Patent Description:
Today, as a consequence of greater social and health awareness with consumers, there is a high demand in the wine world for solutions to decrease the alcohol content in the final wine. As an alternative to full-strength wines, wines with a reduced alcohol content offer a number of potential social and health benefits for consumers. Social benefits may include improved productivity and function after activities involving alcohol (e.g. business lunches), lower risk of accidents while driving and more acceptable social behavior in general. Health advantages may include reduced calorie intake, decreased risk of alcohol-related illness and disease and specific benefits for pregnant women. For the producers of these wines, there exists an incentive of identified markets and market segments, as well as lower sales and duty taxes applicable in many countries (Pickering, <NUM>).

Methods for producing reduced-alcohol wines have been available since the early <NUM>. Although commercial production nowadays relies almost exclusively on systems based on the use of membranes and modified distillation, a number of alternative approaches have been put forward in literature. These approaches are summarized in Table <NUM> (Pickering, <NUM>).

When using unripe fruit and/or diluted juice/must, the flavor of the final wine is compromised, due to the lower concentration of flavor precursors. Mechanical removal of ethanol tends to be a harsh process that affects the overall chemical composition of the wine, and thereby also the sensory profile and experience of the wine.

A lot of work has been done so far in reducing sugar with a biological approach. Five different methods have been explored: <NUM>) aeration of the must during fermentation using Saccharomyces sp. with a conversion of sugar to CO<NUM>, water and biomass, <NUM>) aeration of the must during fermentation using non-Saccharomyces sp. with a conversion of sugar to CO<NUM>, water and biomass, <NUM>) the use of non-Saccharomyces and Saccharomyces sp. in a sequential fermentation, <NUM>) early arrest of fermentation for the production of sweet wines and <NUM>) genetically modifed Saccharomyces cerevisiae wine strains to redirect ethanol production.

Lowering alcohol percentage by aeration of the must during fermentation using Saccharomyces sp. has been explored since <NUM>. A process has been described for the preparation of low-sugar or sugar-free fruit juices based on continuous or semi-continuous culture with yeast, with the conditions resulting in metabolism of sugar to CO<NUM> and water rather than to ethanol and so recovering an alcohol-free, sugar-free or low sugar juice by separation of the biomass (Kaeppeli, <NUM>). A similar system is described by Grossmann et al. (<NUM>) for producing low-alcohol drinks, where oxygen is added to the fermenting liquid in a controlled manner in order to convert the sugars to water and CO<NUM>. The supply of oxygen is interrupted when the required level of residual alcohol is reached, then the yeast is separated from the liquid, and finally the liquid is microfiltered in order to produce a clear liquid product.

By screening a broad collection of yeast species, Kolb et al (<NUM>) found that Pichia stipitis was particularly well suited for juice sugar removal. Their claims include the elimination of more than <NUM>% of juice sugar within <NUM>, and a minimum of adverse effects on the sensory and functional qualities of the juice. Similar results have been obtained by investigating the effect of varying aeration and temperature levels on the reduction of sugar and production of alcohol in Muller-Thurgau grape juice by selected yeast strains (Smith et al. Seven yeast strains showed promising results of which three strains produced significant alcohol reductions: Pichia stipitis, Candida tropicalis and Saccharomyces cerevisiae. By combining short-term controlled aeration, to reduce the sugar content, with anaerobic fermentation using an active dried wine yeast, wines with acceptable taste and <NUM> to <NUM>% less alcohol were produced. However, these wines exhibited the deep golden color indicative of oxidation, which generally is undesired in wine.

More investigations into mixed starter cultures of non-Saccharomyces species with a Saccharomyces cerevisiae wine strain revealed that the alcohol percent (v/v) could be reduced with <NUM>-<NUM>% (Ciani and Ferraro, <NUM>; Ferraro et al. <NUM>; Erten and Campbell, <NUM>; Ciani et al. <NUM>; Garcia et al. <NUM>; Maygar and Toth, <NUM>; Comitini et al. <NUM>; Di Maio et al. <NUM>; Sadoudi et al. In a recent publication of the Australian Wine Research Institute (AWRI), evaluation of <NUM> different non-Saccharomyces isolates, belonging to <NUM> different genera, has been performed for their capacity to produce wine with lower ethanol concentration when used in sequential inoculation regimes with a Saccharomyces cerevisiae wine strain (Contreras et al. A sequential inoculation of Metschnikowia pulcherrima AWRI1149 followed by a Saccharomyces cerevisiae wine strain was the best combination able to produce wine with a lower ethanol concentration than the single-inoculum, wine yeast, control. Sequential fermentations utilizing AWRI1149 produced wines with <NUM>% (v/v) and <NUM>% (v/v) (corresponding to <NUM>/L and <NUM>/L) less ethanol concentration in Chardonnay and Shiraz, respectively.

