HK1146294B - A method for production of a wine with lower content of alcohol - Google Patents

Description

Method for producing wine with low alcohol content

Technical Field

The present invention relates to a method for producing a wine with a low alcohol content comprising treating unfermented grape juice with glucose oxidase and glucose isomerase.

Background

Due to global warming, grapes worldwide contain more polysaccharides. The sugars are converted to alcohol in alcoholic fermentation, resulting in the production of wine with increased levels of alcohol.

Because of this, one of the major problems in today's wine industry is high ethanol levels. Ethanol at levels of 15-16% (V/V) destroys the organoleptic quality of the wine and sometimes places the wine in higher tax classifications than expected. The elevated ethanol levels observed over the last decade have been a result of increased levels of sugar in the harvested glucose. Today, wines with more than 16% (V/V) ethanol content are no longer rare and glucose levels are predicted to increase even more with global warming.

Important wine areas currently considered to be negatively affected by global warming are important areas such as Rioja (spain), Chianti (italy), Hunter Valley (australia) and southern california.

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 wine. Furthermore, the price for reverse osmosis of wine is up to $ 1/gallon, which is a major limitation for the widespread use of this method.

US4675191(Novo Industri, published in 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 glucose 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 related art principle here of this prior art method is schematically illustrated in fig. 1.

Disclosure of Invention

The problem to be solved by the present invention is to provide a method for reducing the alcohol content in wine, wherein the method results in significantly less alcohol in the wine. Furthermore, the process significantly reduces the undesirable stuck alcoholic fermentations.

The solution is based on the fact that the present inventors have identified that glucose oxidase based methods of the art 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 process using only glucose oxidase resulted in a total sugar reduction of around 12%, while the additional addition of glucose isomerase significantly increased this value to a sugar reduction of around 19%. Less sugar in the grape juice means less alcohol content in the wine.

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

The skilled person knows that alcoholic fermentation will be stalled if the glucose/fructose ratio in the grape must is significantly different from 1.1, i.e. the yeast does not ferment all the sugars and will thus result in a too sweet wine.

As known to the skilled person, an important commercially relevant application of glucose isomerase is the conversion of glucose into fructose, for example in order to produce a high fructose syrup (fructose is sweeter than glucose).

Because of this, the inventors are indeed surprised that the addition of glucose isomerase gave such positive results (significantly less sugar — > less alcohol in wine). One reason for this is that glucose isomerase at first glance appears to be an enzyme that can "remove/convert" too much glucose, and glucose oxidase will therefore have less available substrate (glucose oxidase is ineffective on fructose).

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

Without being limited by theory, it is believed that the following is a theoretical explanation for why the addition of glucose isomerase gives very positive results.

Glucose oxidase (EC 1.1.3.4) catalyzes the following reaction in grape juice:

beta-D-glucose + O₂<＝>D-gluconic acid-1, 5-lactone + H₂O₂

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

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+2 H₂O

relevant to the methods described herein is glucose isomerase EC 5.3.1.5 (the legal name xylose isomerase). The legal name for this EC class 5.3.1.5 is xylose isomerase. However, as known to the skilled person, there is also a nickname for the enzyme known as glucose isomerase. Glucose isomerase is the name used in, for example, the relevant commercial product of the enzyme class, such as the commercial product used in the working examples herein.

In grape juice, the reactions catalyzed by glucose isomerase in grape juice that are relevant and well known herein are the following reactions:

d-glucose < ═ D-fructose.

As known to the skilled person, the enzymes also catalyse the reaction:

d-xylose (D-xylulose).

This xylose-related reaction is of little relevance herein.

One theory is that the removal of glucose by glucose oxidase leads to a situation in the grape juice where the glucose/fructose ratio becomes lower than 1: 1 (giving "too much fructose" - "too little glucose"). Thus, glucose isomerase equilibrium is "forced" to the left by > fructose is converted to glucose to "restore" a 1: 1 glucose/fructose ratio > glucose oxidase to "freshly" produced glucose to continue working and thus remove more sugar (both glucose and fructose) from the grape juice as a whole.

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, O₂Is present in unfermented grape must. As known to the skilled person, normal yeast fermentation generally consists of 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 method this process can occur over days to weeks).

Thus, during yeast fermentation, O₂Will disappear sooner or later. Glucose oxygenO is required for the activity of the chemolase₂. However, glucose isomerase with or without O₂Is active in the presence of oxygen. Thus, glucose isomerase can also help maintain a 1: 1 glucose/fructose ratio during the actual yeast alcoholic fermentation process.

