EP1150581A1

EP1150581A1 - Fermented beverage and method for its production

Info

Publication number: EP1150581A1
Application number: EP00901051A
Authority: EP
Inventors: Arne Rytz
Original assignee: Individual
Current assignee: Rytz Arne
Priority date: 1999-01-22
Filing date: 2000-01-21
Publication date: 2001-11-07
Also published as: WO2000045653A1; AU2093000A

Abstract

A method for producing a tasty and sparkling fermented beverage which can be produced on a large scale. The beverage is of the kind in which microbial metabolites, such as e.g. flavour constituents and alcohol, are produced by a combination of microorganisms in form of yeast and bacteria in an aqueous culture medium having a content of plant material, and where the combination of microorganisms comprises at least one yeast and at least one Leuconostoc. Hereby, a tasty beverage is obtained having a sugar content and an alcohol content that can be varied depending on the fermentation conditions and the choice of combination and the ratio of the microorganisms. The sugar content does not exceed the sugar content in fizzy lemonade, and the beverage is produced without preservatives.

Description

1 Fermented beverage and method for its production

The invention relates to a fermented beverage of the kind in which microbial metabolites, such as e.g. flavour constituents and alcohol, are produced by a combination of microorganisms in form of yeast and bacteria in an aqueous culture medium having a content of plant material.

Fermented beverages are known from e.g. cider and beer. Cider has traditionally been produced by letting apples be left alone in water. The microorganisms that are naturally found on the apples will become decisively important for the fermentation, and the flavour and appearance of the finished cider of each fermentation will therefore be different. For industrial production of cider, a Saccharomyces strain is therefore used to ensure that the finished product always obtains a well- defined uniform quality.

A fermented dietetic drink is known from German utility model DE 297 13 010 that is produced by means of a combination of microorganisms. According to the German utility model, the dietetic drink has probiotic qualities as the microorganisms that are used for producing the dietetic drink are neither removed from nor inactivated in the finished drink.

That the content of the dietetic drink of used microorganisms are still active in the drink result in the fact that the drink has an inconveniently short life due to the continuous microbial growth. The possibility of the microorganisms of continuously utilise nutrients and thereby produce different metabolites will furthermore give the drink poor organoleptic qualities .

A fermented beverage is known from US 4,579,739 that is fermented with a combination of at least one yeast and at least one Lactobacillus, in which the yeast is selected from the group of Saccharomyces cerevisiae and Kluyeveromyces lactis and Lactobacillus is selected from the group of Lactobacillus casei and Lactobacillus hilgardii .

The fermented beverage is inoculated with 10-500 times more bacteria than yeast and, after the fermentation has been stopped by cooling, it is centrifuged to separate the finished drink. This method is possible with this combination of microorganisms but it cannot be applied in the cases where dextrin-producing bacteria are used.

The inventor who previously has filed Danish patent application no. PA 1999 00081 which is included as reference in the present application has succeeded in isolating yeast and bacteria from an old kefir culture which has changed over time due to changing growing conditions.

Through several generations, the kefir culture has been used in the daily household for making a tasty and sparkling beverage having a pleasant taste of fruit. However, the drink differed from production to production.

A first object of the invention is to provide a tasty and sparkling fermented beverage which is suited for large scale production, which has a carbohydrate content not exceeding 10%, and which is not added preservatives.

A second object of the invention is to provide a method for producing such a fermented beverage.

According to the invention this is achieved by the fact that the beverage is fermented with a combination of microorganisms that includes one yeast and at least one Leuconostoc .

In an advantageous embodiment, bacteria and yeast can be standard cultures approved for use in food. By using standard cultures, an advantage is furthermore obtained in that the homogeneity of the cultures can easily be maintained. To produce a beverage that organoleptically corresponds to the product made from the old kefir culture, bacteria and yeast can advantageously be isolated from the above kefir culture, after which these microorganisms can be cultivated, identified, multiplied and possibly preserved by using conventionally known techniques .