Another approach of lowering ethanol in wine production is the use of genetically modified yeast strains (Kutyna et al. However, with the current consumer reluctance against the use of GMO yeast strains in wine, this option is not considered to be commercially viable at this moment.

<CIT> discloses a process for the production of a malolactic ferment to produce wine. The malolactic ferment is produced from at least one malolactic bacterium strain (such as Leuconostoc oenos, Lactobavillus brevis or Lactobacillus plantarum) which is cultured in a culture medium containing an assimilable nitrogen source, malic acid and alcohol.

Currently, the mentioned methods have a limited commercial application. The main problem is the reduced sensory quality compared with full-strength wine. In particular, problems with flavor imbalance and a lack of 'body' have been reported. The main flavor compound resulting in an imbalanced flavor profile is ethyl acetate, which is often found in very high concentrations in the reduced alcohol wines produced with the current methods. These altered sensory properties can occur as a result of the processing required to produce the reduced alcoholic wine or as a direct consequence of the reduced ethanol content. The main characteristics of the biological approaches to reduced alcohol wines is the potential off-flavor development (too high acetic acid or ethyl acetate concentrations) with the use of non-Saccharomyces yeast strains or oxidation of the final wine when aeration techniques are used, resulting in undesirable organoleptic characters.

The problem to be solved by the present invention is the provision of an alternative method for reducing the alcohol content in fermented fruit beverages without a loss in sensory quality of the wine.

The solution is based on the surprising finding by the inventors that Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM>, when inoculated prior to or simultaneously with yeast fermentation, can be used to lower the alcohol content in wine with at least <NUM>% (v/v) compared to the wine made by fermentation with the yeast only.

The inventors observed that the lactic acid bacterium uses the sugar in the must to form lactic acid (and other metabolites) without producing ethanol. In this way, ethanol can be reduced in the final wine, without any negative influence on flavor profile.

Moreover, it seems that the fruity notes as well as the round mouth feel are enhanced in these wines.

On top of that, also the malic acid may be converted to lactic acid by the homofermentative or facultative heterofermentative Lactobacillus spp. strains tested, meaning that the malolactic fermentation (MLF) is performed during the alcoholic fermentation.

This means that by using this new technique, winemakers can reduce alcohol and complete MLF at the same time, without compromising flavor.

In comparison to the current state-of-the-art biological approaches to lower alcohol by using either oxygen in combination with a Saccharomyces or non-Saccharomyces yeast or by using non-Saccharomyces strains with lower sugar to ethanol conversion, the present technique of using homofermentative or facultative heterofermentative lactic acid bacterium strains in reverse inoculation (the lactic acid bacterium strain is inoculated prior to fermentation with the yeast) or co-inoculation (the lactic acid bacterium strain is inoculated at the same time as fermentation with the yeast) with a wine yeast does not result in wine oxidation, flavor imbalance or lack of mouth feel, but may even improve the flavor profile of the wine.

One aspect of the present invention relates to a method for producing a beverage with a reduced content of alcohol comprising the steps of:.

In a preferred embodiment, the fermentation of the fruit must with a yeast strain in step b1) is carried out <NUM> hours, such as <NUM> hours, such as <NUM> hours, such as <NUM> hours, after adding Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM>.

In a further preferred embodiment the fermentation of the fruit must with a yeast strain in step b2) is carried out within <NUM> hours, such as within <NUM> hours, such as within <NUM> hours, such as within <NUM> hours, such as within <NUM> hours.

In a preferred embodiment, the yeast strain is Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Torulaspora delbrueckii, or Kluyveromyces thermotolerans.

The term "reduced content of alcohol" in a beverage as used herein relates to a lower concentration of alcohol in the beverage as compared to a beverage prepared under identical conditions but without the addition of the homofermentative or facultative heterofermentative lactic acid bacterium strain.

As used herein, the term "must" designates the unfermented or fermenting juice expressed from grapes or other fruits.

