HK1146295A - Reduced stuck alcoholic fermentations in wine production - Google Patents

Description

Reduction of stuck alcoholic fermentations in wine production

Technical Field

The present invention relates to a process for the production of wine, wherein the process significantly reduces the risk of unwanted stuck alcoholic fermentations. The method comprises adding glucose isomerase to grape juice.

Background

The skilled person knows that a glucose/fructose ratio of grape must in wine production significantly different from 1: 1 results in a stuck alcoholic fermentation, i.e. the yeast does not ferment all the sugars and therefore produces a too sweet wine.

This would be an important problem in the industry of wine production.

To the best of the inventors' knowledge, no practical good industrially relevant solution to this stuck alcoholic fermentation problem is currently available.

Disclosure of Invention

The problem to be solved by the present invention is to provide a new method for producing wine, wherein the method significantly reduces the risk of undesired stuck alcoholic fermentations.

The protocol is based on the finding by the inventors that the addition of glucose isomerase to grape must 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 more details.

As mentioned above, the skilled person knows that glucose/fructose ratios in grape must which differ significantly from 1: 1 lead to a stagnation of alcoholic fermentation, i.e. that the yeast does not ferment all the sugars and therefore produces a too sweet wine.

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, which are relevant and well known herein, are the following reactions:

d-glucose & lt & gt 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.

Before the start of the yeast alcoholic fermentation, the grape must used for wine production usually has a glucose/fructose ratio of around 1: 1.

It is known to the skilled person that during yeast alcoholic fermentation of wine yeast prefers glucose over fructose. In other words, glucose is first preferentially metabolized by yeast and this will result in a glucose/fructose ratio in the grape juice of less than 1: 1.

One theory for the positive effect of using glucose isomerase as described herein is that the removal of glucose by e.g. yeast during alcoholic fermentation leads to a situation in the grape juice where the glucose/fructose ratio becomes below 1: 1 (getting "too much fructose" - "too little glucose"). Thus, glucose isomerase equilibrium is driven to the left > fructose is converted to glucose to "restore" a 1: 1 glucose/fructose ratio > 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. (usually doubling in 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. However, glucose isomerase with or without O₂Is active in the presence and thus the glucose isomerase may play a role before the actual start of the yeast fermentation of alcohol or during the actual yeast fermentation of alcohol.

Accordingly, one aspect of the present invention relates to a method of producing wine comprising the steps of:

(1): treating the must with an effective amount of glucose isomerase during yeast fermentation of alcohol to maintain the glucose/fructose ratio in the must close to 1: 1;

and thereafter,

(2): more appropriate steps to produce the wine of interest.

As shown herein, glucose isomerase is relatively stable under normal wine production conditions. Thus, an effective amount of glucose isomerase may be added before the yeast fermentation of alcohol actually begins and still function satisfactorily during the yeast fermentation of alcohol. See working examples herein, where glucose isomerase was added to unfermented grape juice.

Alternatively, an effective amount of glucose isomerase may be added during yeast fermentation of alcohol. If it is added during the yeast fermentation of alcohol, it is preferably added at the beginning of the fermentation, for example to the grape must at almost the same time as the yeast.

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 "maintaining a glucose/fructose ratio close to 1: 1" in connection with the treatment of the must with an effective amount of glucose isomerase in step (1) of the first aspect can be seen as being directly related to the use of an effective amount of glucose isomerase. As explained above, the herein relevant function of glucose isomerase is to attempt to "re-establish" a 1: 1 glucose/fructose ratio. Thus, the addition of glucose isomerase as described herein automatically allows a 1: 1 ratio to be approached in the must as described herein.

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

Detailed Description

Glucose isomerase

The glucose isomerase to be used in the process as described herein may be obtained from a number of different suitable sources, such as the relevant commercially available enzyme products.

As is known to the skilled person, there are a number of different commercially available enzyme products of glucose isomerase 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 isomerase: products from Sigma (catalog number G4166-50G) -see working examples herein.