The yeast Saccharomyces cerevisiae and the bacterium Leuconostoc mesenteroid.es subsp. mesenteroides/dextranicum are isolated from the original kefir culture, and by combining these two microorganisms and when lemon is used as plant material, a beverage can be produced that by a tasting panel was judged as having a fine lemon and elder flower flavour. The tasting panel also appraised that the drink gave a sparkling and effervesce gustatory sensation resembling the prickly sensation of champagne bubbles on the tongue.

These isolated microorganisms from the kefir culture are of the same genus as the above-mentioned stock cultures, and they are likely to be approved for producing food.

When the beverage is fermented with stock cultures, the gustatory sensation you get is similar to the above, where the fruit flavour of the beverage in both cases can vary depending on the type of plant material.

By combining types and amounts of bacteria and yeast in the starter culture where the bacteria and the yeast e.g. can be in different prechosen stages of growth, beverages can advantageously be produced having very different taste profiles .

The yeast can advantageously be a stock culture of Saccharomyces cerevisiae or the Saccharomyces cerevisiae that the inventor has isolated himself. Saccharomyces cerevisiae utilise under aerobic conditions carbohydrates to CO. and water. In case of absence of oxygen or if the carbohydrate concentration is high, the fermentation will take place forming ethanol and C0₂. Within the scope of the invention, it is possible to use other types of yeast.

The fermentation conditions can therefore advantageously be arranged to provide the desired end product so that if a drink having a high ethanol content is wanted, the fermentation can e.g. take place in batches in oxygen deficit.

The at least one Leuconostoc that is used in combination with yeast can preferably be selected from among the group of Leuconostoc mesenteroides, Leuconostoc lactis, Leuconostoc paramesenteroides and Leuconostoc oenos .

In a preferred embodiment, the stock culture Leuconostoc mesenteroides or the inventor's own isolate Leuconostoc mesenteroides subsp . mesenteroides/dextranicum is selected.

Especially the last-mentioned isolate of Leuconostoc mesenteroides subsp . mesenteroides/dextranicum has surprisingly turned out, in combination with Saccharomyces cerevisiae, to be able to produce a fermented beverage having a so far unknown or undescribed aroma and flavour that can be described as elder flower. Together with flavour constituents originating from the at least one plant material, this drink in total appears as a refreshing, delicate drink.

When the stock culture Leuconostoc mesenteroides or alternatively the isolate Leuconostoc mesenteroides subsp . mesenteroides/dextranicum is used in combination with yeast in a culture medium containing a carbohydrate, an especially advantageous combination of dextrin-producing microorganisms and dextrin-utilising microorganisms is obtained. Dextrin production is normally inappropriate in fermentation of beverage on a large scale as the subsequent separation of the drink itself is hindered considerably when the dextrin content is high.

Leuconostoc mesenteroides are known for producing dextrin in considerable amounts, and this bacterium is therefore not an obvious choice as fermentation organism in a culture medium containing plant material which can have a varying content of types and amounts of carbohydrates.

The dextrin production is highest when the carbohydrate source is sucrose, and even a low content of sucrose in the plant material can result in dextrin production.

The inventor's own tests show that when the concentration of yeast at the beginning of the fermentation is higher than the concentration of bacteria, the presence of dextrin is considerably reduced. In an especially advantageous embodiment, the concentration of yeast is at least twice as high as the concentration of bacteria, preferentially at least five times as high as the concentration of bacteria, and especially at least ten times as high.

When the carbohydrate source also is chosen as either glucose or fructose, or a combination of glucose and fructose, the fermentation advantageously takes place with a very small production of dextrin, without the gustatory sensation being altered.