The term "wine", as used herein, refers to a must, in which the alcohol quantity produced by alcoholic fermentation is at least <NUM>% (v/v), including but not limited to fermented grape must (red wine, white wine, sparkling wine etc.) and cider (fermented fruit must based on juice from apple, pear, peach etc.). The wine may have reached its maximum alcoholic degree if the alcoholic fermentation is ended. The alcohol contents are expressed by the volume of alcohol in relation to the total volume.

Homofermentative lactic acid bacteria ferment glucose and/or fructose with lactic acid as the primary by-product.

Heterofermentative lactic acid bacteria ferment glucose and/or fructose with lactic acid, ethanol/acetic acid and carbon dioxide as by-products. Facultative heterofermentative lactic acid bacteria produce carbon dioxide and other by-products only under certain conditions or from specific substrates.

The group of homofermentative and facultative heterofermentative lactic acid bacterium strains include strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus sake, Lactobacillus salivarius, Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus pentocaceus, Pediococcus parvulus and Streptococcus thermophilus.

In a preferred embodiment the fruit must is inoculated and fermented with at least two or more homofermentative and facultative heterofermentative lactic acid bacterium strain in step a), such as <NUM>, <NUM>, <NUM>, <NUM> or more homofermentative and facultative heterofermentative lactic acid bacterium strains.

It was shown in the Examples below that when the bacteria are inoculated at relatively low concentrations the amount of bacteria inoculated in step a) determines the period of time adequate to convert at least a portion of the sugar to lactic acid. Furthermore, it was shown, that when the bacteria were inoculated at relatively high concentrations, it was possible to co-inoculate the bacteria and the yeast and still achieve a reduction in the content of alcohol in the beverage.

Thus, in a preferred embodiment Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM> is inoculated in an amount of at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, and the fruit must is fermented for a period of time adequate to convert at least a portion of the sugar to lactic acid or other metabolites before the fermentation with the yeast strain.

Preferably, the period of time adequate to convert at least a portion of the sugar to lactic acid or other metabolites is at least <NUM> hours, such as at least <NUM> hours, such as at least <NUM> hours, such as at least <NUM> hours, such as at least <NUM> hours, such as at least <NUM> hours, such as at least <NUM> hours.

In a more preferred embodiment, the portion of the sugar which is converted to lactic acid or other metabolites is at least <NUM>/L, such as at least <NUM>/L, such as at least <NUM>/L, such as at least <NUM>/L.

In another preferred embodiment Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM> is inoculated in an amount of at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, such as at least 1x10<NUM> CFU/ml, and the fruit must is fermented simultaneously with the lactic acid bacterium strain and the yeast strain.

In one preferred embodiment the fruit must is fermented with a yeast strain which is inoculated in an amount of at least 1x10<NUM> CFU/ml, such as at least 5x10<NUM> CFU/ml, at the onset of yeast fermentation in step b2).

In another preferred embodiment the fruit must is fermented spontaneously in step b1) or step b2) with an indigenous yeast.

The yeast which is used may be any type of yeast which is used in the beverage or food industry. Examples include e.g. Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Torulaspora delbrueckii, or Kluyveromyces thermotolerans etc..

Typical yeasts used in wine production are from the yeast family Saccharomycetaceae (ascomycetous yeasts). Yeasts from the genus Saccharomyces (e.g. the species Saccharomyces cerevisiae) are commonly used. Other used yeasts are from the same Saccharomycetaceae family but from other genera such as Kluyveromyces (e.g. the species Kluyveromyces thermotolerans) and the genus Torulaspora (e.g. the species Torulaspora delbrueckii).

For the inoculation of a fruit must, a pure yeast culture may be used (i.e. a culture containing only one type of yeast), but a mixed culture of two or more types of yeast may also be used as inoculant.

In a preferred embodiment the fruit must is inoculated and fermented with at least two or more yeast strains in step b), such as <NUM>, <NUM>, <NUM>, <NUM> or more yeast strains.

The method according to the invention allows for significant reduction in the alcohol concentration of a beverage:.

Accordingly, in a preferred embodiment, the alcohol concentration of the beverage is at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), such as at least <NUM>% (v/v), lower than the alcohol concentration of a beverage prepared under identical conditions but without the addition of the homofermentative or facultative heterofermentative lactic acid bacterium strain.

Furthermore, an aspect of the present invention is directed to Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM>.

An even further aspect relates to use of Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM> in a method for producing a beverage with a reduced content of alcohol comprising the steps of:.

