HK1167873B - A method for production of an alcoholic beverage with reduced content of alcohol - Google Patents

Description

Method for producing alcoholic beverage with reduced alcohol content

Technical Field

The present invention relates to a method of reducing alcohol in alcoholic beverages such as cider, beer, low or no alcohol wine and other low or no alcohol beverages comprising treating the beverage stock with a combination of glucose oxidase and glucose isomerase.

Background

Due to consumer health issues and ethanol intolerance issues, there is an increasing need for alcoholic beverages with reduced ethanol content while having the organoleptic properties characteristic of traditional "high-alcohol" products. Likewise, global warming has resulted in higher sugar content in fruits and berries worldwide, and higher alcohol content in the resulting products when they are used as a stock solution for alcohol fermentation. Such high alcohol content can adversely affect the organoleptic properties of the product.

Thus, there is a need for methods that can control the alcohol content of alcoholic beverages while retaining the organoleptic properties of the specific product.

Current methods of ethanol reduction, like reverse osmosis, rotating cones or dilution, are not satisfactory. These methods can have a detrimental effect on the organoleptic quality of the beverage. Furthermore, reverse osmosis of alcoholic beverages at a cost of up to $ 1/gallon is a major limitation to the widespread use of this method.

US4675191 (published by Novo Industri, denmark-1987) describes a method of reducing the alcohol content of wine which involves the use of glucose oxidase. For the method described, column 2, lines 25-29 write to:

"the process of the invention comprises treating unfermented grape juice with glucose oxidase in the presence of oxygen, thereby converting the glucose in the grape juice to gluconic acid and thereafter fermenting the thus treated grape juice. "

The main relevant technical elements of this prior art method are schematically illustrated in fig. 1 herein.

International PCT application No. PCT/EP2008/068161 was filed on month 12, 2008 and 22. Hansen A/S, and which application was not published at the time of filing of the present application.

PCT/EP2008/068161 describes a method of reducing alcohol in wine made from grape juice comprising the use of an enzyme as described herein. No application of this principle to reduce alcohol in other alcoholic beverages is described in PCT/EP 2008/068161.

Disclosure of Invention

The problem to be solved by the present invention is to provide a new method for reducing the alcohol content in an alcoholic beverage (hereinafter referred to as "alcoholic beverage" or simply "beverage"), wherein the method results in a significant reduction of the alcohol content in the final beverage. Furthermore, the process significantly reduces the risk of unwanted stuck alcoholic fermentations.

The method is based on the findings of the present inventors. They surprisingly found that the art methods based on glucose oxidase can also be significantly improved by including the use of glucose isomerase.

A schematic comparison of the method of US4675191 and the method of the present invention is illustrated in figure 1. It can be seen in the working examples herein that the method using only glucose oxidase results in a total sugar reduction of about 12%. The addition of glucose isomerase increased the sugar reduction significantly to about 19%. Less sugar (substrate of the enzyme) in the beverage stock means less alcohol content in the resulting beverage.

Furthermore, the inventors have found that the additional addition of glucose isomerase helps to maintain the glucose/fructose ratio in the beverage base at around 1: 1, which significantly reduces the risk of unwanted stuck alcoholic fermentations. See working examples herein for more details.

It is known to the skilled person that during alcoholic fermentation the glucose/fructose ratio in any solution containing glucose and fructose should not deviate significantly from 1: 1. If this happens, there is a risk of the alcoholic fermentation stagnating, i.e. the yeast does not ferment all the sugar and the resulting beverage will be too sweet.

An important commercial application of glucose isomerase is the conversion of glucose to fructose, for example to produce high fructose syrup (fructose is sweeter than glucose).

In view of this, it was in fact surprising for the inventors that the addition of glucose isomerase provided such positive results (obviously the less sugar ═ the less alcohol). One reason for this result is that glucose isomerase appears superficially to be an enzyme that can "remove/convert" too much glucose, and therefore less substrate will be available for glucose oxidase (glucose oxidase does not act on fructose).

However, as shown in the working examples herein, the addition of glucose isomerase provided very positive results.

Without being limited by theory, it is believed that the following can theoretically explain why the addition of glucose isomerase provides such positive results:

glucose oxidase (EC 1.1.3.4) primarily catalyzes the following reaction in a beverage stock (also referred to herein as a solution):

beta-D-glucose + O₂(ii) > D-glucono-1, 5-lactone + H₂O₂

The "D-glucono-1, 5-lactone" produced is converted spontaneously in solution to gluconic acid. Thus D-glucono-1, 5-lactone is removed and thus the equilibrium goes to the right-glucose removal from the solution.

If the enzyme preparation also has catalase activity, H is produced₂O₂Also removed > then the equilibrium is more to the right > more glucose is removed. Inclusion of catalase activity is a preferred embodiment herein-discussed below.