It is common knowledge and among others described in "The Yeasts, vol. 5, Edited by A.H. Rose and J.S. Harrison, Academic Press, 1993, pages 7-56" that yeast generally needs metal ions such as zinc, manganese, magnesium, calcium, copper, potassium and iron to assist growth, and ordinary tap water usually contains adequate amounts of metal ions . Tests have shown that it is important to the yeast's growth that the zinc concentration is higher than or equal to 0.1 mg/1 and lower than or equal to 0.6 mg/1. If iron ions and manganese ions also are present, the concentration of these two metal ions and the zinc concentration each have to be higher than 0.6 mg/1.

A zinc concentration lower than 0.1 mg/1 would cause a slow fermentation while a zinc concentration higher than 0.6 mg/1 would slow down the growth unless manganese and iron ions also were present in corresponding concentrations.

When the zinc concentration is kept in the low part of the mentioned interval, the ethanol formation will be small, and the acetaldehyde concentration will be high and contribute to giving the beverage an aroma resembling apples .

The water quality is therefore crucially important for the fermentation result, and the water content of metal ions must be known, and if necessary the zinc concentration must be adjusted.

The combination of microorganisms can furthermore comprise at least one bacterium selected from the I/acfcoJacillus-group that includes Lactobacillus brevis, Lactobacillus pastorianus, Lactobacillus hilgardii , Lactobacillus casei , Lactobacillus kefir and Lactobacillus plantarum, or the actococcus-group that includes Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium and Lactococcus plantarum, or a Bifidobacterium.

By choosing also to add a heterofermentative Lactobacillus, C0₂ and ethanol can be produced in desired amounts at the same time as lactate is produced in an amount that contributes to the characteristic aroma and fragrance of the finished beverage that a tasting panel judged as being lightly fermented in the nature of buttermilk.

Any of the mentioned Lactobacillae can be used but in an especially preferred embodiment, Lactobacillus brevis or Lactobacillus casei is used as both these microorganisms when combined with different Leuconostoc in tests have shown to be especially fitted for giving the finished beverage the above flavour and fragrance in the nature of buttermilk.

In one embodiment of the present invention, said combination can include a Lactococcus, selected from among the group consisting of Lactococcus lactis , Lactococcus garvieae, Lactococcus piscium and Lactococcus plantarum . In an especially advantageous embodiment, Lactococcus lactis is selected as this microorganism in tests also has shown to be fit for giving the finished beverage a taste somewhat like buttermilk.

When the fermentation takes place using the above-mentioned combination of microorganisms and using the above-mentioned ratio between yeast and bacteria concentration for a period of time chosen between 6 and 48 hours at a temperature chosen between 20 and 40°C, preferentially between 25 and 35°C, and especially between 25 and 32°C, a fermented beverage can be produced that has a percentage of alcohol which is higher than

0.1 %vol . and lower than 10 %vol . , and preferentially lower than 5 %vol.

Such an alcohol content is furthermore preservative and contributes therefore in an especially advantageous way to prolonging the life of the beverage.

By adjusting the fermentation duration and temperature, the alcohol percentage can be varied, and the flavour constituents in form of esters, alcohol, ketones and aldehydes be developed to a great or small extent, for in this way being able to, using the same base material, produce a wide range of beverages having different flavour compositions.

The beverage can be produced either as a ready for sale product or as a base drink which can be added further flavour constituents .

These different beverages can be marketed alone or they can be mixed in desired proportions to produce beverages having particular flavour compositions directed especially towards specific user groups.

Both the yeast strain and many of the bacteria of the Lactobacillus strain will produce ethanol as primary metabolite, and the finished beverage can therefore be made to contain alcohol in different concentrations which among others can be controlled by adjusting factors such as applied volume of bacteria, applied volume of yeast, applied volume of carbohydrate, applied volume and type of plant material, fermentation temperature, and fermentation duration and oxygen concentration .

The inventor's own tests have shown that at fermentation with a combination of Lactobacillus brevis, Leuconostoc mesenteroides , and Saccharomyces cerevisiae in an aqueous medium containing sucrose and at least one plant material, a tasty beverage can be produced that a testing panel has found both pleasant and aromatic .