As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The industrially most useful lactic acid bacteria are found within the order "Lactobacillales" which includes Lactococcus spp. , Streptococcus spp. , Lactobacillus spp. , Leuconostoc spp. , Pediococcus spp. , Brevibacterium spp. , Enterococcus spp. , Propionibacterium spp. and Oenococcus spp.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, "a" and "an" and "the" may mean at least one, or one or more.

The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Lab-scale fermentation trials were carried out in <NUM> of grape juice. Four different grape juices were used: Italian white grape juice, Italian red grape juice,Riesling juice and Hungarian white grape juice. All fermentations were performed at <NUM> until completion (sugar levels < <NUM>/L). Lactobacillus plantarum strain CHCC12399 isolated from South African grape juice, another Lactobacillus plantarum strain or a Lactobacillus paracasei strain was inoculated at different inoculation levels, ranging from 5x10<NUM> to 5x10<NUM> CFU/ml. Saccharomyces cerevisiae wine yeast was inoculated at 1x10<NUM> CFU/ml.

Headspace gas chromatography coupled with flame ionisation detection (GC-FID) was used for the measurement of esters and higher alcohols in the fermentation products. Fermentation samples were centrifuged, after which <NUM> was collected in vials. Samples were then analyzed with a calibrated Perkin Elmer GC System with a headspace sampler. The GC was equipped with a DB-WAXETR column (length, <NUM>; internal diameter, <NUM>; layer thickness, <NUM>; Agilent Technologies, Germany). The split-splitless injector was used and held at <NUM>. Samples were heated for <NUM> at <NUM> in the headspace autosampler before injection (needle temperature: <NUM>). Helium was used as the carrier gas. After starting at <NUM>, the oven temperature was raised after <NUM> from <NUM> to <NUM> at <NUM>/min and was finally held at <NUM> for <NUM>. During the GC-program a constant flow rate (<NUM>/min) of the carrier gas (He) was maintained. The FID temperature was kept constant at <NUM> respectively. The results were analyzed with Turbochrom software.

Sensory analysis was performed with a professional tasting panel.

Ethanol, total sugar (glucose + fructose), total acid (TA), volatile acid (VA), pH and malic acid analysis was performed with Oenofoss™ equipment according to the manufacturer's protocol. Lactic acid, glucose, fructose and acetic acid concentrations were measured with HPLC by methods known to the skilled person and as described by e.g. <NPL>).

EXAMPLE <NUM>: Reverse inoculation of grape juice with Lactobacillus plantarum strain CHCC12399 and a wine Saccharomyces cerevisiae yeast strain.

The first experiment is performed with a Lactobacillus plantarum strain CHCC12399 isolated from grape juice, which was below pH <NUM>. Lab trials with the Lactobacillus plantarum strain have been performed in both Italian red and white grape juice. The Lactobacillus plantarum strain used here comes as a product in a frozen format (frozen pellets).

Lactobacillus plantarum was inoculated at several dosages (ranging from 5x10<NUM> to 5x10<NUM> CFU/ml) in a reverse inoculation with a wine Saccharomyces cerevisiae, inoculated after <NUM> at level of 1x10<NUM> CFU/ml. The fermentation volume was <NUM>. The initial parameters of the grape juice are described in Table <NUM>.

In total, <NUM> conditions have been tested for each grape juice:.

When the fermentations were finished (when sugar was depleted), alcohol level and other relevant wine parameters were measured (Table <NUM> and <NUM>).

From Table <NUM> and <NUM>, it is clear that the wine inoculated with 5x10<NUM> CFU per ml Lactobacillus plantarum had ≤ <NUM>% less alcohol (v/v), compared to the control wine and this was the case for both the red and white wine. Interestingly, the dosage of Lactobacillus plantarum seems of high importance to reach the lower alcohol percent, as inoculation of 1x10<NUM> CFU/ml has no significant effect on the alcohol level. From Table <NUM>, it is can also be seen that addition of Lactobacillus plantarum has no negative effect on the acetic acid production, as the concentrations are similar between the control and all dosages of Lactobacillus plantarum. However, it is believed that lower dosages of Lactobacillus plantarum will also reduce alcohol, depending on the time of inoculation of yeast afterwards; the longer Lactobacillus plantarum are allowed to ferment alone, the more sugar will be converted to lactic acid or other metabolites. It is also clear that no more acetic acid is produced when adding the Lactobacillus plantarum.

