v ethanol. Enzyme 2 showed the highest activity level at 5 ppm in 5% v / v ethanol and 10 ppm in 20% v / v ethanol.

[0097] Spectrophotometry and gas chromatography analysis indicated that enzyme 2 exhibited strong ethanol tolerance and no decrease in activity was observed at an ethanol concentration of 20% v / v. A high activity level could still be maintained in enzyme 2 when only 5 - 10 ppm of various aldehyde substrates were present. In summary, these results suggest that one or more of the enzymes evaluated (including all enzymes) may be effective under conditions relevant to alcohol beverage production (such as distilled spirit production), and enzyme 2 showed the highest catalytic activity in this specific experiment.

[0098] Experiment 2 - Aldehyde Dehydrogenase Characterization

[0099] This experiment aimed to select a specific enzyme for further testing in a craft distillery to demonstrate the positive sensory impact of eliminating beverage pungency. Enzyme powders of enzymes 1 - 4 (corresponding to SEQ ID NO:5, 6, 8, and 14 respectively) and a negative control were prepared synchronously in the experiment, and the relative contents of soluble and insoluble enzyme powders were determined. As Figure 7As shown by Western blot, the relative contents of soluble proteins produced by Enzyme 2 (54.1 kDa) and Enzyme 3 (54.0 kDa) are the highest. Based on the relatively high content of soluble proteins in the enzyme powder, Enzyme 2 and Enzyme 3 may have the best functions when added to the liquid fermentation broth and / or distilled samples in powder form.

[0100] Subsequently, the activity levels of Enzymes 1-4 in 10% v / v and 15% v / v ethanol were evaluated in the pH range of 3.5 - 7.5 by the aforementioned NAD(P)H spectrophotometry to verify their functions in the high alcohol content and low pH environment common in fermentation. Figure 8 It was shown that in 15% v / v ethanol, Enzymes 1 and 4 exhibited lower overall activity at all pH values compared to Enzymes 2 and 3, and they had the best stability under low pH conditions. Figure 9 It was shown that in 15% v / v ethanol, Enzyme 2 was more stable than Enzyme 3 at most pH levels (especially at pH 3.5 and 4.0).

[0101] The cofactor preferences of Enzymes 2 and 3 for NAD+ and NADP+ were detected by NAD(P)H spectrophotometry. As Figure 10 shown, both enzymes showed activity with both cofactors. The activity of Enzyme 2 with NAD+ was about 1.5 times that of Enzyme 3, while the activity of Enzyme 3 with NADP+ was about 1.2 times that of Enzyme 2. Therefore, although both are effective in the absence of cofactors (including NAD+ and NADP+), the presence of NAD+ can maximize the activity of Enzyme 2, while the presence of NADP+ has a more significant effect on enhancing the activity of Enzyme 3.

[0102] Under the conditions of 15% ethanol and pH 5.5, using 100 ppm octanal, nonanal or dodecanal as substrates, Enzymes 2 and 3 were evaluated head-to-head at 30 °C and 37 °C respectively. The test concentration range for each enzyme was from 0.001 g / L to 11 g / L of the enzyme powder (enzyme:aldehyde molar ratio from 1:100000 to 1:10). The activity levels of each enzyme were measured at 1 hour, 4 hours and 21 hours, with Enzyme 2 using NAD+ as the cofactor and Enzyme 3 using NADP+ as the cofactor. The reaction was quenched and terminated by extraction with ethyl acetate, and analyzed by gas chromatography. The substrate conversion rate was calculated by dividing the product peak area by the sum of the product peak area and the substrate peak area.

[0103] As Figure 11 shown, at the 4-hour time point, Enzyme 2 showed a higher activity level than Enzyme 3 under all test conditions. The activity level generally increased with the increase in the enzyme addition amount, up to about 0.1 g / L of the enzyme, after which the enzyme activity remained relatively stable. The highest activity of Enzyme 2 occurred under the conditions of pH 5.5 and 15% v / v ethanol. Figure 12The enzyme activity data at the 21-hour time point are shown. As depicted, the activity levels of the two enzymes at 21 hours are basically equivalent to those at 4 hours. The activities of both enzymes are higher at 30 °C than at 37 °C.