Accordingly, one aspect of the present invention relates to a method for producing a wine having a low alcohol content, comprising the steps of:

(1): treating unfermented grape juice with effective amounts of the following two enzymes:

(a) glucose oxidase, treated in the presence of oxygen for a period of time sufficient to convert at least a portion of the glucose in the grape juice to gluconic acid; and

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

and subsequently,

(2): the so treated must with reduced amounts of glucose and fructose is fermented to produce a wine with a low alcohol content.

Definition of

All definitions of terms used herein are to be accorded to such terms as would be understood by a person of ordinary skill in the relevant art in view of the background herein.

The term "low alcohol content" in relation to wine produced according to the process of the first aspect means herein that the alcohol content in wine is lower compared to wine 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 grape juice and thus less alcohol in the wine. Similarly, for an effective amount of glucose isomerase, it converts at least part of the fructose in the grape juice to glucose > the glucose thus produced is then removed by glucose oxidase > and thus less alcohol in the wine.

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

Drawings

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

Detailed Description

Reducing/reducing the alcohol content:

indeed, the methods described herein may be used to produce wine having virtually any desired low alcohol content.

For example, it may be so-called mild liquor having an alcohol content of about 6 to 7%.

As mentioned above, due to global warming, grapes around the world contain more sugar, resulting in wine with increased levels of alcohol. This would be undesirable for different reasons, such as poor mouth feel or different tax categories.

Thus, in a preferred embodiment, the wine produced as described herein with a low alcohol content actually has an alcohol content which is considered normal for today's red/white wine-e.g. 12-14%.

In other words, the same wine will have a much higher percentage of alcohol (e.g., 15-17%) if it is not made by the process described herein.

The wine may be any relevant type of wine, such as white wine, red wine, still wine or sparkling wine.

Gluconic acid:

treatment of unfermented grape juice with glucose oxidase produces gluconic acid, which is not fermentable by yeast and thus occurs in wine. As is known to the skilled person, gluconic acid gives wine with unpleasant organoleptic properties.

Thus, an embodiment of the process of the first aspect comprises the optional step (3) of removing at least part of the gluconic acid to obtain a wine having satisfactory organoleptic properties.

In one embodiment gluconic acid is removed by neutralisation means by addition of a material forming a sparingly soluble salt of gluconic acid, preferably calcium carbonate. Calcium carbonate is inexpensive and has been used as a chemical deacidification agent for wine, and precipitated gluconates, mainly calcium gluconate, can be easily removed by filtration. Since wine is filtered anyway, this neutralization does not introduce any additional filtering step into the wine manufacturing process.

As known to the skilled person, wines with a high alcohol content often lack acidity. This means that it may be preferable not to actually remove gluconic acid for such wines.

Glucose oxidase and glucose isomerase

The glucose oxidase and glucose isomerase enzymes to be used in the process as described herein may be obtained from a variety of different suitable sources, such as related 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 under normal wine making conditions (e.g. relevant pH, temperature, etc.).

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

glucose oxidase:(from Amano)

Glucose isomerase: products from Sigma (catalog number G4166-50G) -see working examples herein.

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

Catalase Activity

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

Hydrogen peroxide (H)₂O₂) Can produce an objectionable wine color and is therefore not a desirable ingredient in wine.

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, the grape must is also treated with an effective amount of a preparation having catalase activity for a period sufficient to convert at least part of the H in the grape must₂O₂Conversion to O₂+H₂And O time.

Preferred production parameters-step 1 of the first aspect

As is known to the skilled person, variations in the wine making process change the organoleptic properties of the wine product. Therefore, a close fit between the usual wine making process and the practice of the methods described herein is preferred. To the extent possible, the taste and aroma of the wine can be altered by practicing the invention without doing anything.

The enzyme-catalyzed processes generally proceed within the optimum pH of the enzyme. The preferred practice of the invention is to treat unfermented must without adjusting its pH. Fortunately, suitable commercially available relevant enzyme products, as used herein, exhibit sufficient activity and stability in the usual pH range 3-4 of unfermented must.

It will be appreciated that any of the enzymes described herein may be used in the process of the present invention, provided that they exhibit the appropriate relative activity and stability at the pH and temperature commonly used during wine production processes. Thus, although soluble enzyme preparations are generally preferred, both soluble and immobilized enzyme preparations may be used.

The person skilled in the art will easily determine how much of a given type of enzyme is needed for a given grape must and for the 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 h1 grape juice 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 h1 must would be appropriate.