As more lactic acid is produced than is theoretically possible from malic acid, part of the glucose and fructose is converted to lactic acid, leaving less sugar to be converted to ethanol (Table <NUM>). As can be seen in Table <NUM>, more than <NUM>/L sugar (glucose and fructose) has been consumed by Lactobacillus plantarum in the first <NUM> hours. This was very surprising, as it was expected that only malic acid would have been consumed under these conditions. <NUM>% of ethanol (v/v) corresponds to <NUM>/L of sugar consumed by Lactobacillus plantarum. If all sugar would be converted to lactic acid, at least <NUM>/L of lactic acid should be formed. As this is not the case, either other metabolic products are formed from glucose or the lactic acid reacts with other metabolic products present during fermentation to form yet another set of metabolic products.

To confirm that 5x10<NUM> CFU/ml of Lactobacillus plantarum is needed to reduce the final alcohol percentage in wine with <NUM>% (v/v), another set of experiments was performed. In this experiment, inoculation levels between 1x10<NUM> CFU/ml and 5x10<NUM> CFU/ml Lactobacillus plantarum were tested for their effect on ethanol production. As adding the wine yeast after <NUM> is not commercially viable, the wine yeast was added after <NUM>. In this case, Riesling juice was used for the fermentation experiments. The initial parameters of the Riesling juice are given in Table <NUM>.

Lactobacillus plantarum was added in <NUM> different inoculation dosages: 2x10<NUM> CFU/ml, <NUM>. 5x10<NUM> CFU/ml, 3x10<NUM> CFU/ml, <NUM>. 5x10<NUM> CFU/ml, 4x10<NUM> CFU/ml and 5x10<NUM> CFU/ml in the Riesling grape juice. After <NUM>, a wine Saccharomyces cerevisiae strain (1x10<NUM> CFU/ml) was added to the juice to finish the fermentation. The fermentation volume was <NUM> and the fermentation temperature was <NUM>. The final wine parameters are shown in Table <NUM>. The malolactic fermentation (MLF) performance and final ethanol levels are depicted in <FIG>.

The results in Table <NUM> and <FIG> clearly show that 5x10<NUM> CFU/ml Lactobacillus plantarum is needed to decrease the ethanol percent with <NUM>% (v/v). However, from an inoculation level of <NUM>. 5x10<NUM> CFU/ml, alcohol level is decreasing in the final wine. On top of that, <FIG> shows that MLF is performed during the first three days of fermentation by inoculating Lactobacillus plantarum in all dosages.

EXAMPLE <NUM>: Co-inoculation of grape juice with Lactobacillus plantarum strain CHCC12399 and a wine Saccharomyces cerevisiae yeast strain.

As it is easier to add both Lactobacillus plantarum and the yeast together in a so called co-inoculation, it was tested if the ethanol percentage could be reduced in final wine by co-inoculating the Lactobacillus plantarum with a Saccharomyces wine yeast. In this way, winemakers can add both the Lactobacillus plantarum and the wine yeast at the start of a wine fermentation, which makes the new technique easier to use in a commercial way.

For this experiment, the same Riesling juice was used as described in Table <NUM>. Lactobacillus plantarum was added at an inoculation dosage of 5x10<NUM> CFU/ml, together with the Saccharomyces cerevisiae wine yeast (1x10<NUM> CFU/ml). The fermentation was performed at <NUM>. Malolactic fermentation (MLF) activity and alcohol production are shown in <FIG>.

<FIG> clearly show that co-inoculation of Lactobacillus plantarum with a Saccharomyces cerevisiae wine yeast results in a final alcohol reduction of <NUM>% (v/v). Also malic acid can be completely degraded when using co-inoculation. This means that co-inoculation of Lactobacillus plantarum together with Saccharomyces cerevisiae in the correct dosage results in a final alcohol reduction of <NUM>% (v/v) with a completed MLF during the alcoholic fermentation.

EXAMPLE <NUM>: Reverse inoculation of grape juice with Lactobacillus plantarum strain CHCC12399, Lactobacillus plantarum strain LPL-<NUM> or Lactobacillus paracasei strain LPA-<NUM>, respectively, and a wine Saccharomyces cerevisiae yeast strain.