Catalase (EC 1.11.1.6) catalyzes the reaction:

2H₂O₂＜＝＞O₂+2H₂O

an enzyme of particular interest in the process of the invention is glucose isomerase EC 5.3.1.5. The formal name of this class EC5.3.1.5 is xylose isomerase. However, as known to the skilled person, it may also be referred to as glucose isomerase. Glucose isomerase is the name used in related commercial products such as the class of enzymes, for example the commercial products used in the working examples herein.

In a beverage stock, the reactions catalyzed by glucose isomerase that are relevant and well known herein are the following reactions:

d-glucose & lt & gt D-fructose.

As known to the skilled person, the enzymes may also catalyze the reaction:

d-xylose ≦ D-xylulose.

This xylose-related reaction is less relevant herein.

One theory is that the removal of glucose by glucose oxidase creates a situation in solution where the glucose/fructose ratio becomes less than 1: 1 ("too much" fructose- "too little" glucose). Thus, the glucose/fructose equilibrium of the glucose isomerase reaction is "forced" to the left-side, > fructose is converted to glucose to "restore" a 1: 1 glucose/fructose ratio > glucose oxidase to obtain "fresh" produced glucose to continue working and thus remove more total sugar (both glucose and fructose) from the solution.

As mentioned above, maintaining a 1: 1 glucose/fructose ratio also has the advantage of significantly reducing the risk of stuck alcoholic fermentations.

Before alcoholic fermentation of yeast, O₂Present in the unfermented solution (beverage stock). As known to the skilled person, normal yeast fermentation typically comprises two parts:

part 1

Aerobic growth (presence of oxygen)

This is the initial rapid growth process in which the number of yeast cells doubles approximately every 4 hours. (typically 24-72 hours)

Section 2

Anaerobic fermentation (absence of oxygen)

The slower activity and yeast ferment sugars (both glucose and fructose) to convert them to alcohol (sugars > 2 ethanol +2 CO)₂) Rather than increasing the number of yeast cells. (depending on the yeast and formulation, this process can be performed for days to weeks).

Thus, during yeast fermentation, O₂Will disappear sooner or later. The activity of glucose oxidase requires O₂. However, glucose isomerase in the presence or absence of O₂Is active in this case. Thus, glucose isomerase can also help maintain a 1: 1 glucose/fructose ratio during actual yeast alcoholic fermentation.

Accordingly, a first aspect of the present invention relates to a method of producing an alcoholic beverage with a reduced alcohol content, comprising the steps of:

(1): treating an unfermented beverage base with effective amounts of the following two enzymes:

(a) treating with glucose oxidase in the presence of oxygen for a period of time sufficient to convert at least a portion of the glucose in solution to gluconic acid; and

(b) treating with glucose isomerase for a time sufficient to convert at least a portion of the fructose in solution to glucose;

and thereafter,

(2): fermenting said treated solution having a reduced amount of glucose and fructose to obtain an alcoholic beverage having a reduced alcohol content.

Definition of

All definitions of related terms are consistent with their commonly understood meanings by those skilled in the relevant art.

The term "reduced alcohol content" in a beverage produced according to the method of the first aspect of the invention relates to a beverage having a lower alcohol content compared to a beverage produced under the same conditions but without treatment with the two enzymes of step (1) of the first aspect. In fact, the term can be seen as directly related to the use of an effective amount of both enzymes.

If an effective amount of glucose oxidase is used, at least part of the glucose will be removed from the solution and thus less alcohol in the beverage. Similarly, for an effective amount of glucose isomerase, it converts at least part of the fructose in solution to glucose > the glucose so produced is then removed by glucose oxidase > and thus less alcohol in the beverage.

Embodiments of the present invention are described below by way of example only.

Drawings

FIG. 1 shows a schematic view of a: the method of US4675191 is schematic illustration/comparison of the method of the present invention.

Detailed Description

Reduction/reduction of alcohol content:

indeed, the methods described herein may be used to produce alcoholic beverages with virtually any desired reduced or lower alcohol content.

For example, it may be a so-called light beverage (light beer) with an alcohol content of about 10%, for example 5-10% alcohol content by volume (ABV), 6-7% ABV or even lower alcohol content, for example 1-5% ABV, including 2-4% ABV.

As noted above, due to global warming, fruits and berries around the world contain more sugar, resulting in beverages with increased alcohol levels. This may be undesirable for different reasons, such as poor taste. In addition, due to consumer health concerns, the demand for low alcohol beverages has increased. Finally, low-alcohol beverages are needed for target populations suffering from ethanol intolerance.

Thus, the present invention addresses the problem of reducing the alcohol content of a beverage while maintaining the characteristics of the particular beverage, such as taste, flavor, color, etc.

