# Patent

## Title
Method for production of an alcoholic beverage with reduced content of alcohol

## Abstract

A method for production of an alcoholic beverage with reduced content of alcohol involving treating unfermented beverage starting solution with glucose oxidase and glucose isomerase.

## Background
FIELD OF THE INVENTION
The present invention relates to a method for reduction of alcohol in an alcoholic beverage such as cider, beer, low- or non alcoholic wine, and other low or non alcoholic beverages involving treating the beverage starting solution with a combination of the enzymes glucose oxidase and glucose isomerase.
BACKGROUND ART
Due to consumer health issues and ethanol intolerance problems there is an increasing demand for alcoholic beverages with reduced alcohol content which at the same time possess the characteristic sensory attributes as the conventional “high-alcohol” products. Also, global warming has led to higher sugar content in fruits and berries worldwide which, when used as the starting solution for alcoholic fermentation, results in higher alcohol content in the resulting product. Such high alcohol content may negatively impact the sensory characteristics of the product.
Accordingly, there is a need for methods allowing for control of the alcohol content of alcoholic beverages and at the same time maintaining the sensory characteristics of the particular product.
Current methods for ethanol reduction like reverse osmosis, spinning cone or dilution are not satisfactory. These methods may have adverse effects on the sensorial quality of the beverage. Furthermore, the price of up to 1 USD/gallon for reverse osmosis of alcoholic beverages is a major limitation for a wider application of this approach.
U.S. Pat. No. 4,675,191 (Novo Industri, Denmark—published 1987) describes a method for reducing alcohol content in wine that involves use of the enzyme glucose oxidase. With respect to the described method column 2, lines 25-29 read:

    
    
        “The method of this invention comprises treating unfermented grape juice with glucose oxidase in the presence of oxygen, thereby converting glucose in the grape juice into gluconic acid and thereafter fermenting the so-treated grape juice.”
    
    


The herein main relevant technical elements of this prior art method are schematically illustrated in FIG. 1.
International PCT application with application number PCT/EP2008/068161 was filed 22 Dec. 2008. Applicant is Chr. Hansen A/S and the application was not published at the filing date of this present application.
PCT/EP2008/068161 describes a method for reduction of alcohol in wine manufactured from grape juice involving the use of enzymes as described herein. Use of this principle for the reduction of alcohol in other alcoholic beverages is not described in PCT/EP2008/068161.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a new method for reducing the alcohol content in alcoholic beverages (hereafter referred to as “alcoholic beverage” or just “beverages”), wherein the method results in significantly reduced content of alcohol in the final beverage. Further, the method significantly decreases the risk of unwanted stuck alcoholic fermentations.
The method is based on a discovery of the present inventors. They surprisingly found that a glucose oxidase based method of the art can be significantly improved by also including use of glucose isomerase.
A schematic comparison of the method of U.S. Pat. No. 4,675,191 and the method of the invention is illustrated in FIG. 1. In a working example herein can be seen that a method only using glucose oxidase resulted in a total sugar reduction of around 12%. Addition of glucose isomerase significantly increased this to a sugar reduction of around 19%. Less sugar in the beverage starting solution (substrate for the enzymes) implies less alcohol content in the resulting beverage.
Further, the inventors found that the extra addition of glucose isomerase helped to maintain the ratio glucose/fructose in the beverage starting solution at around 1:1, which significantly decreases the risk of unwanted stuck alcoholic fermentations. For further details, see working example herein.
It is known to the skilled person that the glucose/fructose ratio in any glucose fructose containing solution should not differ significantly from 1:1 during alcohol fermentation. If this happens there is a risk of stuck alcoholic fermentations, i.e. the yeast is not fermenting all the sugar and the resulting beverage may appear too sweet.
An important commercial application of glucose isomerase is to convert glucose into fructose for instance in order to make high fructose syrup (fructose is sweeter than glucose).
In view of this, the present inventors were actually surprised that addition of glucose isomerase provided such a positive result (significantly less sugar=>less alcohol). One reason for this was that glucose isomerase may prima facie be seen as a enzyme that can “remove/convert” too much glucose and the glucose oxidase would therefore have less available substrate (glucose oxidase does not work on fructose).
However, as shown in the working examples herein, addition of glucose isomerase provided very positive results.
Without being limited by theory, it is believed that the following could theoretically explain why addition of glucose isomerase provides such positive results:
Glucose oxidase (EC 1.1.3.4) primarily catalyzes the following reaction in the beverage starting solution (also referred to herein as solution):