The fermentation can advantageously be made using sucrose as carbohydrate source and lemon and orange as the plant material which is to provide the rest of the nutrients, especially assimilable nitrogen which is a condition for the microbial activity which is to give the beverage its final form and character, e.g. in form of colour and flavour. A low concentration of carbon in relation to nitrogen will mean that esters are produced that have a fruity aroma.

When the carbohydrate source is sucrose, the yeast content of the enzyme invertase will quickly hydrolyse sucrose into equal parts glucose and fructose which quickly will enter as culture substrate in the further fermentation process. Glucose is converted more quickly than fructose which is sweeter than glucose.

The use of different types and combinations of carbohydrates for fermentation can advantageously give the beverage different degrees and types of sweetness.

The carbohydrate source can for that matter be any easily utilised carbohydrate and is in this way not limited to sucrose. Of alternative preferred carbohydrate, e.g. the monosaccharides glucose and fructose, and the disaccharide maltose or arabinose can be mentioned. Invert sugar and confectioners' glucose can furthermore be used.

The finished beverage can preferably have a carbohydrate content of no more than 10%, and especially no more than 5%. This content can e.g. be compared with the average carbohydrate content of 9,8% of fizzy lemonade. The beverage is an attractive and new alternative to fizzy lemonade.

The invention also relates to a method for producing a fermented beverage .

The method comprises a preparatory treatment of the raw plant material from which large components possibly have been removed first by e.g. sifting.

The purpose of the preparatory treatment is to inactivate microorganisms that would contaminate the fermentation and that exist in or on the plant material, or merely to reduce the concentration of microorganisms on the plant material by e.g. using one or more of the following processes: rinsing, blanching, heat treatment, such as pasteurisation, or irradiation. Some of the enzyme activity of the plant material might possibly be maintained either when an inactivation is limited to the surface of the plant material, as for example by blanching, or by inactivation processes in which the temperature is not so high that the protein is denatured. Another purpose of the preparatory treatment is to extract specific parts of the plant material, such as pulp, oils, rind parts etc., before the plant material is to be used in the fermentation .

After this, the pretreated plant material is transferred to a fermentation reactor together with mineral containing water and no or at least one type of carbohydrate which can be e.g. sucrose, glucose or fructose depending on the demands determined by the selected specific combination of microorganisms. The microorganisms are added to the fermentation reactor in form of a combination of microorganisms at least comprising at least one yeast and at least one Leuconostoc .

The concentration of yeast is chosen to be higher than the concentration of bacteria. The concentration of yeast is preferably twice as high as the concentration of bacteria, preferentially five times higher, and especially ten times higher, and is determined in such a way that the start concentration of both yeast and bacteria preferably is no lower than 10⁵ Cfu/ml and preferably no higher than 10¹⁰ Cfu/ml .

The fermentation can be carried out on a large scale as a batch fermentation or as a fed-batch fermentation in which the carbohydrate source is added continuously. The fermentation temperature is determined in advance and will be around 20 and 40°C, preferentially between 25 and 35°C, and especially between 25 and 32°C. The oxygen supply is set at a level at which the yeast grows faster than the bacteria and/or at a level at which the production of specific desirable aroma constituents is encouraged. The fermentation time is determined in advance to between 6-48 hours depending on the alcohol content, the flavour constituents and the sugar content wanted in the finished drink.

The fermentation is advantageously stopped by in random order treating the fermentation mixture with one or more of the processes filtration, e.g. depth filtration, filtration with filter aid, high-pressure filtration, rotary vacuum filtration or cross-flow filtration, centrifugation, irradiation, high- pressure preservation, pasteurisation, high-temperature pasteurisation or UHT treatment.

Each of the processes can be used alone but in connection with some of the combinations of microorganisms, especially where the especially preferred dextrin-producing microorganisms are used, one process alone cannot always remove biomass and dextrin sufficiently to be able to produce a clear drink that is not siimy .