As known to the skilled person, the international unit is defined as the amount of enzyme catalyzing the conversion of 1. mu.M substrate per minute. The conditions must also be specified. As known to the skilled person, one typically uses a temperature of 30 ℃ and a pH and substrate concentration that results in a maximum substrate conversion.

Here, 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, determining the international units of an enzyme as described herein is routine work for the skilled person and can be determined within relatively little uncertainty herein.

For example, as known to the skilled artisan, the optimum pH and optimum substrate concentration will vary with the particular enzyme of interest (e.g., a 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.

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 h1 grape juice; and

(ii) the method comprises the following steps A glucose isomerase activity of approximately between about 5,000 and 500,000 international units per h1 grape juice.

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 increased from, for example, around 25 ℃ to 40 ℃, the overall reaction rate will increase with increasing temperature. However, in this case, if the temperature is increased 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 longer treatment at 40 ℃ is also detrimental to the quality of the must.

In general, preferably, the only changes made in the wine making mechanism imply short term storage of unfermented grape juice while the (aerated) grape juice is treated with glucose oxidase, isomerase and optionally other enzymes as described herein. In this regard, it should be noted that the control parameter is the treatment time, not the enzyme activity. For example, sufficient glucose oxidase should be employed to convert a 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 grape must, it was found that the 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 practice of the invention also takes into account the fact that the glucose-reduced must from step 1 is continuously supplied as acid (acid storage) and mixed with must lacking acid to improve the organoleptic characteristics of the wine obtained.

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. This has the advantage that the pH in the grape juice increases and this generally improves the activity of glucose oxidase, since glucose oxidase generally has an optimum pH around neutral pH.

In a preferred embodiment, the relevant enzyme preparation is a solid, water-soluble preparation, preferably a non-dusting preparation. 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. It is recommended that the user dissolve the solid form reagent in a small amount of water immediately before use.

In a preferred embodiment of the method described herein, the supply of oxygen to the must 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. It is desirable that the oxygen is supplied by an air pump, which is the most efficient means of introducing oxygen into the must.

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 grape must, 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, the effective amount and period of time for both glucose oxidase/isomerase enzymes during step (1) is such that:

(A) the method comprises the following steps The sugar content in the grape must 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 the aroma, taste and aroma of wine are extremely sensitive properties, it would not be predictable whether a lower alcohol wine produced according to the present invention would possess the desired properties, since a lower alcohol wine produced according to the present invention with a soluble glucose oxidase/isomerase preparation would contain trace amounts of inactive glucose oxidase/isomerase and may also differ from conventional wine in the concentration of other components. However, it has been found that the lower alcohol wines produced according to the invention possess all of the normal characteristics of wine, including taste and flavour, with the exclusion of characteristics directly related to alcohol concentration.

Preferred production parameters-step 2 of the first aspect

The step 2 of the first aspect, i.e. the fermentation of the must treated, is carried out as an essential step of the process of the invention. However, a detailed discussion of the fermentation step need not be provided herein, as the development of conventional wine making practices is expressly contemplated and are well known to those skilled in the art of wine brewing (oenology). It has been emphasized herein that in the preferred practice of the present invention any unnecessary changes to the mechanisms of the wine making process are avoided.

As mentioned above, in the course of yeast fermentation, O₂Disappear sooner or later. Glucose oxidase requires O₂Can work. However, glucose isomerase may be in the presence or absence of O₂The situation works. Thus, glucose isomerase can also help maintain a 1: 1 glucose/fructose ratio in the actual yeast alcohol fermentation process.

In practice, this will typically occur in a process as described herein. The glucose isomerase added to the unfermented grape must 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 step (2).

Examples

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

One possible way to reduce the final alcohol content in wine is to reduce the sugar concentration in the must before the alcoholic fermentation. Thus, enzymatic treatment of grape must 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) or 100mg of glucose oxidase and 1G of glucose isomerase (Sigma, G4166-50G, > 350u/G) 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

Days treatment Total sugar (g/l) Total sugar reduction (%)

0 GOX 230 0

GOX + isomerase 2350

3 GOX 202 12

GOX + isomerase 19019

Conclusion

These results of this example 1 show that the process using only glucose oxidase gives a total sugar reduction of around 12%, and the additional addition of glucose isomerase significantly increased this value to a sugar reduction of around 19%. Less sugar in the grape juice means a lower alcohol content in the wine.

Example 2: yeast fermentation of the grape juice treated-step (1) of the first aspect

Examples of step (2)

A complete simulation of the normal wine making process was carried out on a laboratory scale. In this experiment it was shown that the enzyme treatment did have an effect on the final alcohol level without negatively affecting the main wine production parameters, such as alcoholic fermentation or lactic acid malate fermentation.