[0104] Therefore, aldehyde dehydrogenases (including one or more enzymes having the same or similar sequences as any of the sequences in SEQ ID NOs: 1-17, such as enzyme 2 corresponding to SEQ ID NO: 6) can effectively improve the pungency of potable beverages and beverage components. The above results further indicate that one or more of the enzymes evaluated (including all the enzymes) can maintain effective activity under the conditions related to the production of alcoholic beverages (such as the production of distilled spirits), and enzyme 2 showed the highest activity in a specific experiment.

[0105] Experiment 3 - Distillation Analysis of Enzyme 2

[0106] When making distilled spirits using a pot still, the initial portion of the distillate is usually discarded or not collected ("the heads"). After discarding the heads, the "heart" of the collected distillate is used to make the beverage. Finally, the portion of the distillate containing off-flavors ("the tails") is usually also discarded.

[0107] In Experiment 3, gas chromatography-mass spectrometry was used to characterize the distillate heads, the final 40% v / v ethanol product, and the post-fermentation / pre-distillation samples obtained during the production of the distilled spirits of the present disclosure, with and without treatment with enzyme 2. Specifically, for each intermediate and final product in the fermentation / distillation process, control samples, samples treated with enzyme 2, and samples treated with enzyme 2 and NAD+ cofactor were obtained respectively. By identifying the analyte peaks in each sample, it was determined whether enzyme 2 could reduce the presence of potentially pungent compounds at one or more stages of the distilled spirit production process.

[0108] Figure 13 The analyte peaks detected in the 40% ethanol samples are shown. The control samples (marked with asterisks) had the highest number and height of peaks, indicating that there were more analytes and higher concentrations of analytes in the samples without aldehyde dehydrogenase treatment. The peaks of the samples treated with enzyme 2 (marked with circular dots) were smaller than those of the control group, while the samples treated with enzyme 2 and NAD+ cofactor (marked with squares) had the fewest peaks, indicating that the effect was best when enzyme 2 was used in combination with the NAD+ cofactor.

[0109] Figure 14Shows the analyte peaks detected in the head sample. The control group sample shows the largest number and tallest peaks (marked with asterisks). The peaks of the sample treated with Enzyme 2 (marked with circles) are significantly smaller, while the peaks of the sample treated with Enzyme 2 and NAD+ cofactor (marked with squares) are again the fewest, which further indicates that in some examples, Enzyme 2 may be most effective when used in combination with the NAD+ cofactor, and Enzyme 2 alone may also be effective. Similarly, any one or other aldehyde dehydrogenase having at least about 80%, 85%, 90%, 95%, 99% or 100% homology with any of SEQ ID NOS: 1-17 may also be effective.

[0110] Figure 15 Shows the analyte peaks detected in the fermentation sample. As shown, the total number of detected peaks is more than five times that of the 40% ethanol and head samples, indicating the presence of more diverse analytes in the fermentation broth. Enzyme 2 with and without the NAD+ cofactor (peaks marked with circles and squares respectively) both reduced the number of peaks relative to the control group (marked with asterisks), which further indicates that in the fermentation broth, Enzyme 2 may be effective with or without the cofactor. Similarly, any one or other aldehyde dehydrogenase having at least about 80%, 85%, 90%, 95%, 99% or 100% homology with any of SEQ ID NOS: 1-17 may also be effective, which is at least partly attributed to their common activity and homology.

[0111] Experiment 4 - First Distillation Test with Enzyme 2: Testing Unaged Whiskey

[0112] In a distillation experiment for producing whiskey samples for tasting, the ability of Enzyme 2 (with or without cofactor) to act on very low concentrations of various aldehydes within a specific pH and temperature range was further evaluated. This experiment was conducted at a commercial craft distillery. By collecting sensory data generated after consumers drank the enzyme-treated distilled alcoholic beverages, the effective oxidation effect of aldehyde dehydrogenase on the target aldehydes was determined.