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 implementation of the method 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.

Basically, the wine makers of the prior art will preferably make no changes in their preferred wine making process other than the addition of the glucose isomerase enzyme as described herein.

For example, enzymatic processes typically 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 during the relevant steps herein of the wine making process.

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.

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.

The person skilled in the art will easily determine how much of a given type of enzyme is needed for a given grape must.

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

(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 gives 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 person, the optimum pH and the optimum substrate concentration for a particular enzyme of interest (e.g.a particular glucose isomerase) will vary. 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 conditions are used:

(i) the method comprises the following steps A glucose isomerase activity of approximately between about 5,000 and 500,000 international units per h1 must would be suitable.

In a preferred embodiment, the effective amount for glucose isomerase during step (1) is such that:

(A) the method comprises the following steps The sugar content of the grape must at the end of the yeast alcoholic fermentation is less than 4g/l, more preferably less than 1g/l and even more preferably less than 0.1 g/l.

It can be seen in table II of working example 2 herein that the addition of glucose isomerase resulted in no detectable ("0") of sugar in the must after fermentation-in other words, the addition of glucose isomerase virtually completely prevented the undesired stagnation of fermentation. As the skilled person understands, yeast will not utilize all sugars and a significant amount of sugars will remain in the grape must at the end of the yeast alcoholic fermentation if there is fermentation stagnation.

In other words, if the specified remaining sugars are as little as point (a) above, there is no significant undesirable fermentation lag.

Due to the fact that the aroma, taste and aroma of wine are extremely sensitive properties, it would not be predictable whether the lower alcohol wine produced according to the invention would possess the desired properties, since the lower alcohol wine produced according to the invention with a soluble isomerase preparation would contain traces of inactive isomerase and would 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 carrying out of step 2 of the first aspect, i.e. the more appropriate steps for producing the wine of interest, is 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).

For example, these further steps would be the relevant storage steps

Wine with low alcohol content-glucose oxidase added

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.

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 described method, column 2, acts 25-29:

"the process of the invention comprises treating unfermented glucose with glucose oxidase in the presence of oxygen, whereby

The glucose in the must is converted to gluconic acid and the must thus treated is then fermented. "

Thus, US4675191 describes that glucose oxidase can remove some glucose from unfermented must. Less sugar in the must means less alcohol content in the final wine.

Glucose oxidase has been used in some working examples herein to produce wine with low alcohol content. It is clear from these examples that the presence of glucose isomerase as described herein significantly improves the wine making process, which involves the use of glucose oxidase to reduce the alcohol content.

One reason for the glucose isomerase-related improvement is that glucose isomerase significantly reduces fermentation lag, as discussed herein (see examples 2 and 3 herein).

Thus, in an embodiment of the first aspect, the following steps are arranged before the start of the alcoholic yeast fermentation of step (1) of the first aspect:

(A) the method comprises the following steps Treating the unfermented grape juice with an effective amount of 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.

The glucose oxidase addition of step (a) is carried out substantially as described in US 4675191. In fact, wine makers typically do not change anything related to normal practice-except for the addition of glucose oxidase.

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

beta-D-glucose + O₂(ii) > 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 a catalase activity is a preferred embodiment herein. Catalase (EC 1.11.1.6) catalyzes the reaction:

2H₂O₂＜＝＞O₂+2H₂O

if glucose oxidase is used as described in step (A) above, it is preferred that glucose isomerase is added to the unfermented grape juice together with the glucose oxidase. Doing so in the working examples herein yields very positive results.

Preferred embodiments related to this step (A) of treating unfermented grape juice with an effective amount of glucose oxidase are discussed below.

One theory that the combined use of glucose oxidase and isomerase will remove a significant portion of the glucose is as follows. The removal of glucose by glucose oxidase leads to a situation in the grape juice where the glucose/fructose ratio becomes below 1: 1 (giving "too much fructose" - "too little glucose"). Thus, glucose isomerase equilibrium is "forced" to the left-side > 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.