However, it is possible to choose fermentation conditions which allow for keeping the dextrin production low.

In cases where a specific desirable aroma constituent is to be fermented, it can however be necessary to apply fermentation conditions that mean that dextrin is also fermented which subsequently is removed through one or more processes.

In this case, it is necessary to e.g. centrifuge first. To preserve the drink, it can subsequently be heat-treated. As the heat treatment will produce sediment in form of protein depositions, a final centrifugation might prove necessary for which reason it in some cases is especially preferred to heat- treat first and then centrifuge.

Filtration can be used alone or in combination with initial centrifugation. Centrifugation can e.g. be carried out as ultracentrifugation or as continuous centrifugation. Out of consideration for the life of the beverage, centrifugation must however be followed by a treatment that also stops the enzyme activity.

The microbial activity is advantageously stopped by e.g. heat inactivation, high-pressure pasteurisation, high-pressure preservation or irradiation.

When high-pressure preservation is chosen for stopping microbial activity, it has, at pressures over 6000 bar, turned out that it is possible also to completely stop a possible enzyme activity from the inactivated microorganisms or from the plant material. If the enzyme activity is not stopped, the taste of the beverage will gradually change character, and the conservability will be shortened considerably.

By stopping and/or removing the microbial activity, it is ensured that there is no further growth in the finished beverage. ^l The product can moreover be kept at specific, predetermined carbohydrate and alcohol levels.

The beverage is an alternative to known beverages and can be produced on the basis of e.g. ecological ingredients. The drink can be bottled in packaging in form of e.g. tightly sealed bottles or cartons which are quickly sealed to preserve the sparkling character of the drink. It is furthermore possible to enrich the drink with e.g. a vitamin or mineral supplement whereby the drink can be aimed at particular target groups, such as sportsmen.

By using different types of plant material, the drink can be given different flavour compositions and colours. The plant material can be from any plant at all, e.g. apple, pear, lemon, orange, raisin, fig, grape or carrot, but other fruits or vegetables can be used.

The plant material can advantageously initially be processed by e.g. blending, comminution or pressing.

By using the mentioned types of plant material, it is ensured that the pH in the fermentation liquid does not reach such a high value that the problems known in the art relating to bad taste in connection with a high fermentation pH will not arise at any time.

pH in the finished drink will typically be in the area of under 4.5. At higher pH values the fermentation will easily be exposed to microbial contamination, the result of which is change in flavour, aroma and life of the product.

Examples :

Example 1 :

Isolation of strains from kefir culture

From the original kefir culture, a number of dilution steps were carried out for 10^"5, 10^"6, 10^"7 and 10^"8. From each dilution step, 2 x 1 ml were dispersed on four different culture substrates MRS (Merck 1.10660), YM (DIFCO 0711-17-1 + lOg agar- agar (Merck 1.01614) per 500ml), Malt (Merck 1.05391 + lOg agar-agar (Merck 1.01614 per 500ml) and PCA (Merck 1.05463), and the plates were incubated both anaerobically and aerobically at 25°C for 48 hours. After 48 hours, the plates were examined for absence of contaminants and by means of microscopy, a suitable number of colonies were examined for purity and species. A total number of eighteen colonies were selected that represented both yeast, cocci and rods and that appeared to be pure and having different morphology on the agar surface. These colonies were spread on the same type of culture substrate that was used for isolation, and were incubated once more at 25°C until good growth. Of the eighteen colonies, several were estimated as being alike, and fifteen colonies were spread once more on the same culture substrate, incubated for 48 hours, icroscoped and examined for purity. These fifteen colonies were then cultivated in bouillon.