A complete experiment was performed at room temperature of approximately 22 ℃.6 experiments were carried out, 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 substances other than the enzymes described in this example were added.

The grape must was pre-incubated with the enzyme for three days as described below, followed by 11 days of alcoholic fermentation and 10 days of malic acid lactic acid 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) 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) for three days, and the grape juice in the control group was not treated with enzyme. After addition of the enzyme, the flask was vigorously aerated for three days in the presence of the enzyme, after which alcoholic fermentation was started. Aeration is important because oxygen is required for glucose oxidase-mediated enzymatic conversion.

Alcohol fermentation

The alcoholic fermentation was started by inoculating 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, OxoidL42, 9g/l NaCl, 1.14g/l 2% antifoam 1510, BHD 63215) for 10 minutes at room temperature.

Aeration was stopped at this point and oxygen consumed light due to yeast metabolism over the next few days. The alcoholic fermentation was run at room temperature for 11 days, resulting in almost complete conversion of all sugars to alcohol.

Lactic acid fermentation of malic acid

After the alcoholic fermentation, the malic acid lactic acid 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 the wine production process. Lactic acid fermentation of malic acid is mainly achieved by the bacterium Oenococcus oeni. If the growth of the wine coccus is impaired by the enzymatic treatment of the must, it will be highly undesirable.

After 11 days from the start of alcoholic fermentation, lactic acid fermentation of malic acid was started by adding wine coccus (vinillora, chr. hansen. batch: 2711097) to the fermented grape juice. Freeze-dried wine yeast (0.7g of 8.2E +11CFU/g) was rehydrated in 100ml peptone water (15g/L tryptone, Oxoid L42.9g/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. The mixture was left at room temperature for another 10 days.

Results

Effect of enzyme treatment on alcohol levels

Samples were analyzed for glucose and fructose levels using a commercial UV-based assay system supplied by Boehringer Mannheim/R-biopharm (Cat. No. 10139106035) and following the manufacturer's protocol.

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 desalkohlgeholts nach Dr. Rebelein. by C Schliesmann Kellerie-Chemie GmbH& Co.KG，Auwiesenstrasse 5，74523 Rebelein titration method described in Ha11 publication (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 grape juice was pretreated with only glucose oxidase, which is the result of incomplete fermentation. In this experiment, glucose oxidase treated grape juice was not usable in normal wine making because the residual sugars at the end of fermentation, in particular the fructose levels, were high (table II).

Thus, the additional addition of glucose isomerase helps to maintain the glucose/fructose ratio in the must around 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 isomerase substantially prevents fermentation arrest.

Table III, alcohol levels during fermentation. Lactic acid fermentation of malic acid 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 (vol%)
			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

[0150] 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 must around 1: 1, which significantly reduces the risk of unwanted stuck alcoholic fermentations when compared to the use of GOX alone.

Low alcohol levels (9.3%) were found when grape juice was pretreated with only glucose oxidase, which was the result of incomplete fermentation, in other words an undesired stuck alcoholic fermentation. Glucose oxidase treated grape juice was not used for normal wine making in this experiment because the residual sugars, especially the fructose levels, were 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 grape must). This demonstrates that the isomerase itself prevents fermentation arrest.

Example 3: yeast growth-isomerase addition in alcohol fermentation process

Obviously reduces the stagnation of alcoholic fermentation.

It is known to the skilled person that when fructose concentrations are considerably higher than glucose concentrations, fermentation stagnation typically occurs. A glucose/fructose ratio of 1/1 at the beginning of alcoholic fermentation becomes negative during alcoholic fermentation, resulting in 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 wine production was performed as described in example 2 herein. Grape juice was pre-incubated with enzymes for three days, followed by 11 days of alcoholic fermentation and 10 days of malic acid lactic acid 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) or 100mg of glucose oxidase and 1G of glucose isomerase (Sigma, G4166-50G, > 350u/G) 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 L42.9g/l NaCl, 1.14g/l 2% antifoam 1510, BHD 63215) for 10 minutes at room temperature. In that11 days after the start of alcoholic fermentation, lactic acid fermentation of malic acid was started by adding wine coccus (Viniflora, Chr. Hansen. batch: 2711097) to the fermented grape juice. Freeze-dried wine yeast (0.7g of 8.2E +11CFU/g) was rehydrated in 100ml peptone water (15g/L tryptone, Oxoid L42.9g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes. Adding 3ml to 4000ml fermented grape juice to obtain final concentration of 4.3 × 10⁶CFU/ml. The mixture was left at room temperature for another 10 days.