Gluconic acid:

treatment of the unfermented beverage stock with glucose oxidase produces gluconic acid, which cannot be fermented by yeast and thus is present in the beverage. As is known to the skilled person, gluconic acid gives beverages with unsatisfactory organoleptic properties.

Accordingly, an embodiment of the method of the first aspect comprises the optional step (3) of removing at least part of the gluconic acid to obtain a beverage with satisfactory organoleptic properties.

In one embodiment, the gluconic acid is removed by neutralization means by adding a material that forms a sparingly soluble salt of gluconic acid, preferably calcium carbonate. Calcium carbonate is inexpensive and has been used as a chemical deacidification agent for beverages, and precipitated gluconates, mainly calcium gluconate, can be easily removed by filtration.

Glucose oxidase and glucose isomeraseThe glucose oxidase and glucose isomerase enzymes to be used in the methods as described herein may be obtained from a variety of different suitable sources, such as the relevant commercially available enzyme products.

As known to the skilled person, there are numerous different commercially available glucose oxidase/isomerase products on the enzyme market that work at relevant pH, temperature, etc. Another useful enzyme is hexose oxidase, which is capable of converting other hexoses.

In the following working examples, the following commercially available enzyme products were used:

glucose oxidase: hyderase(from Amano)

Glucose isomerase: product from Sigma (# G4166-50G). Catalog number-see working examples herein.

Hyderase One advantage of the product is that it also contains catalase activity.

The "beverage stock" is a substrate for the above-mentioned enzymes and is ultimately used for alcoholic fermentation. Suitable substrates for enzymes include, but are not limited to: glucose, fructose, xylose, mannose, galactose, any other hexose, and any combination thereof. The present invention includes any carbohydrate that can serve as a substrate for the enzyme. Obviously, in order to achieve the best results of the invention, the substrate preferably comprises glucose and fructose as the main sugar components.

Useful sources of such sugars are a range of fruits, berries and cereal grains. Such products in liquid form are widely used as "beverage liquids" and are commonly referred to as "unfermented grape juice" (must), "juice" (juice) or "juice" (worth) in the production of alcoholic beverages. Conventional examples include grapes used in the production of wine, apples and pears used in the production of cider, and barley used in the production of beer. However, more exotic (exotic) products are often used as raw materials to create new beverages, examples of which include, but are not limited to, pomegranates, pineapples, strawberries, and mangoes.

In one embodiment, the beverage base is grape juice, as described in PCT/EP 2008/068161.

Catalase Activity

As noted above, the use of an enzyme preparation having catalase activity in the methods described herein may also result in the removal of H produced by glucose oxidase₂O₂。

Hydrogen peroxide (H)₂O₂) Can produce objectionable colors and, therefore, is not a desired ingredient in the beverage.

Further, as described above, by removing H₂O₂The glucose oxidase equilibrium can be shifted even further to the left-more glucose is removed.

Thus, in a preferred embodiment of step (1) of the first aspect of the invention, the solution is also treated with an effective amount of a preparation having catalase activity sufficient to remove at least part of the H in the juice₂O₂Conversion to O₂+H₂A period of time of O.

Preferred production parameters-step 1 of the first aspect

As is known to the skilled person, variations in the beverage manufacturing process change the organoleptic properties of the beverage product. Thus, a close fit between the usual beverage manufacturing process and the implementation of the method described herein is preferred. Thus, by practicing the present invention, no adverse effects are observed on the taste and flavor of the beverage.

The enzyme-catalyzed processes generally proceed within the optimum pH of the enzyme. A preferred practice of the invention is to treat the unfermented solution (beverage stock) without adjusting its pH. Fortunately, suitably related commercially available enzyme products, as used herein, exhibit sufficient activity and stability in the normal pH range of the unfermented beverage bulk.

Depending on the specific substrate (beverage stock), the sugar content and the ratio between the different sugar components may vary. For example, in apples the ratio of glucose to fructose is 30: 70, in mangos the ratio is 24: 76, in pineapples the ratio is 43: 57, and in strawberries the ratio is 20: 80. These ratios may vary depending on the climate and growth conditions and the harvest time, as known to those skilled in the art. This ratio is related to the optimal dose of the chosen enzyme. Based on this information, which is readily available, the skilled person can easily select the optimal dosage of the enzyme for a particular application.

It will be appreciated that any of the enzymes described herein may be used in the present method, provided that they exhibit reasonably relevant activity and stability at the pH and temperature commonly used during the process of producing a particular beverage. Thus, both soluble and immobilized enzyme preparations can be used, even though soluble enzyme preparations are generally preferred.

One skilled in the art can readily determine how much of a particular type of enzyme is needed for a particular application and desired sugar conversion.