Beta-D-glucose+O2<=>D-glucono-1,5-lactone+H2O2 

Within the solution the generated “D-glucono-1,5-lactone” is spontaneously converted into gluconic acid. Accordingly, D-glucono-1,5-lactone is removed and the equilibrium is therefore going to the right=>glucose is removed from the solution.
If the enzyme preparation also has catalase activity the created H2O2 is also removed=>equilibrium is therefore going even more to the right=>more glucose is removed. Involvement of catalase activity is a preferred embodiment herein—see discussion below.
Catalase (EC 1.11.1.6) catalyzes the reaction:

2H2O2<=>O2+2H2O

The enzyme of particular relevance in the method of the present invention is glucose isomerase EC 5.3.1.5. The official name for this EC 5.3.1.5 class is xylose isomerase. However, as known to the skilled person, it may also be termed glucose isomerase. Glucose isomerase is for instance the name used in relevant commercial products of this enzyme class, such as e.g. the commercial product used in working examples herein.
The herein relevant and well known reaction catalyzed by glucose isomerase in the beverage starting solution is the following:

D-glucose<=>D-fructose.

As known to the skilled person this enzyme class may also catalyze the reaction:

D-xylose<=>D-xylulose.

This xylose related reaction is less relevant herein.
One theory is that the glucose removed by the glucose oxidase then creates a situation in the solution, where the glucose/fructose ratio becomes lower than 1:1 (“too much” fructose—“too little” glucose). The glucose/fructose equilibrium on which the glucose isomerase reacts is consequently “forced” to the left=>fructose is converted to glucose to “recover” the glucose/fructose ratio of 1:1=>the glucose oxidase receives “newly” created glucose to work on and thereby more of the total sugar is removed (both glucose and fructose) from the solution.
As discussed above, the maintenance of the glucose/fructose ratio of 1:1 also has the advantage of significantly reducing the risk of stuck alcoholic fermentations.
Before yeast alcoholic fermentation O2 is present in the unfermented solution (beverage starting solution). As known to the skilled person, normal yeast fermentation generally consists of two parts:
Part 1

    
    
        Aerobic growth (Oxygen is present)
        This is the initial rapid growth process where the yeast doubles its cell number roughly every 4 hours. (Usually 24-72 hours)
    
    


Part 2

    
    
        Anaerobic fermentation (No oxygen present)
        Slower activity and the yeast ferments sugar (both glucose and fructose), converting it to alcohol (sugar=>2 ethyl alcohol+2 CO2) rather that increasing the number of yeast cells. (This process can take from days to weeks depending on the yeast and the recipe).
    
    


Accordingly, during the yeast fermentation the O2 will sooner or later disappear. The glucose oxidase requires O2 for activity. However, the glucose isomerase is active with or without the presence of O2. Accordingly, glucose isomerase can also help to maintain the glucose/fructose ratio of 1:1 during the actual yeast alcohol fermentation.
Accordingly, a first aspect of the invention relates to a method for production of an alcoholic beverage with reduced content of alcohol, comprising the following steps:

    
    
        (1): treating unfermented beverage starting solution with an effective amount of the following two enzymes:
        
            (a) glucose oxidase in the presence of oxygen for a period of time adequate to convert at least a portion of the glucose in the solution into gluconic acid; and
            (b) glucose isomerase for a period of time adequate to convert at least a portion of the fructose in the solution into glucose;

and thereafter,

        
        
        (2): fermenting said treated solution with reduced amounts of glucose and fructose to obtain the alcoholic beverage with reduced content of alcohol.
    
    


DEFINITIONS
All definitions of relevant terms are in accordance with the general understanding of the person of skill in the relevant technical area.
The term “reduced content of alcohol” in a beverage produced according to the method of the first aspect of the present invention relates to a lower content of alcohol in the beverage as compared to a beverage produced under identical conditions but without the treatment with both of the two enzymes of step (1) of the first aspect. In fact this term may be seen as directly relating to using an effective amount of the two enzymes.
If an effective amount of glucose oxidase is used at least a portion of the glucose will be removed from the solution and thereby less alcohol in the beverage is obtained. Similarly, for an effective amount of glucose isomerase, which converts at least a portion of the fructose in the solution into glucose=>this created glucose is then removed by the glucose oxidase=>thereby less alcohol in the beverage.
Embodiment of the present invention is described below, by way of examples only.



DRAWINGS
FIG. 1: A schematic illustration/comparison of the method of U.S. Pat. No. 4,675,191 and the method of the invention.