A culture medium made of 1 litre boiled, cooled tap water, 50g sucrose (not analytic grade) which is not dissolved but merely spread, and 32g lemon slices were inoculated with 1% yeast and 1% bacterium from bouillon and incubated at 25°C for 48 hours, after which the fermented beverage was sensorial judged by a tasting panel with a view to select the combination of microorganisms that gave the same gustatory sensation as the original kefir culture. The testing panel judged that this was the case when the fermentation had been carried out using microorganisms from a medium-sized colony of yeast which was isolated and cultivated on PC, in combination with a small colony of bacteria which was isolated and cultivated on PC. The tasting panel appraised the taste as sweet lemonade which further was experienced as being slightly sour and sparkling and with a mild fragrance of buttermilk and lemon.

The two isolated cultures were spread again on PCA plates and cultivated in a so-called PC-medium made of 5 g/1 casein peptone, 2.5 g/1 yeast extract, 1.0 g/1 sucrose. After incubation at 25°C for 48 hours, the strains were identified as the yeast Saccharomyces cerevisiae and the bacterium Leuconostoc mesenteroides subsp. mesenteroides/dextranicum.

These types of microorganisms are both known cultures which are often used in food and stimulants like bread, beer, wine, acidulated dairy products and cheese.

Using these two isolated strains, it was possible to reproduce the taste, but the beverage had a high content of dextrin.

Example 2 :

Tests with varying ratios between yeast and bacterium concentrations

Under the same fermentation conditions as in example 1 and using the isolated Saccharomyces cerevisiae and Leuconostoc mesenteroides subsp . mesenteroides/dextranicum, fermentations were carried out in which the ratio between the yeast and bacterium volume concentrations were 1:1, 3:1, and 9:1 respectively. Hereby, beverages were produced having the same taste as the beverage in example 1. It was furthermore demonstrated that the higher the concentration of yeast was in proportion to the concentration of bacteria, the lower was the dextrin production.

Example 3 :

Tests with various carbohydrate sources

Culture substrates were made from the following formula: 5 g/1 casein peptone, 2.5 g/1 yeast extract and 40 g/1 carbohydrate. Three different kinds of carbohydrate were used: sucrose, fructose and glucose respectively, which resulted in three different culture substrates. Each type of substrate was sterilised partly by autoclave treatment for 15 min. at 121°C, partly by sterilisation by filtration. The thus made six culture substrates were all inoculated with 1% Leuconostoc mesenteroides subsp . mesenteroides/dextranicum and were allowed to incubate at 25°C and 32°C for 48 hours, after which the viscosity was estimated visually and by centrifugation. The degree of slime development together with the viscosity was taken as a demonstration of the dextrin production. It appears from the results in Table 1 below that the slime development and thus the dextrin production is reduced considerably when the fermentation temperature rises.

Table 1

Example 4 :

Tests with different sucrose concentrations

Basic culture substrates were made of 5 g/1 casein peptone, 2.5 g/1 yeast extract and 1 g/1, 10 g/1, 30 g/1, and 50 g/1 respectively sucrose. The substrates were inoculated with 1% of the isolated Saccharomyces cerevisiae for fermentation with agitation, 1% of the isolated Saccharomyces cerevisiae for fermentation without agitation, and 1% of the isolated Leuconostoc mesenteroides subsp . mesenteroides/dextranicum respectively, which in total gave twelve fermentation reactors that were incubated at 25°C. After 0, 8, 24 and 32 hours, OD and pH were determined. At pH lower than 5.5, adjustment was made with ammonia to pH 5.5. If pH was higher than 5.5, no adjustment was made. The results are shown in table 2 below:

Table 2

Both of the isolated strains, Saccharomyces cerevisiae and Leuconostoc mesenteroides subsp. mesenteroides/dextranicum show good growth at sucrose concentrations right up to 50 g/1. For production at an industrial scale, it is desirable to reduce slime development, and the experiences from the test in example 3 can be used as the dextrin production in this example proved possible to reduce or completely avoidable when the fermentation temperature was increased to 32°C. Example 5 :

Tests using a combination of standard cultures

A culture medium was made by adding % slice of blanched orange and V. slice of blanched lemon to an autoclaved mixture of 30g sucrose in half a litre of water. To the culture medium was added 1 ml of the standard cultures Leuconostoc mesenteroides and Lactobacillus brevis so that the start bacterial count in the culture medium was 2-10* Cfu/ml. 1 ml Saccharomyces cerevisiae was also added so that the start bacterial count was 1-10⁵ Cfu/ml. The mixture was incubated at 25°C for 48 hours without air supply. To remove the microorganisms in the drink, the mixture was finally high-pressure filtered by means of conventionally known techniques.