The glucose oxidase to be used in the methods 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 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).

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

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 suitable.

In a preferred embodiment, the following conditions 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.

In a preferred embodiment, the effective amount and period of time for both glucose oxidase/isomerase enzymes during step (a) 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%.

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.

Either 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 enzyme addition. Samples were analyzed for the presence of glucose and fructose using a commercial UV-based assay system 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 that the additional addition of glucose isomerase significantly increases 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 treated grape must-examples of step (1) and step (2) of the first aspect

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, Oxoid L42.9g/l NaCl, 1.14g/l 2% antifoam 1510, BHD 63215) for 10 minutes at room temperature.

At this point aeration is stopped and oxygen is depleted in the following days due to yeast metabolism. 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 sensory 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. It would be very unpleasant if the growth of the wine cocci were impaired by the enzymatic treatment of the grape juice.

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，Auwiesenstrass 5，74523Rebelein titration method described in Hall 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%).

The low levels of alcohol found when grape juice is pretreated with glucose oxidase alone are the result of fermentation stagnation. 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 the arrest of fermentation.

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: not determined

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

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.

The low level of alcohol (9.3%) found when grape juice was pretreated with glucose oxidase alone was the result of fermentation stagnation, in other words, undesirable alcohol fermentation stagnation. 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: the yeast growth-isomerase addition during the alcoholic fermentation process significantly reduces the alcoholic fermentation stagnation.

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 the alcoholic fermentation becomes negative during the alcoholic fermentation, resulting in incomplete 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 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.

Either 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. After 11 days from the start of alcoholic fermentation, lactic acid fermentation of malic acid was started by adding wine coccus (Vinflora, Chr. Hansen. batch: 2711097) to the fermented grape juice. Freeze-dried wine yeast (0.7g of 8.2E +11CFU/g) in 100ml peptone water(15g/L tryptone, Oxoid L42.9g/L NaCl, 1.14 g/L2% antifoam 1510, BHD 63215) for 10 minutes. 3ml of fermented grape juice was added to 4000ml of fermented grape juice to give a final concentration of 4.3X 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 (fermentation was arrested) 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 the presence of fermentation lag.

Sugar analysis supported the results. In untreated 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 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 unfermented must. 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
				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.

Example 4:action of glucose isomerase in fermentation of artificial grape juice

In order to study the effect of the individual glucose isomerases under defined conditions, the fermentation of artificial grape must was carried out. Grape juice consisted of yeast nitrogen source (YNB), tartaric acid and varying amounts of glucose and fructose.

The role of glucose isomerase was studied by analyzing yeast growth and glucose/fructose reduction and ethanol production during fermentation.

The experiment was performed in 11 autoclaved fermentation flasks with 500ml of artificial grape juice in each flask and all fermentations were performed in duplicate. An artificial grape juice medium (0.67% YNB, 2.0g/l tartaric acid, varying amounts of glucose and fructose, miliQ water, and pH adjusted with 50% w/w KOH) was inoculated with rehydrated freeze-dried wine yeast (Saccharomyces cerevisiae Merit. Ferm, Chr. Hansen, 0.1g/l) to a final concentration of 9E +05 CFU/ml.

Immediately before inoculation of the yeast, glucose isomerase EC 5.3.1.5(Sigma G4166, > 350U/G) was added (0.5G/l) to the flask. The fermentation was carried out at room temperature (approximately 23 ℃) for 41 days without stirring.

The number of Saccharomyces cerevisiae colony forming units and sugar levels at a given time were determined as described in example 3. The ethanol concentration was measured according to the enzymatic UV method and the protocol supplied by Boehringer Mannheim/R-biopharm (Cat. No. 10176290035).