The number of colony forming units of s.cerevisiae was determined by taking samples from fermented must at different time points and plating serial dilutions onto YGC solid medium agar plates, followed by incubation overnight at 30 ℃.

Sugar levels were determined using a commercial UV-based assay system supplied by Boehringer Mannheim/R-biopharm (catalog number 10139106035) and following the manufacturer's protocol.

Results

Effect of isomerase on alcohol fermentation retardation

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

Treatment with glucose oxidase alone showed that alcohol fermentation was delayed (stagnant fermentation) due to the delay in growth of saccharomyces cerevisiae (as shown in table IV). In unfermented grape juice pre-treated with glucose oxidase, yeast growth was very poor a few days before the alcoholic fermentation. The CFU number was below the limit of detection on the first day of alcoholic fermentation and below approximately 3log units 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 the pretreatment and alcoholic fermentation, the fermentation process behaves almost the same as the fermentation of untreated grape juice. The remaining sugar levels and the number of Saccharomyces cerevisiae CFU's (Table IV) were comparable to untreated grape juice. In other words, glucose isomerase was able to overcome the fermentation lag caused by GOX treatment.

Table IV, viable saccharomyces cerevisiae cell count during alcoholic fermentation. The grape must has been pretreated for 3 days as described above. Yeast was added at day t-0. Nd is below the limit of detection.

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

[0172]

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 would cause significant undesirable fermentation stagnation.

The results of this example 3 show that the addition of isomerase can help to overcome the negative impact of the addition of GOX on the growth of saccharomyces cerevisiae, which is commonly used for wine production.

Reference to the literature

US4675191(Novo Industri, published in Denmark, 1987)

Claims (16)

1. A method for producing a wine with a low alcohol content comprising the steps of:

(1): treating unfermented grape juice with effective amounts of the following two enzymes:

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

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

and thereafter,

(2): the so treated must with reduced amounts of glucose and fructose is fermented to produce a wine with a low alcohol content,

wherein the term "low alcohol content" means that the alcohol content of the wine is lower compared to the wine produced under the same conditions but without the treatment with the two enzymes of step (1).

2. The process of claim 1, wherein the wine produced in step (2) of claim 1 with low alcohol content has an alcohol content of 12-14% which is considered normal for red/white wine, and wherein the wine would have too high an alcohol percentage, i.e. 15-17%, if it were made without enzymatic treatment in step (1) of claim 1.

3. 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 wine having satisfactory organoleptic properties; and wherein gluconic acid is removed by neutralization with the addition of a material which forms a sparingly soluble salt of gluconic acid.

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

5. The method of claim 3 wherein the material that forms a sparingly soluble salt of gluconic acid is calcium carbonate.

6. The method of claim 4 wherein the material that forms a sparingly soluble salt of gluconic acid is calcium carbonate.

7. The method of any one of claims 1-6, wherein said effective amount and said 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 grape must is reduced by at least 10%.

8. The method of claim 7 wherein the sugar content of the grape must is reduced by at least 17%.

9. The method of claim 7, wherein

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

Effective amounts for both enzymes glucose oxidase/isomerase are:

(i) the method comprises the following steps A glucose oxidase activity between 1,000 and 50,000,000 international units per hl grape juice; and

(ii) the method comprises the following steps A glucose isomerase activity between 100 and 5,000,000 international units per hl grape juice; and

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

10. The method of claim 8, wherein

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

Effective amounts for both enzymes glucose oxidase/isomerase are:

(i) the method comprises the following steps A glucose oxidase activity between 1,000 and 50,000,000 international units per hl grape juice; and

(ii) the method comprises the following steps A glucose isomerase activity between 100 and 5,000,000 international units per hl grape juice; and

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

11. The process of claim 9 or 10, wherein the temperature during step (1) of claim 1 is between 3 and 30 ℃.

12. A method according to any one of claims 1 to 6, wherein oxygen is continuously supplied to the must during step (1) of claim 1.

13. The method of claim 12, wherein the oxygen is supplied by an air pump.

14. The method according to any one of claims 1 to 6, wherein in step (1) of claim 1 the grape must is also treated with an effective amount of a preparation having catalase activity for a period sufficient to convert at least part of the H in the grape must₂O₂Conversion to O₂+H₂And O time.

15. The method of any one of claims 1-6, wherein additional glucose isomerase is added during the yeast alcoholic fermentation of step (2) of claim 1.

16. The method of any one of claims 1-6, wherein the wine is white wine, red wine, still wine or sparkling wine.