Hereby, a slightly sour, clear beverage was obtained which a testing panel judged as extremely tasty and having a pleasant fragrance .

Metabolites were measured by means of Boehringer Mannheim test kits and are shown in Table 3 below:

Table 3

Example 6 :

Tests using a combination of standard cultures

A culture medium was made by adding ^x slice of blanched orange and 2 slice of blanched lemon to an autoclaved mixture of 30g sucrose in half a litre of water. To the culture medium was added 1 ml of the standard cultures Leuconostoc mesenteroides and Lactobacillus brevis and Lactococcus lactis so that the start bacterial count in the culture medium was 2-10^* Cfu/ml. 1 ml Saccharomyces cerevisiae was also added so that the start bacterial count was 1-10⁵ Cfu/ml. The mixture was incubated at 25°C for 48 hours with air supply. To remove the microorganisms in the drink, the mixture was finally high-pressure filtered by means of conventionally known techniques.

Hereby, a clear beverage was obtained which a testing panel judged as extremely tasty and having a pleasant fragrance.

Metabolites were measured by means of Boehringer Mannheim test kits and are shown in Table 4 below:

Table 4

Example 7 :

Tests with addition of probiotic microorganisms

The isolated Saccharomyces cerevisiae is cultivated by inoculation with 1% from the previous cultivation in an autoclaved culture substrate made of 5 g/1 casein peptone, 2.5 g/1 yeast extract and 30 g/1 sucrose. Leuconostoc mesenteroides subsp . mesenteroides/dextranicum is cultivated by inoculation with 1% from the previous cultivation in an autoclaved culture substrate made of 5 g/1 casein peptone, 2.5 g/1 yeast extract and 40 g/1 sucrose. Leuconostoc mesenteroides subsp . mesenteroides/dextranicum is incubated at 32°C and Saccharomyces cerevisiae at 25°C for 24-30 hours. In addition, the probiotic standard cultures Lactobacillus casei and Bifidobacterium are cultivated in MRS (Merck 1.10660) at 32°C.

Inoculation is made with 1% of each of the isolates Saccharomyces cerevisiae and Leuconostoc mesenteroides subsp . mesenteroides/dextranicum in 5 1 of the lemon culture substrate used in example 1, and it is incubated at 25°C.

40ml of this cultivation is centrifuged and concentrated 10 times by decanting 9ml per 10ml after centrifugation. The sediment is microscoped and used for fermentation of 2 1 beverage by inoculation with 0.1% of the concentrated sediment in the lemon culture substrate from example 1 and incubate at 25°C.

The above process is repeated using an additional 1% of the cultivation of Lactobacillus casei and 1% of the cultivation of Bifi doba cterium.

In a sensorial way, it is tested if the product made from cultivation and the product made from centrifuged culture are 22 identical , and if addition of probiotic strains have any inf luence at all on the product .

The sensorial judgment of the products appears from table 5 below :

Table 5

Product Judgment

Yeast + Leuconostoc mesenteroides Good fragrance of lemon and mild culture . subsp. mesenteroides/dextranicum Sparkling and fresh with a good taste of

( standard product) lemon and culture . pH 3 . 65

The product is like the standard product .