To investigate if this property of lowering the alcohol content is strain- and/or species-specific, another strain of Lactobacillus plantarum, LPL-<NUM>, and a Lactobacillus paracasei strain, LPA-<NUM>, were tested for reducing alcohol (see Table <NUM>). The same protocol was used as before, where the Saccharomyces cerevisiae wine yeast was added after <NUM>. The Lactobacillus strains were inoculated in at a rate of 5x10<NUM> CFU/ml and the yeast was added at a rate of 1x10<NUM> CFU/ml.

It is very clear from Table <NUM> that all the tested Lactobacillus sp. can reduce alcohol with at least <NUM>%. However, it seems that only Lactobacillus plantarum has the combined property of reducing alcohol and degrading the malic acid completely.

EXAMPLE <NUM>: Reverse inoculation with Lactobacillus plantarum strain CHCC12399 and a wine Saccharomyces cerevisiae yeast strain - Field trials.

In these experiments, it was tested if this also works in the field. Two trials were set-up in wineries, where the wine Lactobacillus plantarum CHCC12399 strain was tested in two different grape varieties: a white grape variety, Chardonnay and a red grape variety, Merlot. In the field trials, it was also tested how the alcohol reduction with Lactobacillus sp. affect the flavor profile of the final wine, as this is the main negative issue with using either non-Saccharomyces yeast or aeration for reducing alcohol.

In all cases, Lactobacillus plantarum was added on the grapes or in the grape juice at the start of fermentation at a dosage of 5x10<NUM> CFU/ml and yeast was added after <NUM> hours. The Chardonnay trial was done in <NUM> hL tanks and the Merlot trial was performed in <NUM> hL tanks. In all cases, the initial sugar content was approximately the same and there was a control fermentation carried out with only adding Saccharomyces wine yeast. The results are shown in Table <NUM>.

The results in Table <NUM> show that in all field trials, the wine Lactobacillus plantarum was able to reduce the final alcohol percent with at least <NUM>% (v/v) and this with a very low inoculation rate (5x10<NUM> CFU/ml). This means the proposed technique of using a Lactobacillus strain to reduce alcohol with at least <NUM>% also works in big scale.

The wines from Table <NUM> were analyzed for flavor profile. Furthermore, the Chardonnay wines were analyzed by a professional sensory panel to investigate if the final wines are acceptable with regard to flavor profile compared to the control wines. The sensory panel consisted of <NUM> people who scored the wines <NUM> to <NUM> for each characteristic.

The flavor analysis results, performed with headspace-GC-FID, are shown in <FIG>. Sensory analysis results are shown in <FIG>.

As can be seen in both <FIG>, the flavor profile of the Chardonnay control compared with the Chardonnay inoculated with Lactobacillus plantarum look very similar. Especially ethyl acetate, which is a main trouble component when non-Saccharomyces strains are added, is very similar in both wines and even a bit less in the Chardonnay with addition of Lactobacillus plantarum. The same is true for the Merlot wines, also here, the flavor profiles look very similar.

It is very clear from the sensory analysis that the Chardonnay wine with addition of Lactobacillus plantarum has certainly no defects compared to the control wine. The test wine is hotter, as shown in <FIG>, but the overall aromatic intensity and complexity are more preferred in the test wines, compared to the control wine. This is confirmed in the descriptive analysis, where the Chardonnay with the addition of Lactobacillus plantarum has a more fruity character (grapefruit, pineapple, mango), compared to the control wine.

EXAMPLE <NUM>: Reverse inoculation with different time periods between inoculation with Lactobacillus plantarum strain CHCC12399 and inoculation with Merit wine yeast in Hungarian white grape juice.

To investigate the potential of Lactobacillus plantarum strain CHCC12399 to reduce alcohol by reducing glucose and/or fructose into lactic acid at the beginning of wine fermentation, experiments were performed in Hungarian white grape juice with the following characteristics (Table <NUM>).

The fermentation set-up is described in Table <NUM>. Here it is shown that <NUM> different setups were used: addition of Lactobacillus plantarum strain CHCC12399 (NoVA) at the start of fermentation with addition of yeast at different time points afterwards. As control fermentations only yeast were inoculated (exp <NUM> and <NUM>). The yeast used is Merit inoculated at a concentration of 1x10<NUM> CFU /ml. Fermentations were carried out at <NUM>.

Fermentations were carried out until sugar was depleted. During the fermentations, samples were taken for Oenofoss measurements, as well as HPLC measurements for sugars and acids.