[0113] To prepare whiskey ("white dog" whiskey), a mash mixture composed of corn, rye malt, barley malt and dextrin was first formed and added to a 5.5-gallon fermenter. No acids were added. The starting temperature was 75°F (about 23.9°C) and the initial pH was 5.7. Subsequently, fermentation was carried out using brewer's yeast. Approximately 2.75 pounds of glucose equivalent were added to the fermentation broth at about 24 hours and 48 hours after the start of fermentation. The pH value of the fermentation broth changed continuously during fermentation. After 48 hours, the measured pH was about 3.5. After successive additions of teaspoonfuls of calcium carbonate for adjustment, the final pH reached 6.7. This pH regulator is a commonly used ingredient by distillers and brewers. Fermentation continued until the ethanol content in the fermentation broth reached about 15.6%.

[0114] The fermented broth was then divided into three 5-liter samples, with one set as a negative control and the other two as test samples. The control sample was not treated with Enzyme 2; the first test sample was treated with Enzyme 2 at a ratio of 1:1000 at 90°F and pH 6.7; the second test sample was treated with Enzyme 2 (at a ratio of 1:1000) and the dinucleotide cofactor (NAD+) at 91°F and pH 6.7. The control sample had an ABV of 15.6%, the first test sample had an ABV of approximately 12%, and the second test sample had an ABV of approximately 13%.

[0115] All samples were distilled at an initial pressure of approximately 2.5 psi and a boiling temperature of approximately 130°F. The same volume percentage of the core in the 125 - 135 proof range was collected for each sample, and the final distillate was diluted to 80 proof.

[0116] Subsequent sensory tests were conducted to determine if the effects on pungency were perceptible. Thirty subjects tasted the control sample and the test samples separately. Compared to the control sample, the pungency of the first test sample and the second test sample was reduced by 85% and 100% respectively. Therefore, adding aldehyde dehydrogenase to the fermented broth can significantly reduce the final pungency level of unaged whiskey, and the combined use of aldehyde dehydrogenase and cofactor is particularly effective. This data confirms that by adding aldehyde dehydrogenase to the fermented broth, and by adding aldehyde dehydrogenase and cofactor together to the fermented broth, and preparing distilled alcoholic beverages from the enzyme-treated fermented broth, those compounds that cause pungency among the hundreds of compounds produced during fermentation can be eliminated or reduced to levels below sensory detection.

[0117] Regarding the ability of these enzymes to act on multiple aldehydes at very low concentrations within a specific pH and temperature range during a typical fermentation process, we further evaluated this through the tasting of other subjects who tasted the samples independently and reported the results separately. By collecting the sensory data generated by the subjects after drinking the distilled alcoholic beverages treated or untreated with the enzyme, we determined the effective oxidation effect of aldehyde dehydrogenase on the target aldehydes.

[0118] All 30 subjects were not only able to distinguish between the treated and untreated samples, but also able to clearly identify which samples were treated and which were untreated based on the relatively stronger pungency of the untreated samples.

[0119] Experiment 5 - Second Distillation Test of Enzyme 3: Vodka Test

[0120] This experiment demonstrated that enzyme 3 (corresponding to SEQ ID NO: 8), in the presence of cofactor NADP+, can significantly reduce the pungent compounds and sensory perception in vodka. Vodka mash was prepared using white winter wheat malt by heating with water and calcium chloride to 152°F and holding for 1 hour. After cooling to 70°F and aerating, yeast starter and yeast nutrient salts were added to initiate fermentation. At the end of fermentation, sodium bicarbonate was used to control the pH to a stable value of 6.5. Before distillation, part of the fermented broth was treated with enzyme 3 (at a ratio of 1:1000) and cofactor (NADP+) at 86°F (30°C) for 24 hours. At the end of fermentation, the alcohol by volume (ABV) of the fermented broth entering the distillation stage was 9.4%. The fermented broth was distilled, and the heads were collected at 184 proof (92% ABV), and finally diluted to 80 proof (40% ABV) for tasting.

[0121] Sensory evaluations were performed by 10 people, tasting the control sample and the enzyme-treated sample. All 10 subjects were able to clearly identify the treated sample, with its smoothness increased by at least 80%, specifically manifested as completely eliminating the pungent bite experienced when drinking the control sample.