For example, depending on the details of the treatment time and temperature:

(i) the method comprises the following steps A glucose oxidase activity of approximately between about 1,000 and 50,000,000 international units per hundred liters of solution would be appropriate; and

(ii) the method comprises the following steps A glucose isomerase activity of approximately between about 100 and 5,000,000 international units per hundred litres of solution would be appropriate.

Thus, no additional values are provided for the present invention to specify the appropriate enzyme dosage, as this will be relatively simple to determine experimentally under specific process parameters. As set forth below, the relevant amounts used in the examples are within the above levels and the above ranges are assumed to cover any relevant application.

In this context, the international units are as described above and are determined according to the definition in the art, i.e.at a temperature of 30 ℃ and at a pH value and substrate concentration at which maximum substrate conversion is obtained.

As known to the skilled person, the optimum pH and the optimum substrate concentration will vary with the particular enzyme of interest (e.g.the particular glucose oxidase). However, such optimum pH and substrate concentration can be easily identified, since it is for example usually given in the product documentation of the relevant commercially available enzyme products. Furthermore, it is often routine work to identify parameters such as pH optimum and substrate concentration for a particular enzyme of interest. Thus, when applying the principles of the present invention in the production of a desired alcoholic beverage, the best match between the available enzyme product and the process parameters should be considered.

In a preferred embodiment, the following are used:

(i) the method comprises the following steps A glucose oxidase activity of approximately between about 15,000 and 5,000,000 international units per hundred liters of solution; and

(ii) the method comprises the following steps A glucose isomerase activity of approximately between about 5,000 and 500,000 international units per hundred liters of solution.

In a preferred embodiment, the temperature during step (1) of the first aspect is between 1 and 35 ℃, preferably between 3 and 30 ℃. Generally, in an enzymatic reaction, if the temperature is raised from, for example, about 25 ℃ to 40 ℃, the overall reaction rate will increase with temperature. However, in this case, if the temperature is raised from 25 ℃ to 40 ℃, more oxygen will be released from the liquid and thus the overall reaction rate will be reduced in this case. Prolonged treatment at 40 ℃ is also detrimental to the quality of the solution and thus to the resulting beverage.

Overall, preferably, the only change made during beverage making is short term storage of the unfermented solution while the (aerated) solution is being treated with glucose oxidase, isomerase, and optionally other enzymes described herein. In this regard, it should be noted that the control parameter will be the treatment time, not the enzyme activity. For example, sufficient glucose oxidase should be used to convert the desired proportion of glucose within an appropriate treatment time, which typically does not exceed 72 hours, and most of the time does not exceed 48 hours. Even if some yeast is present in the unfermented solution, it was found that fermentation did not start to any appreciable extent during the first 48 hours and thus it was possible to remove relevant amounts of sugar.

As regards step 1 of the first aspect, it should be noted that the degree of conversion is very easy to control, since the reaction from glucose to gluconic acid by cutting off the oxygen supply stops almost immediately.

The implementation of the invention also takes into account the case: i.e. the reduced sugar solution from step 1 is continuously supplied as acid (acid storage) and mixed with the acid-deficient solution to improve the organoleptic properties of the resulting beverage.

It will be appreciated that the removal of gluconic acid may be carried out at any time, for example after the fermentation step.

Alternatively, the removal of gluconic acid may be performed by adding e.g. calcium carbonate before adding glucose oxidase as described herein. The advantage is that the pH in the beverage bulk is increased and this generally improves the activity of glucose oxidase, since glucose oxidase generally has an optimum pH at around pH neutrality.

In a preferred embodiment, the one or more relevant enzyme preparations are solid, water-soluble preparations, preferably non-dusting preparations. The storage stability of the solid formulation is superior to that of the liquid formulation, and it is also unnecessary to add any preserving agent. Although it is recommended that the user dissolve the solid form agent in a small amount of water immediately before use.

In a preferred embodiment of the process described herein, the supply of oxygen to the solution is continued during step 1 of the first aspect. Oxygen supply has a very large influence on the reaction rate of the enzymatic reaction of glucose oxidase in particular. Thus, the continuous introduction of oxygen ensures a high reaction rate. Desirably, the oxygen is supplied by an air pump, which is the most efficient means of introducing oxygen into the solution.

In a preferred embodiment of the method according to the invention, the amount of glucose oxidase/isomerase preparation added in step 1 is sufficient to produce the desired reduction in sugar concentration in a period of time not exceeding about 72 hours. As already indicated, even if some yeast is present in the solution, the fermentation does not progress to any appreciable extent during the first 48 hours, and therefore no appreciable amount of glucose/fructose is simultaneously fermented to alcohol during the enzymatic conversion of glucose to gluconic acid.