DETAILED DESCRIPTION OF THE INVENTION
Lowering/Reducing Alcohol Content:
In essence the method as described herein may be used to make a alcoholic beverage with virtually any desired reduced or lower content of alcohol.
For instance it may be a so-called light beverage with an alcohol content of about 10% alcohol, such as a content of 5-10% alcohol by volume (ABV), 6-7% ABV or even a lower content of alcohol such as from 1-5% ABV including 2-4% ABV.
As discussed above, due to global warming fruits and berries worldwide contain more sugar resulting in beverages with increased alcohol levels. This may be unwanted for different reasons e.g. poor taste. Additionally, due to consumer health considerations there is an increasing demand for low alcohol beverages. Finally, there is a need for low-alcohol beverages targeting the population of people suffering from ethanol intolerance.
Thus, the present invention addresses the problem of lowering the alcohol content in beverages while maintaining the characteristic of the particular beverage e.g. taste, bouquet, color etc.
Gluconic Acid:
Treatment of the unfermented beverage starting solution with glucose oxidase generates gluconic acid, which is non-fermentable by yeast and therefore gluconic acid appears in the beverage. As known to the skilled person gluconic acid may give unsatisfactory organoleptic properties to the beverage.
Accordingly, an embodiment of the method of the first aspect comprises an optional step (3) of removing at least a portion of the gluconic acid to obtain a beverage with satisfactory organoleptic properties.
In an embodiment, the gluconic acid is removed by means of neutralization by addition of a substance forming a sparingly soluble salt of gluconic acid, preferably calcium carbonate. Calcium carbonate is inexpensive and is already used as a chemical deacidification agent for beverages, and precipitated gluconic acid salts, mainly calcium gluconate, can easily be removed by filtration.
Glucose Oxidase and Glucose Isomerase
The glucose oxidase and glucose isomerase to be used in the method as described herein may be obtained from numerous different suitable sources such as relevant commercially available enzyme products.
As known to the skilled person there are numerous different commercially available glucose oxidase/isomerase enzyme products on the market with enzymes that work within the relevant pH values, temperature etc. Another useful enzyme is hexose oxidase, which is capable of converting other hexose sugars.
In the working examples below the following commercially available enzyme products were used:

    
    
        glucose oxidase: Hyderase® (from Amano)
        glucose isomerase: Product from Sigma (# G4166-50 g). Catalogue number—see a working example herein.
    
    


An advantage of the Hyderase® product is that is also comprises catalase activity.
The “beverage starting solution” is the substrate for the above mentioned enzymes and eventually for the alcoholic fermentation. Suitable substrates for the enzymes include but are not limited to: glucose, fructose, xylose, mannose, galactose, any other hexose and any combinations thereof. Any sugar compound which can act as a substrate for the mentioned enzymes are encompassed by the present invention. Obviously, for achieving the optimal result according to the present invention the substrate preferable comprises, as the main sugar component, glucose and fructose.
Useful sources of the above mentioned sugars are a range of fruit, berries, and cereal grains. The liquid form of such products is widely used as “beverage starting solution” and often referred to as “must”, “juice” or “worth” in the production of alcoholic beverages. Conventional examples include grapes in the production of wine, apple and pears in the production of cider, and barley in the production of beer. However, new beverages are constantly invented using more exotic products as starting materials examples include but are not limited to pomegranates, pineapples, strawberries and mango's.
In one embodiment, the beverage starting solution is grape juice as described in PCT/EP2008/068161.
Catalase Activity
As discussed above, the use of an enzyme preparation with catalase activity in the method as described herein may also result in the removal of the H2O2 created by glucose oxidase.
Hydrogen peroxide (H2O2) may result in an unwanted color and may therefore not be a desirable component in the beverage.
Furthermore, as discussed above, by removing H2O2 one may get the glucose oxidase equilibrium moved even further to the left=>more glucose is removed.
Accordingly, 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 with catalase activity for a period of time adequate to convert at least a portion of the H2O2 in the juice into O2+H2O.
Preferred Production Parameters—Step 1 of First Aspect
As known to the skilled person, changes in the beverage production procedures alter the organoleptic properties of the beverage product. Therefore, a close-fit between the usual beverage production procedures and practice of the method as described herein is preferred. Consequently, through practicing of this invention no adverse effect are observed with respects to taste and bouquet of the beverage.
Enzyme catalyzed processes are usually conducted within the pH optimum of the enzyme. Preferred practice of this invention is to treat the unfermented solution (beverage starting solution) without adjusting the pH. Fortunately, suitable relevant commercially available products of the enzymes as used herein exhibit adequate activity and stability in the pH range normal for unfermented beverage starting solutions.
Depending on the specific substrate (beverage starting solution) the sugar content and ratios between different sugar components may vary. In e.g. apples the ratio glucose:fructose is 30:70, in mango the ratio is 24:76, in pineapple the ratio is 43:57, and in strawberry the ratio is 20:80. As known to the person skilled in the art these ratios may vary depending on climate and growth conditions as well as time of harvest. The ratios will be relevant for selecting optimal dosages of the enzymes. Based on this information, which is readily available, the skilled person will have no difficulties in selecting the optimal dosages of enzymes for the particular application.
It is to be understood that any enzyme as described herein can be used in the method according to the invention, provided that it exhibits a reasonable relevant activity and stability at the pH and temperature prevailing during the method for producing a specific beverage. Thus, both soluble and immobilized enzyme preparations may be used, even if soluble enzyme preparations are usually preferred.
It is easy for the worker skilled in the art to establish how much enzyme of a given kind is needed for a given application and a desired sugar conversion.
For instance, depending on the details of treatment times and temperatures:

    
    
        (i): a glucose oxidase activity roughly between about 1,000 and 50,000,000 international units per hl of solution will be appropriate; and
        (ii): a glucose isomerase activity roughly between about 100 and 5,000,000 international units per hl of solution will be appropriate.
    
    


Thus, it provides no additional value to the present invention specifying appropriate dosages of the enzymes as this will be relatively easy to test under the specific process parameters. As illustrated hereinafter the relevant amounts applied in the examples are within the above levels and it is assumed that the above ranges cover any relevant applications.
Herein the international units are defined as described above and according to the art, i.e. determined at a temperature of 30° C. and the pH value and substrate concentration that yield the maximal substrate conversion rate.
As known to the skilled person, the optimal pH value and optimal substrate concentration may vary for a specific enzyme of interest (e.g. a specific glucose oxidase). However, it is easy to identify this optimal pH and substrate concentration since it is e.g. generally given on the product documentation for a relevant commercial enzyme product. Furthermore, for a specific enzyme of interest it is in general routine work to identify parameters such as optimal pH and substrate concentration. Hence an optimal match between available enzyme products and process parameters should be considered when applying the principle of the present invention in the production of a desired alcoholic beverage.
In a preferred embodiment the following is used:

    
    
        (i): a glucose oxidase activity roughly between about 15,000 and 5,000,000 international units per hl of solution; and
        (ii): a glucose isomerase activity roughly between about 5,000 and 500,000 international units per hl of solution.
    
    


In a preferred embodiment, the temperature during step (1) of first aspect is between 1 and 35° C., preferably between 3 and 30° C. Generally, in enzymatic reactions, the overall reaction rate will increase with temperature, if the temperature is raised from e.g. around 25 to 40° C. In this case, however, more oxygen will be liberated from the liquid, if the temperature is raised from 25 to 40° C., and thus, the overall reaction rate will be reduced under such circumstances. Extended treatment at 40° C. could also be detrimental to the quality of the solution and thus the resulting beverage.
Overall speaking, preferably the only change made in the mechanics of beverage making amounts to a short-term storage of the unfermented solution while the (aerated) solution is being treated with glucose oxidase, isomerase and optionally other herein described enzymes. In this connection, it is noted that treatment time rather than enzyme activity may be the controlling parameter. For instance, enough glucose oxidase should be employed to convert the desired proportion of glucose within reasonable treatment time, in which time generally does not exceed 72 hours and many times does not exceed 48 hours. Even if some yeast is present in the unfermented solution, it has been found that the fermentation will not initiate to any appreciable degree during the first 48 hours, and thus, it is possible to remove relevant amounts of sugar.
In regard to Step 1 of the first aspect, it is to be noted that the conversion degree is very easily controlled as the reaction of glucose to gluconic acid is stopped almost immediately by cutting the oxygen supply.
Practice of this invention also contemplates instances when the sugar reduced solution resulting from Step 1 is kept as an acid supply (acid reserve) and blended with an acid deficient solution to improve the organoleptic properties of the resulting beverage.
It is to be understood that the removal of the gluconic acid can be performed at any time, such as after the fermentation step.
Alternatively, it may be done by addition of e.g. calcium carbonate before addition of glucose oxidase as described herein. An advantage of this is that the pH increases in the beverage starting solution, and this may generally improve the activity of the glucose oxidase, since it generally has optimal pH around pH neutral.
In a preferred embodiment the relevant enzyme preparation(s) is a solid water soluble preparation, preferably a non-dusting preparation. The storage stability of a solid preparation is better than the storage stability of a liquid preparation, and also, it is unnecessary to add any conservation agents. It is recommended, though, that the user dissolve the solid form agent in a small amount of water immediately before use.
In a preferred embodiment of the method as described herein oxygen is supplied continuously to the solution during step 1 of the first aspect. The supply of oxygen has a remarkably influence on the reaction rate of particularly the glucose oxidase enzymatic reaction. Thus, continuous introduction of oxygen ensures a high reaction rate. Desirably, the oxygen is supplied by means of an air pump, a most efficient means for 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 for generation of the wanted reduction of sugar concentration in a period of time not exceeding around 72 hours. As it has already been pointed out, even if some yeast is present in the solution, fermentation will not progress to any appreciable degree during the first 48 hours, and thus, no appreciable amount of glucose/fructose is simultaneously fermented to alcohol during enzymatic conversion of glucose to gluconic acid.
In a preferred embodiment the effective amount and the period of time for the two glucose oxidase/isomerase enzymes during step (1) is so that:

    
    
        (A): the sugar content in the solution is reduced by at least 10%, more preferably at least 14% and even more preferably at least 17%.
    
    


As discussed above, in working examples herein the sugar content (both glucose and fructose) was reduced by 19%.
In a preferred embodiment of the method according to the invention, the pH value in step 1 is not controlled. This embodiment is particularly preferred in instances where a longer treatment time e.g., up to around 72 hours is feasible and is employed.
Due to the fact that the aroma, the taste and the bouquet of alcoholic beverages are properties which are extremely sensitive, it could not be predicted whether the lower alcohol beverages produced according to the invention would possess the wanted properties. Additionally, it was considered whether the alcoholic beverages produced according to the invention, with soluble glucose oxidase/isomerase preparations, would contain traces of inactive glucose oxidase/isomerase and therefore differ from conventionally produced beverages. However, it has been found that the lower alcohol beverages produced according to the invention possesses all normal properties of the conventionally produced product, including taste and bouquet, except, of course, for properties related directly to the alcohol concentration and the production of gluconic acid.
Preferred Production Parameters—Step 2 of First Aspect
Conduct of step 2 of first aspect, i.e., fermentation of the treated solution, is an obligatory step of the method of the invention. However, no detailed discussion of the fermentation step needs to be provided herein since conduct of conventional beverage production practices are contemplated expressly and those practices are well known to persons skilled in the art hereof. It has already been emphasized herein that changes in the mechanics of the beverage production process are avoided according to preferred practice of this invention.
As discussed above, during the yeast fermentation the O2 will sooner or later disappear. The glucose oxidase needs O2 to work. However, the glucose isomerase can work with or without the presence of O2. Accordingly, glucose isomerase can also help to maintain the glucose/fructose ratio of 1:1 during the actual yeast alcohol fermentation.
In fact this will generally happen during the method as described herein. The glucose isomerase added to the unfermented solution during step (1) is normally still active during the yeast alcohol fermentation of step (2).
However, optionally one could add additional glucose isomerase during the yeast alcohol fermentation of step (2).
Accordingly, in an embodiment of the invention of the first aspect additional glucose isomerase is added during the yeast alcohol fermentation step (2).
EXAMPLES
Example 1
Enzymatic Sugar Reduction in Grape Juice—Example of Step (1) of First Aspect
One possible way to reduce the final alcohol content in beverages is to reduce the sugar concentration in the solution before the alcoholic fermentation. Therefore an enzymatic treatment of the solution, in this case grape juice, was performed in order to reduce the total sugar content.
Three independent experiments were performed using two replicates in each case. In each sample 200 ml grape juice (Pinot Blanc 2007, Germany, pasteurized) was added to a glass flask and continuously mixed with a magnetic stirrer. The samples were aerated throughout the experiment.
Either 100 mg glucose oxidase (Hyderase, Amano, >15,000 u/g, corresponding to 150,000 u per hl solution) or both 100 mg glucose oxidase and 1 g glucose isomerase (Sigma, G4166-50 g, >350 u/g, corresponding to 35,000 u per hl solution) were added to the flasks. The incubation was allowed to run at room temperature for 3 days.
Samples were taken just before addition of the enzymes and after 3 days. Samples were analyzed for the presence of glucose and fructose using a commercial UV based assay supplied by Boehringer Mannheim/R-biopharm (catalog number 10 139 106 035) following the protocol provided by the manufacturer. The results of this experiment are summarized in Table I below.