Viability of Lactobacillus was analysed by anaerobic plating on grape juice agar (GJ5; <NUM>/L grape juice concentrate (K V Saft Vallø), <NUM>/L yeast extract (Bio Springer), <NUM>/L Tween® <NUM> (Sigma-Aldrich), <NUM>/L MnSO<NUM>, H<NUM>O (Merck), <NUM>/L agar (SO-BI-GEL) in tap water) with natamyxin (<NUM>/L; Delvocide from DSM Food Specialities B. The plates were incubated for <NUM> days at <NUM> and then counted.

Viability of yeast was analysed by plating on standard YGC (yeast extract, glucose, chloramphenicol) agar plates. The plates were incubated for <NUM> days at <NUM> and then counted.

The results shown do not contain experiment number <NUM> and <NUM>, as spontaneous fermentation with yeast started in these samples and they are therefore considered to be inaccurate. Spontaneous fermentation also started in exp number <NUM> and <NUM>, but was over seeded with Merit yeast at <NUM> hours after addition of Lactobacillus plantarum strain CHCC12399 (NoVa). The Lactobacillus and yeast cell counts are given in <FIG>, respectively. The sugar and acid measurements are shown in <FIG>.

From the results shown in <FIG>, it is clear that Lactobacillus plantarum grew in all experiments, except for the experiment with co-inoculation, where the cell count does not change over time. This could be explained by the fact that yeast counts are already very high at day <NUM>, meaning that Lactobacillus plantarum strain CHCC12399 does not have the time to grow. Lactobacillus plantarum strain CHCC12399 (NoVA) is still active, though, as malic acid is completely consumed after day <NUM> (<FIG>).

Ethanol concentrations during fermentation are shown in <FIG> and final ethanol concentrations are shown in Table <NUM>.

It is very clear from the final ethanol concentrations in Table <NUM> that adding Lactobacillus plantarum strain CHCC12399 (NoVA) in a concentration of 5x10<NUM> CFU/ml and fermenting <NUM> or <NUM> days before the addition of yeast can reduce alcohol levels. Highest alcohol reduction was with yeast inoculation after <NUM> days in this case, where the final alcohol reduction is <NUM>,<NUM>% ethanol (v/v).

From <FIG>, it is also clear that malic acid needs to be consumed first, before Lactobacillus plantarum strain CHCC12399 (NoVA) starts eating sugar. Malic acid is consumed within <NUM> days for all different scenarios. Even with the co-inoculation of Lactobacillus plantarum strain CHCC12399 and yeast, malic acid gets consumed within <NUM> days after the start of fermentation. Lactic acid is produced from both malic acid and sugar (fructose), resulting in a higher lactic acid production when the yeast is added later. Almost no lactic acid is produced in the control fermentation, where only yeast is added.

It is also clear from the single sugar results in Table <NUM> that Lactobacillus plantarum strain CHCC12399 (NoVA) prefers fructose over glucose.

Lactobacillus plantarum strain CHCC12399 consumes fructose and no glucose before the yeast is added, but must also consume more fructose (and/or glucose) during the start of alcoholic fermentation, in order to decrease the alcohol level with <NUM>%.

It is clear from this experiment that Lactobacillus plantarum strain CHCC12399 can reduce alcohol in final wines by reverse inoculation. The results show that malic acid is assimilated first, before Lactobacillus plantarum strain CHCC12399 starts assimilating sugar. It was also surprising to find that Lactobacillus plantarum strain CHCC12399 consumes fructose as the main sugar, and does not seem to consume glucose in this case.

The strain of Lactobacillus plantarum CHCC12399 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-<NUM> Braunschweig, Germany on <NUM> August <NUM> under the accession number DSM <NUM>.

The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

Claim 1:
A method for producing a beverage with a reduced content of alcohol comprising the steps of:
a) inoculating and fermenting a fruit must with Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM <NUM>; and
b1) within a period of time after adding Lactobacillus plantarum strain CHCC12399, fermenting the fruit must with at least one yeast strain to obtain the beverage with a reduced content of alcohol compared to the beverage fermented with the at least one yeast strain alone, wherein Lactobacillus plantarum strain CHCC12399 is inoculated in an amount of at least 1x10<NUM> CFU/ml and the fruit must is fermented for a period of time of at least <NUM> hours before the fermentation with the at least one yeast strain; or
b2) simultaneously fermenting the fruit must with at least one yeast strain to obtain the beverage with a reduced content of alcohol compared to the beverage fermented with the yeast strain alone.