In a preferred embodiment, during step (1), the effective amounts and time periods for both glucose oxidase/isomerase enzymes are such that:

(A) the method comprises the following steps The sugar content in the solution is reduced by at least 10%, more preferably by at least 14% and even more preferably by at least 17%.

As described above, in the working examples herein, the sugar content (both glucose and fructose) was reduced by 19%.

In a preferred embodiment of the process according to the invention, the pH in step 1 is not controlled. This embodiment is particularly preferred where longer processing times, for example up to about 72 hours, are feasible and employed.

Due to the fact that aroma, taste and aroma of alcoholic beverages are extremely sensitive properties, it is not possible to predict whether a lower alcohol beverage produced according to the present invention will have the desired properties. In addition, it is contemplated that alcoholic beverages produced using the soluble glucose oxidase/isomerase formulation according to the present invention will contain trace amounts of inactive glucose oxidase/isomerase and thus differ from conventional beverages. However, it has been found that the lower alcohol beverages produced according to the present invention have all of the normal characteristics of conventionally produced products, including taste and aroma, except, of course, those directly related to alcohol concentration and gluconic acid production.

Preferred production parameters-step 2 of the first aspect

It is an essential step of the process of the invention to carry out step 2 of the first aspect, i.e. the fermentation of the treated solution. However, a detailed discussion of the fermentation step need not be provided herein, as it is expressly contemplated that conventional beverage production practices may be developed and those practices are well known to those skilled in the art. It has been emphasized herein that the preferred practice according to the present invention avoids modifications to the beverage production process.

As mentioned above, during yeast fermentation, O₂Will disappear sooner or later. Glucose oxidase requires O₂To function. However, glucose isomerase may be in the presence or absence of O₂The situation is functional. Thus, glucose isomerase can also help maintain a 1: 1 glucose/fructose ratio during the actual yeast alcoholic fermentation.

In practice, this will typically occur during the process as described herein. The glucose isomerase added to the unfermented solution during step (1) is normally still active during the yeast alcoholic fermentation of step (2).

However, optionally, additional glucose isomerase may be added during the yeast alcoholic fermentation of step (2).

Thus, in an embodiment of the invention of the first aspect, additional glucose isomerase is added during the yeast alcoholic fermentation of step (2).

Examples

Example 1: enzymatic sugar reduction in grape juice-step (1) example of the first aspect

One possible way to reduce the final alcohol content in a beverage is to reduce the sugar concentration in the solution prior to alcoholic fermentation. Thus, in the case of this grape must, an enzymatic treatment of the solution is carried out to reduce the total sugar content.

Three independent experiments were performed, using two replicates in each case. In each sample, 200ml of grape juice (Pinot Blanc 2007, germany, pasteurized) was added to a glass flask and mixed continuously using a magnetic stirrer. The samples were aerated throughout the experiment.

100mg of glucose oxidase (Hyderase, Amano, > 15,000u/G, corresponding to 150,000 u/hl solution) or 100mg of glucose oxidase and 1G of glucose isomerase (Sigma, G4166-50G, > 350u/G, corresponding to 35,000 u/hl solution) were added to the flask. Incubate at room temperature for 3 days.

Samples were taken immediately before and 3 days after the addition of the enzyme. Samples were analyzed for the presence of glucose and fructose using a commercial UV-based assay supplied by Boehringer Mannheim/R-biopharm (Cat. No. 10139106035) and following the manufacturer's protocol. The results of this experiment are summarized in table I below.

TABLE I: enzymatic sugars (glucose and fructose) decrease in grape juice, GOX ═ glucose oxidase

Conclusion

These results of this example 1 show that the process using only glucose oxidase produced a total sugar reduction of about 12%, and the additional addition of glucose isomerase significantly increased this value to a sugar reduction of about 19%. Less sugar in the juice means a lower alcohol content in the final fermented beverage product.

Example 2: yeast fermentation of the treated grape must-an example of both step (1) and step (2) of the first aspect

In the case of this wine production, a full simulation of a normal beverage production process was performed on a laboratory scale. In this experiment it was shown that the enzyme treatment did have an effect on the final alcohol level without adversely affecting major wine production parameters, such as alcoholic fermentation or malolactic fermentation.

The complete experiment was performed at room temperature, approximately 22 ℃.6 experiments were performed, each using 4 litres of grape juice in a fermentation flask (Pinot Blanc 2007, Germany, pasteurised). The pH of the grape must was not adjusted and no other substances were added in addition to the enzymes described in this example.

Grape juice was preincubated with enzymes for 3 days as described below, followed by 11 days of alcoholic fermentation and 10 days of malolactic fermentation.

Enzyme treatment

The 6 flasks were divided into three groups of two flasks each.