Centrifuged yeast In the two batches produced, there was a

+ Leuconostoc mesenteroides subsp . difference between the smell of the mesenteroi des/dextrani cum culture of the two products . pH 3 . 75

The product has a fainter smell of lemon

Yeast + Leuconostoc mesenteroides than the standard product. The new culture subsp. mesenteroides/dextranicum + has a suggestion of acetic acid taste but Bifidobacterium is otherwise like the standard product. pH 3.30

The product has a strong taste of lemon

Yeast + Leuconostoc mesenteroides and is bitter. There is also a good subsp. mesenteroides/dextranicum + sweetness in the product but it has Lactobacillus casei changed character by the addition of Lactobacillus casei

Claims

A fermented beverage of the kind in which microbial metabolites, such as e.g. flavour constituents and alcohol, are produced by a combination of microorganisms in form of yeast and bacteria in an aqueous culture medium having a content of plant material, characterised in that the combination of microorganisms comprises at least one yeast and at least one Leuconostoc .

2 . A fermented beverage according to claim 1, characterised in that the at least one Leuconostoc is selected from among the group Leuconostoc mesenteroides , Leuconostoc mesenteroides subsp. mesenteroides/dextranicum, Leucono- stoc lactis, Leuconostoc parames enter o ides and Leuconostoc oenos .

3. A fermented beverage according to claim 1, characterised in that the at least one yeast is a Saccharomyces cerevisiae.

4. A fermented beverage according to claim 1, characterised in that at the start of the fermentation, the concentration of yeast is higher than the concentration of bacteria, preferably twice as high, preferentially five times higher, and especially ten times higher.

5. A fermented beverage according to claim 1, characterised in that the aqueous culture medium has - a concentration of zinc ions that is higher than or equal to 0.1 mg/1 and lower than or equal to 0.6 mg/1, and a concentration of zinc ions, iron ions and manganese ions that each are higher than 0.6 mg/1.

6. A fermented beverage according to claim 1, characterised in that the combination of microorganisms furthermore comprises at least one bacterium selected from at least one of the groups consisting of: - Lactobacillus brevis, Lactobacillus pastorianus, Lactobacillus hilgardii , Lactobacillus casei,

Lactobacillus kefir and Lactobacillus plantarum, Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium and Lactococcus plantarum, or - Bifidobacterium

. A fermented beverage according to claim 1-6, characterised in that the finished beverage has a percentage of alcohol which is higher than 0.1 %vol . and lower than 10 %vol .

8. A fermented beverage according to claim 1-7, characterised in that the finished beverage has a carbohydrate content of less than 10%, and especially under 5%.

9. A method for producing a fermented beverage according to claim 1-7, characterised in that the method comprises the steps of: pretreating the raw plant material by either rinsing, blanching, pasteurisation or filtration to reduce the concentration of microorganisms on the plant material, transferring the pretreated plant material to a fermentation reactor together with mineral containing water and no or at least one type of carbohydrate, - adding to the fermentation reactor a combination of microorganisms which at least comprises at least one yeast and at least one Leuconostoc so that the concentration of yeast is higher than the concentration of bacteria, and so that the start concentration of both yeast and bacteria is no lower than 10⁵ Cfu/ml and preferably no higher than 10¹⁰ Cfu/ml, adjusting the fermentation temperature to a determined temperature between 20 and 40°C, preferentially between 25 and 35°C, and especially between 25 and 32°C, adjusting the oxygen supply to a level at which the yeast grows faster than the bacteria or to a level at which specific desirable aromatic compounds are created, fermenting for a determined period of time of between 6-48 hours, and stopping the fermentation by in random order treating the fermentation mixture with one or more of the processes centrifugation, irradiation, high-pressure preservation, pasteurisation, high-temperature pasteurisation or UHT treatment.

10. A method according to claim 9, characterised in that the aqueous medium is adjusted so that the concentration of zinc ions in the aqueous medium is higher than or equal to 0.1 mg/1 and lower than or equal to 0.6 mg/1, or to adjust the concentration of zinc ions, iron ions and manganese ions so that the concentrations mainly are equally high and higher than or equal to 0.6 mg/1