TABLE I







Enzymatic sugar (glucose and fructose) reduction in grape juice.












total sugar
Reduction total


Day
Treatment
(g/l)
sugar (%)













0
GOX
230
0



GOX + isomerase
235
0


3
GOX
202
12



GOX + isomerase
190
19





GOX = glucose oxidase






Conclusion

These results of this example 1 show that a process only using glucose oxidase resulted in a total sugar reduction of around 12% and extra addition of glucose isomerase significantly increased this to a sugar reduction of around 19%. Less sugar in the juice implies less alcohol content in the final fermented beverage product.
Example 2
Yeast Fermentation of Treated Grape Juice—Example of Both Step (1) and Step (2) of First Aspect
A full simulation of a general beverage production process, in this case wine production, was done at laboratory scale. In this experiment it was shown that the enzymatic treatment did have an effect on the final alcohol level without negatively influencing major wine production parameters like the alcoholic fermentation or the malolactic fermentation.
The complete experiment was carried out at room temperature, approximately 22° C. Six experiments were performed with each four liters of grape juice (Pinot Blanc 2007, Germany, pasteurized) in fermentation flasks. The pH of the grape juice was not adjusted and no material was added other than the enzymes described in this example.
The grape juice was preincubated for three days with enzymes as described below, followed by the alcoholic fermentation of 11 days and a malolactic fermentation of 10 days.
Enzymatic Treatment
The six flasks were divided in three groups of two flasks.
The grape juice in group 1 was preincubated for three days with 0.5 g/l glucose oxidase (Hyderase, Amano, >15,000 u/g, corresponding to 750,000 u per hl solution), the grape juice in the second group with 0.5 g/l glucose oxidase and 2 g/l Glucose Isomerase (Sigma, G4166-50 g, >350 u/g, corresponding to 70,000 u per hl solution) and the grape juice in the control group was not treated with enzymes. Following enzyme addition, the flasks were vigorously aerated for three days in the presence of the enzymes, before the alcoholic fermentation was started. Aeration is important since oxygen is required in the glucose oxidase mediated enzymatic conversion.
Alcoholic Fermentation
The alcoholic fermentation was initiated by inoculation with rehydration of freeze dried wine yeast (Saccharomyces cerevisiae Merit.Ferm, Chr. Hansen, 0.1 g/l) to a final concentration of 9E+05 CFU/ml. Rehydration was performed in peptone water (15 g/l Tryptone, Oxoid L42, 9 g/l NaCl, 1.14 g/l 2% antifoam 1510, BHD 63215) for 10 minutes at room temperature.
At this point the aeration was stopped and the process became depleted for oxygen during the following days as a result of the yeast metabolism. The alcoholic fermentation was allowed to run for eleven days at room temperature which resulted in almost complete conversion of all sugar to alcohol.
Malolactic Fermentation
Following the alcoholic fermentation, the malolactic fermentation was started.
The aim of this part of the process is to convert malate into lactate which results in a more pleasant sensoric sensation and thus is an important part of ex. cider or wine producing process. The malolactic fermentation is mostly performed by the bacteria Oenococcus oeni. It would be highly undesirable if growth of O. oeni would be impaired by the enzymatic treatment of the juice.
Eleven days after the start of the alcoholic fermentation the malolactic fermentation was started by addition of O. oeni (Viniflora, Chr. Hansen. Batch no.: 2711097) to the fermented grape juice. Freeze dried O. oeni (0.7 g of 8.2 E+11 CFU/g) was allowed to rehydrate for 10 minutes in 100 ml of peptone water 15 g/l Tryptone, Oxoid L 42.9 g/l NaCl, 1.14 g/l 2% antifoam 1510, BHD 63215). Three ml were added to 4000 ml of fermented grape juice, resulting in a final concentration of 4.3*106 CFU/ml. This was allowed to stand for another 10 days at room temperature.
Results
Effect of Enzymatic Treatment on Alcohol Levels
Glucose and fructose levels were measured using a commercial UV based assay supplied by Boehringer Mannheim/R-biopharm (catalog number 10 139 106 035), using the protocol supplied by the provider.







TABLE II







Sugar levels at the start end of the alcoholic fermentation.













Glucose
Fructose
Total sugar


day
Treatment
(g/l)
(g/l)
(g/l)