The grape juice in group 1 was preincubated with 0.5G/l glucose oxidase (Hyderase, Amano, > 15,000u/G, corresponding to 750,000 u/hl solution) for three days, the grape juice in group 2 was preincubated with 0.5G/l glucose oxidase and 2G/l glucose isomerase (Sigma, G4166-50G, > 350u/G, corresponding to 70,000 u/hl solution) for three days, and the grape juice in the control group was not treated with enzyme. After the enzyme addition, the flasks were vigorously aerated for three days in the presence of the enzyme, after which alcoholic fermentation was started. Aeration is important because oxygen is required in the glucose oxidase-mediated enzymatic conversion.

Alcohol fermentation

The alcoholic fermentation was started by inoculation of rehydrated freeze-dried wine yeast (Saccharomyces cerevisiae) Merit. Ferm, Chr. Hansen, 0.1g/l) to a final concentration of 9E +05 CFU/ml. Rehydration was performed in peptone water (15g/L tryptone, Oxoid L42, 9g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes at room temperature.

At this point aeration is stopped and during the following days the process consumes oxygen due to yeast metabolism. The alcoholic fermentation was run at room temperature for 11 days, which allowed almost complete conversion of all sugars to alcohol.

Lactic acid fermentation of apples

After the alcoholic fermentation, the malolactic fermentation was started. The goal of this part of the process is to convert malic acid to lactic acid which produces a more pleasant organoleptic sensation and is therefore an important part of, for example, a cider or wine production process. The lactic acid fermentation of apples is mainly performed by the bacterium Oenococcus oeni. If the growth of the hop bacteria is impaired by enzymatic treatment of the juice, it is highly undesirable.

After 11 days from the start of alcoholic fermentation, malolactic fermentation was started by adding wine coccus (vinifera, chr. hansen. batch: 2711097) to the fermented grape juice. Freeze-dried distillers yeast (0.7g of 8.2E +11CFU/g) was rehydrated in 100ml peptone water (15g/L tryptone, Oxoid L42.9 g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes. To 4000ml of fermented grape juice 3ml were added, resulting in a final concentration of 4.3X 10⁶CFU/ml. It was allowed to stand at room temperature for another 10 days.

Results

Effect of enzyme treatment on alcohol levels

Glucose and fructose levels were measured using a commercial UV-based assay supplied by Boehringer Mannheim/R-biopharm (catalog number 10139106035) using a protocol supplied by the supplier.

Table II:sugar levels at the beginning and end of alcoholic fermentation.

At different days during the alcoholic fermentation, the literature is used, for example (Bestimung des koholgelhals nach Dr. Rebelein. issued by: C Schliesmann Kellerie-Chemie GmbH)& Co.KG，Auwiesenstrasse 5，74523 Rebelein titration method described in Hall (2001)) measures alcohol. In untreated grape must, the fermentation is almost complete, reaching a final alcohol level of 12.7% at the end of the fermentation process. When the grape juice was pre-treated with glucose oxidase and glucose isomerase, the sugar fermentation was complete, but the final level of alcohol was significantly lower (11.8%).

Low levels of alcohol were found when the juice was pretreated with only glucose oxidase, which was the result of incomplete fermentation. In this experiment, the glucose oxidase treated juice was not usable in normal fermentation because the residual sugars, in particular fructose, were high at the end of the fermentation (table II).

Thus, the additional addition of glucose isomerase helps to maintain the glucose/fructose ratio in the must at about 1: 1, which significantly reduces the risk of unwanted stuck alcoholic fermentations as shown when using GOX alone.

Furthermore, the experiment with isomerase removed all sugars, while some fructose (8g/l) was still present in the control (untreated grape must). This demonstrates that the isomerase enzyme also prevents fermentation arrest.

Table III:alcohol levels during fermentation. Malolactic fermentation began on day 11. The alcohol levels in the Glucose Oxidase (GOX) pretreated samples are indicated in italics to indicate that these values are the result of severe retardation of the alcoholic fermentation. Nd: can not determine

Number of days	Treatment of	Alcohol (volume%)
			0	Control	0
	GOX	0
				GOX + isomerase	0
7	Control	10.9±0.3
				GOX	Nd
	GOX + isomerase	10.7±0.5
			11	Control	12.3±0.1
	GOX	7.5±1.3
				GOX + isomerase	11.7±0.1
16	Control	12.7±0.1

	GOX	9.3±0.6
				GOX + isomerase	11.8±0.01

Conclusion

The results of this example 2 show that GOX + isomerase significantly reduced the percentage of alcohol to 11.8% compared to 12.7% for the control.

Furthermore, the additional addition of glucose isomerase helps to maintain the glucose/fructose ratio in the juice at about 1: 1, which significantly reduces the risk of unwanted stuck alcoholic fermentations compared to the use of GOX alone.