TABLE V overview of fermentation and expectations

Results

Action of glucose isomerase in fermentation of artificial grape juice

Fermentation of grape must with high sugar levels (total sugars 260g/l) with balanced and unbalanced glucose/fructose ratios results in fermentation stagnation when not treated with glucose isomerase. See tables V and VI. This suggests that only high sugar levels can lead to fermentation stalls. However, fermentation is much faster after treatment with isomerase and all sugars are fermented.

Table VI, reduction of glucose and fructose during alcoholic fermentation in the first 4 experiments. The yeast was inoculated at day t-0.

Settings with low and unbalanced sugars (60/100) are believed to represent production of wine with reduced final alcohol concentration. Here, the effect of GI was observed at two different pH values of pH 3.6 and pH 5.2, respectively. At elevated pH, the enzyme proved to be more efficient as expected, comparing the fermentation of 60/100 with isomerase at pH 3.6, day 6 with pH 5.2, day 6, showing a total of 18 and 5g/l of sugar remaining, respectively. However, fermentation with GI treatment at these two pH values was more effective than fermentation without treatment, in which the yeast took 4 more days to fully ferment.

When the effect of glucose isomerase is tested in a better standard grape juice, such as a grape juice with balanced glucose/fructose ratio and total sugar of 200g/l, the results are about the same and the alcoholic fermentation is improved when the enzyme is included.

Table VII, glucose and fructose were reduced during the alcoholic fermentation in settings 5-10. Yeast was inoculated on day t-0.

The cell count of s.cerevisiae supports these data from the measurement of sugars. During the first two weeks of fermentation, yeast growth was seen to be almost similar in all settings, but thereafter, yeast tended to die faster in fermentations treated with glucose isomerase. This indicates that fermentation is completed more quickly when treated with the enzyme.

Table VII, viable CFU counts of saccharomyces cerevisiae during alcoholic fermentation. Yeast was added at day t-0. The open symbols (. smallcircle.) represent experiments without glucose isomerase, and the closed symbols (●) represent experiments with enzyme.

Conclusion

As shown in example 4, the use of glucose isomerase results in more efficient alcoholic fermentation and considerably reduces the risk of fermentation stagnation. This applies to grape juices with high or low sugar levels and balanced and unbalanced glucose and fructose ratios.

Reference to the literature

US4675191(Novo Industri, published in Denmark, 1987)

Claims (10)

1. A method of producing wine comprising the steps of:

(1): treating the must with an effective amount of glucose isomerase during yeast fermentation of alcohol to maintain the glucose/fructose ratio in the must close to 1: 1;

and thereafter,

(2): further suitable steps to produce the wine of interest.

2. The method of claim 1, wherein

An effective amount of glucose isomerase is added before the actual start of the yeast fermentation of alcohol-i.e. it is added to the unfermented grape must;

or

An effective amount of glucose isomerase is added during the yeast fermentation of alcohol, preferably at the beginning of the fermentation, e.g. approximately at the same time as the yeast is added to the grape must.

3. The method of claim 1 or 2, wherein the effective amount of glucose isomerase during step (1) is such that:

(A) the method comprises the following steps At the end of the yeast alcoholic fermentation, the sugar content of the grape must is less than 4 g/l.

4. The method according to claim 3, wherein the sugar content in the grape must is less than 0.1 g/l.

5. The method of any one of the preceding claims, wherein the effective amount of glucose isomerase has:

(i) the method comprises the following steps Between 100 and 5,000,000 international units per h1 grape must.

6. The process according to any of the preceding claims, wherein the following steps are arranged before the start of the alcoholic yeast fermentation of step (1) of claim 1:

(A) the method comprises the following steps Treating the unfermented grape juice with an effective amount of glucose oxidase 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.

7. The method of claim 6 wherein glucose isomerase is added to the unfermented grape juice along with glucose oxidase.

8. The method of claim 6 or 7, wherein the effective amount of glucose oxidase has:

(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.

9. The method of claim 7, wherein 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 of the unfermented grape juice is reduced by at least 17%.

10. The method of any one of the preceding claims, wherein the wine is white wine, red wine, still wine or sparkling wine.