0
Control
110 ± 3 
118 ± 1
229 ± 4



GOX
96 ± 5
124 ± 3
225 ± 9



GOX + Isomerase
106 ± 22
119 ± 5
 225 ± 25


11
Control
 0 ± 0
 8 ± 2
 8 ± 2



GOX
 24 ± 17
 59 ± 11
 83 ± 27



GOX + Isomerase
0
0
0









Alcohol was measured at different days during the alcoholic fermentation using the Dr. Rebelein titration method as described in the literature (Bestimmung des alkoholgehalts nach Dr. Rebelein. Issued by: C Schliesmann Kellerie-Chemie GmbH & Co. KG, Auwiesenstrasse 5, 74523 Schwabische Hall (2001)). In the untreated grape juice the fermentation was almost complete, reaching a final alcohol level of 12.7% at the end of the process. When the juice was pretreated with both glucose oxidase and glucose isomerase the sugar fermentation was complete but the final level of alcohol was still significantly lower (11.8%).
The low levels of alcohol found when the juice was pretreated with glucose oxidase only, are a result of incomplete fermentation. The glucose oxidase treated juice in this experiment is not usable in normal fermentation due to the high levels of residual sugars—especially fructose—at the end of fermentation (Table II).
Accordingly, the extra addition of glucose isomerase helped to maintain the ratio glucose/fructose in the juice at a ratio around 1:1, which significantly decreases the risk of unwanted stuck alcoholic fermentations as shown when using only GOX.
Further, the experiment with isomerase removed all sugar while some fructose sugar (8 g/l) still was present in the control (untreated grape juice). This demonstrates that isomerase as such prevents stuck fermentations.







TABLE III







Alcohol levels during the fermentation.









Day
Treatment
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





At day 11 the malolactic fermentation was started. The alcohol levels in the glucose oxidase (GOX) pretreated samples are in italics to indicate that these values are the result of a severely delayed alcoholic fermentation.


Nd: not determined






Conclusion

The results of this example 2 shows that GOX+Isomerase significantly reduced alcohol percentage to 11.8% as compared to 12.7 of control.
Further, the extra addition of glucose isomerase helped to maintain the ratio glucose/fructose in the juice at a ratio around 1:1, which significantly decreases the risk of unwanted stuck alcoholic fermentations as compared to using GOX alone.
The low levels of alcohol (9.3%) found when the juice was pretreated with glucose oxidase only, are a result of incomplete fermentation—in other words unwanted stuck alcoholic fermentations. The glucose oxidase treated juice in this experiment is not usable in normal fermentations due to the high levels of rest sugar—especially fructose—at the end of fermentation (Table II).
Further, the experiment with isomerase removed all sugar while some fructose sugar (8 g/l) still was present in the control (untreated juice). This demonstrates that isomerase as such prevents stuck fermentations.
Example 3
Growth of Yeast During the Alcoholic Fermentation—Addition of Isomerase Significantly Reduces Stuck Alcoholic Fermentation
It is known to the skilled person that stuck fermentations typically arise when fructose concentrations are considerably higher than glucose concentrations. During the alcoholic fermentation the glucose/fructose ratio may change resulting in a delayed fermentation.
In this example 3 a delayed (stuck) fermentation was induced by treatment of the must with Glucose oxidase alone.
In order to investigate the effect of glucose isomerase on the ability of yeast to grow and survive during an alcoholic fermentation a simulated beverage production, in this case wine, was performed as described in Example 2 herein. The juice was pre-incubated for three days with enzymes as described below, followed by the alcoholic fermentation of 11 days and a malolactic fermentation of 10 days.
Three independent experiments were performed using two replicates in each case. In each sample 200 ml grape juice (Pinot Blanc 2007, Germany, pasteurized) was added to a glass flask and continuously mixed with a magnetic stirrer. The samples were aerated throughout the experiment.
Either 100 mg glucose oxidase (Hyderase, Amano, >15,000 u/g, corresponding to 150,000 u per hl of solution) or both 100 mg glucose oxidase and 1 g glucose isomerase (Sigma, G4166-50 g, >350 u/g, corresponding to 35,000 u per hl solution) were added to the flasks. The incubation was allowed to run at room temperature for 3 days. After this time point the alcoholic fermentation was started by inoculation with re-hydrated freeze dried wine yeast (Saccharomyces cerevisiae Merit.Ferm, Chr. Hansen, 0.1 g/l) to a final concentration of 9E+05 CFU/ml. Re-hydration was performed in peptone water (15 g/l Tryptone, Oxoid L 42.9 g/l NaCl, 1.14 g/l 2% antifoam 1510, BHD 63215) for 10 minutes at room temperature. Eleven days after the start of the alcoholic fermentation the malolactic fermentation was started by addition of O. oeni (Viniflora, Chr. Hansen. Batch no.: 2711097) to the fermented juice. Freeze dried O. oeni (0.7 g of 8.2 E+11 CFU/g) was allowed to re-hydrate for 10 minutes in 100 ml of peptone water 15 g/l Tryptone, Oxoid L 42.9 g/l NaCl, 1.14 g/l 2% antifoam 1510, BHD 63215). Three ml were added to 4000 ml of fermented juice, resulting in a final concentration of 4.3*106 CFU/ml. This was allowed to stand for another 10 days at room temperature.
The number of S. cerevisiae colony forming units (CFU) was determined at different time points by taking samples from the fermented juice and plating serial dilutions on YGC solid medium agar plates followed by an overnight incubation at 30° C.
Sugar levels were determined using a commercial UV based assay supplied by Boehringer Mannheim/R-biopharm (catalog number 10 139 106 035), using the protocol supplied by the provider.
Results
Effect of Isomerase on a Stuck Alcoholic Fermentation
During the alcoholic fermentation the sugars in the juice are converted to ethanol by the yeast S. cerevisiae. 
Treatment with glucose oxidase alone was shown to result in a delayed alcoholic fermentation (stuck fermentation) due to delayed growth of S. cerevisiae (as shown in Table IV). In the must pretreated with glucose oxidase, growth of yeast was very poor during the first days of the alcoholic fermentation. The number of CFUs was below the detection limit at day 1 and was approximately 3 log units lower at day 2 of the alcoholic fermentation. This is a clear indication of a stuck fermentation.
This result was supported by the sugar analysis. While in the non-treated must approx. 60% of the sugar was fermented after 3 days of yeast fermentation, less than 10% was fermented in the GOX pre-treated must.
However, when glucose isomerase was present during the pretreatment and alcoholic fermentation, the fermentation process behaved almost identical to the fermentation of untreated must. Both the remaining sugar levels and the S. cerevisiae CFU numbers (Table IV) were comparable to the untreated must. In other words; glucose isomerase was able to overcome the stuck fermentation caused by GOX treatment.