Low levels of alcohol (9.3%) were found when the juice was pretreated with only glucose oxidase, which was the result of incomplete fermentation, in other words, undesirable stuck alcoholic fermentation. The glucose oxidase treated juice was not used for normal fermentation in this experiment because the residual sugar, especially fructose, was high at the end of the fermentation (table II).

Furthermore, the experiment with isomerase removed all sugars, while some fructose (8g/l) was still present in the control (untreated juice). This demonstrates that isomerase as such prevents fermentation arrest.

Example 3: yeast growth during alcohol fermentation-addition of isomerase significantly reduced alcohol fermentation stasis.

It is known to the skilled person that fermentation stalls typically occur when fructose concentrations are much higher than glucose concentrations. During alcoholic fermentation, the glucose/fructose ratio may change, which leads to a delay in fermentation.

In this example 3, delayed (stagnant) fermentation was induced by treating unfermented grape juice with only glucose oxidase.

To investigate the effect of glucose isomerase on yeast growth and viability during alcoholic fermentation, simulated beverage production-in this case wine-was carried out as described in example 2 herein. Grape juice was preincubated with enzymes for three days, followed by 11 days of alcoholic fermentation and 10 days of malolactic fermentation, as described below.

Three separate experiments were performed, using two replicates in each experiment. In each sample, 200ml of grape juice (Pinot Blanc 2007, germany, pasteurized) was added to a glass flask and mixed continuously using a magnetic stirrer. The samples were aerated throughout the experiment.

100mg of glucose oxidase (Hyderase, Amano, > 15,000u/G, corresponding to 150,000 u/hl solution) or 100mg of glucose oxidase and 1G of glucose isomerase (Sigma, G4166-50G, > 350u/G, corresponding to 35,000 u/hl solution) were added to the flask. Incubate at room temperature for 3 days. After this time point, alcoholic fermentation was started by inoculation of rehydrated freeze-dried wine yeast (Saccharomyces cerevisiae Merit. Ferm, Chr. Hansen, 0.1g/l) to a final concentration of 9E +05 CFU/ml. Rehydration was performed in peptone water (15g/L tryptone, Oxoid L42.9 g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes at room temperature. Malolactic fermentation was started 11 days after the start of alcoholic fermentation by adding oenococcus vinifera (vinifera, chr. hansen. batch: 2711097) to the fermented juice. Freeze-dried distillers yeast (0.7g of 8.2E +11CPU/g) was rehydrated in 100ml peptone water (15g/L tryptone, Oxoid L42.9 g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes. To 4000ml of fermented juice 3ml were added, yielding 4.3X 10⁶Final concentration of CPU/ml. Let stand at room temperature for another 10 days.

The number of Saccharomyces cerevisiae Colony Forming Units (CFU) was determined as follows: samples were removed from the fermented juice at different time points and serial dilutions were plated on YGC solid medium agar plates, followed by overnight incubation at 30 ℃.

Sugar levels were measured using a commercial UV-based assay provided by Boehringer Mannheim/R-biopharm (catalog number 10139106035) using a protocol provided by the supplier.

Results

Effect of isomerase on alcohol fermentation retardation

During alcoholic fermentation, the sugars in the juice are converted into ethanol by saccharomyces cerevisiae.

Treatment with glucose oxidase alone showed that alcohol fermentation was delayed (fermentation was arrested) due to the delayed growth of saccharomyces cerevisiae (as shown in table IV). In unfermented grape juice pre-treated with glucose oxidase, yeast growth was very poor during the first few days of alcoholic fermentation. The CPU number was below the detection limit on the first day of alcoholic fermentation and approximately 3 log units lower on the second day. This clearly indicates fermentation arrest.

Sugar analysis supported the results. In untreated unfermented grape juice, approximately 60% of the sugars were fermented after 3 days of yeast fermentation, whereas less than 10% of the sugars were fermented in GOX-pretreated unfermented grape juice.

However, when glucose isomerase is present during pretreatment and alcoholic fermentation, the fermentation process behaves almost the same as the fermentation of untreated unfermented must. The remaining sugar levels and the Saccharomyces cerevisiae CPU numbers (Table IV) were comparable to untreated unfermented grape juice. In other words, glucose isomerase was able to overcome the fermentation lag caused by GOX treatment.

TABLE IVViable Saccharomyces cerevisiae cells were counted during the alcoholic fermentation. The juice had been pretreated for 3 days as described above. Yeast was added at day t-0. And ND is lower than the detection limit.