[0122] Experiment 6 - Comparative Test of Natural Enzyme and Genetically Engineered Enzyme

[0123] To demonstrate that both native and engineered oxidoreductases are effective alcohol pungency reducers, we conducted a series of tests to show the efficacy of the engineered enzyme in catalyzing the conversion of pungent compounds in specific reactions. As Figure 16 shown, both the native dehydrogenases corresponding to SEQ ID NOS: 1 - 17 and the engineered dehydrogenases based on the enzymes of SEQ ID NOS: 1 - 17 exhibited effective substrate catalytic activity, as evidenced by the strong rates of target substrate catalysis shown by the native enzyme group (marked by dotted triangles) and the engineered enzyme group (marked by dotted parallelograms). In contrast, the sample without any form of enzyme treatment showed little substrate catalytic activity (marked by dotted circles). These data further indicate that a variety of native or engineered aldehyde dehydrogenases can be used according to the methods of the present disclosure to catalyze those substrates that are determined to generally cause the pungency associated with potable alcoholic beverages and their components.

[0124] The foregoing description of the embodiments is by way of example only and is in no way intended to limit the disclosed embodiments or their application or use. Refer to the accompanying drawings, which form a part of this invention and show the systems and methods in the manner of illustrating specific embodiments. The description of these embodiments has been detailed enough to enable those skilled in the art to implement the currently disclosed systems and methods. It should be understood that other embodiments may be adopted and structural and logical modifications may be made without departing from the spirit and scope of the present system. For example, it should be understood that any example, embodiment, and / or process described herein may be combined with one or more other examples, embodiments, and / or processes, or separated and / or performed between different devices or components of the device according to the present system, device, and method. In addition, for the sake of clarity, certain features that are obvious to those skilled in the art are not discussed in detail so as not to obscure the description of the present system. Therefore, this detailed description should not be construed as a restrictive interpretation, and the scope of the present system is only defined by the appended claims.

Claims

1. A method for producing a potable alcohol product, characterized in that, The method includes: forming a mash mixture; fermenting the mash mixture to form a fermentation broth; mixing at least one oxidase with the fermentation broth; and collecting the fermentation broth for addition to the potable alcohol product.

2. The method according to claim 1, wherein The at least one oxidase includes aldehyde dehydrogenase.

3. The method according to claim 2, wherein The aldehyde dehydrogenase is a native aldehyde dehydrogenase.

4. The method according to claim 2, wherein The aldehyde dehydrogenase is an engineered or modified aldehyde dehydrogenase.

5. The method according to claim 2, characterized in that, The aldehyde dehydrogenase selectively oxidizes fatty aldehydes.

6. The method according to claim 5, wherein The fatty aldehydes include C2-C10 fatty aldehydes.

7. The method according to claim 6, wherein The potable alcohol product has a lower content of fatty aldehydes compared to a potable alcohol product produced by the method of claim 1 but without mixing the at least one dehydrogenase with the fermentation broth.

8. The method according to claim 7, wherein The concentration range of the content of fatty aldehydes in a potable alcohol product produced by the method of claim 1 but without mixing the at least one oxidase with the fermentation broth is from ppm to ppb.

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

10. The method according to claim 9, wherein The dinucleotide cofactor includes NAD + , NADP + or both.

11. The method of claim 1, further comprising distilling the fermentation broth to form distilled ethanol, the distilled ethanol being formulated to be included in the potable alcohol product.

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

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

14. The method according to claim 1, characterized in that, The potable alcohol product includes beer.

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

0.

16. The method according to claim 1, wherein The pH of the fermentation broth is not adjusted.

17. The method according to claim 1, wherein The addition amount of the at least one oxidase is about 5 mg / L to about 2 g / L of the fermentation broth volume.

18. The method according to claim 1, characterized in that, The fermentation mash mixture includes the fermentation mash mixture for at most about 5 days.

19. The method according to claim 1, characterized in that, The mash mixture includes one or more of corn, rye, rice, barley, wheat, agave, dextrin, potato, fruit, molasses, water, enzyme, sugar source, or sucrose.

20. The method according to claim 1, wherein The ethanol concentration of the fermentation broth is about 1% to about 20%.

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