TABLE IV







Viable S. cerevisiae cell count during the alcoholic fermentation.













Total sugar


Days
Treatment
CFU/ml (average)
(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





The juice had been pretreated for three days as described. Yeast was added at t = 0 days.


Nd = below detection limit.






Conclusion

As shown in this example 3 use of GOX alone may induce significant unwanted stuck fermentation.
The results of this example 3 show that addition of isomerase can help to overcome the negative effects of addition of GOX on the growth of S. cerevisiae generally used for fermentation of beverages.
REFERENCES


    1. U.S. Pat. No. 4,675,191 (Novo Industri, Denmark—published 1987)

## Claims

1. A method for production of an alcoholic beverage with reduced content of alcohol, comprising the following steps:
(1): treating unfermented apple, pear, barley, pomegranate, pineapple, strawberry, or mango beverage starting solution with an effective amount of:
(a) glucose oxidase in the presence of oxygen for a period of time adequate to convert at least a portion of glucose in the solution into gluconic acid, and
(b) glucose isomerase for a period of time adequate to convert at least a portion of fructose in the solution into glucose;

and thereafter
(2): fermenting said treated solution to obtain the alcoholic beverage with reduced content of alcohol.

2. The method of claim 1, wherein the produced beverage has an alcohol content of about 10%.

3. The method of claim 1 further comprising removing at least a portion of the gluconic acid to obtain a beverage with satisfactory organoleptic properties; and wherein the gluconic acid is removed by adding a substance that forms a sparingly soluble salt of gluconic acid.

4. The method of claim 1, wherein the effective amount and the period of time for glucose oxidase/isomerase enzymes during step (1) are such that:
(A): the sugar content in the unfermented beverage starting solution is reduced by at least 5%.

5. The method of claim 4, wherein the sugar content in the unfermented beverage starting solution is reduced by at least 17%.

6. The method of claim 1, wherein
the period of time is 72 hours or less; and
the effective amounts of glucose oxidase/isomerase enzymes are:
(i): a glucose oxidase activity between 1,000 and 50,000,000 international units per hl of solution; and
(ii): a glucose isomerase activity between 100 and 5,000,000 international units per hl of solution; and

the temperature during step (1) is between 1 and 35° C.

7. The method of claim 1, wherein oxygen is supplied continuously to the solution during step 1 of claim 1.

8. The method of claim 1, wherein step (1) further comprises treating the solution with an effective amount of a preparation with catalase activity for a period of time adequate to convert at least a portion of the H2O2 in the juice into O2+H2O.

9. The method of claim 1, further comprising adding additional glucose isomerase before or during step (2).

10. The method of claim 1, wherein the beverage is cider or beer.

11. The method of claim 3, wherein the substance is calcium carbonate.

12. The method of claim 6, wherein the temperature during step (1) of claim 1 is between 3° C. and 30° C.

13. The method of claim 7, wherein the oxygen is supplied by means of an air pump.

14. The method of claim 2, wherein the alcohol content is at least 3% lower than if the beverage starting solution had not been treated with glucose oxidase and glucose isomerase.

15. The method of claim 1, wherein the produced beverage has an alcohol content of 5% to 10%.

16. The method of claim 1, wherein the produced beverage has an alcohol content of 1% to 5%.