Number of days	Treatment of	CFU/ml (average)	Total sugar (g/l)
				0	Control	7.0±1.4E+05	229±4
	GOX	9.0±4.2E+05	225±9
					GOX + isomerase	9.0±1.4E+05	225±25
1	Control	6.5±2.1E+05
					GOX	Nd
	GOX + isomerase	1.0±0.9E+06
				2	Control	2.2±0.2E+07	212±6
	GOX	5.0±7.1E+04	220±6
					GOX + isomerase	1.1±0.9E+07	209±11
3	Control	6.9±0.9E+07	86±64
					GOX	2.7±3.3E+06	207±5
	GOX + isomerase	5.1±1.7E+07	96±80
				7	Control	3.1±0.9E+07	22±10
	GOX	3.2±1.1E+07	141±55
					GOX + isomerase	4.3±0.3E+07	11±8
9	Control	2.7±0.2E+07

	GOX	2.0E±0.8+07
					GOX + isomerase	1.1±1.6E+07
16	Control	6.9±6.6E+06	2±1
					GOX	2.9±1.0E+06	53±12
	GOX + isomerase	9.5±9.2E+05	0±0
				18	Control	2.5±0E+05
	GOX	4.0±2.1E+06
					GOX + isomerase	2.0E+05

Conclusion

As shown in this example 3, the use of GOX alone can cause significant undesirable fermentation stagnation.

The results of this example 3 show that the addition of isomerase can help to overcome the adverse effect of the addition of GOX on the growth of saccharomyces cerevisiae commonly used for beverage production.

Reference to the literature

US4675191(Novo Industri, Denmark-1987 publication)

Claims (15)

1. A method of producing an alcoholic beverage with a reduced alcohol content, comprising the steps of:

(1): treating an unfermented beverage base with effective amounts of the following two enzymes:

(a) treating with glucose oxidase in the presence of oxygen for a period of time sufficient to convert at least a portion of the glucose in the solution to gluconic acid; and

(b) treating with glucose isomerase for a time sufficient to convert at least a portion of the fructose in the solution to glucose;

and thereafter,

(2): fermenting said treated solution having a reduced amount of glucose and fructose to obtain an alcoholic beverage having a reduced alcohol content; and is

Wherein the beverage stock solution is stock solution of apple, pear, barley, pomegranate, pineapple, strawberry or mango.

2. The process of claim 1 wherein the process of claim 1 comprises the additional step (3) of removing at least part of the gluconic acid to obtain a beverage with satisfactory organoleptic properties; and wherein gluconic acid is removed by neutralisation means by addition of a material which forms a sparingly soluble salt of gluconic acid.

3. The method of claim 2 wherein the sparingly soluble gluconate salt forming material is calcium carbonate.

4. The method of any one of claims 1-3, wherein the effective amount and the period of time for both glucose oxidase/isomerase enzymes during step (1) are such that:

(A) the method comprises the following steps The sugar content of the juice is reduced by at least 5%.

5. The method of claim 4, wherein the sugar content of the juice is reduced by at least 17%.

6. The method of claim 4, wherein

-said period of time does not exceed 72 hours; and is

-the effective amounts of the two glucose oxidase/isomerase enzymes are:

(i) the method comprises the following steps (ii) a glucose oxidase activity between 1,000 and 50,000,000 international units per hundred liters of solution;

(ii) the method comprises the following steps (ii) a glucose isomerase activity between 100 and 5,000,000 international units per hundred litres of solution; and

-the temperature during step (1) of claim 1 is between 1 ℃ and 35 ℃.

7. The method of claim 5, wherein

-said period of time does not exceed 72 hours; and is

-the effective amounts of the two glucose oxidase/isomerase enzymes are:

(i) the method comprises the following steps (ii) a glucose oxidase activity between 1,000 and 50,000,000 international units per hundred liters of solution;

(ii) the method comprises the following steps (ii) a glucose isomerase activity between 100 and 5,000,000 international units per hundred litres of solution; and

-the temperature during step (1) of claim 1 is between 1 ℃ and 35 ℃.

8. The process of claim 6, wherein the temperature during step (1) of claim 1 is between 3 ℃ and 30 ℃.

9. The process of claim 7, wherein the temperature during step (1) of claim 1 is between 3 ℃ and 30 ℃.

10. The method of any one of claims 1-3, wherein oxygen is continuously supplied to the solution during step (1) of claim 1.

11. The method of claim 10, wherein the oxygen is supplied by means of an air pump.

12. The process of any of claims 1-3, wherein during step (1) of claim 1, the solution is further treated with an effective amount of an agent having catalase activity sufficient to bind at least a portion of the H in the juice₂O₂Conversion to O₂+H₂A period of time of O.

13. The method of any one of claims 1-3, wherein additional glucose isomerase is added before or during the yeast alcoholic fermentation of step (2) of claim 1.

14. The method of any one of claims 1-3, wherein the beverage base is an apple, pear or barley base.

15. The method of any one of claims 1-3, wherein the alcoholic beverage is cider or beer.