FIELD OF THE INVENTION

The present invention relates to the use of lactic acid bacteria as bioprotective agents against unwanted microorganisms, such as mold, and spoilage bacteria, which may be present during the preparation of wine. Especially the present invention relates to inhibition of molds and/or gram-negative bacteria, such as acetic acid bacteria, with Lactobacillus plantarum in winemaking. It also relates to a specific Lactobacillus plantarum strain, a method for preparing a wine and a wine obtainable by the method.

BACKGROUND OF THE INVENTION

Wine is a fermented beverage produced from fruits, mainly grapes. The fermentation of the sugars into alcohol is performed by yeast, mainly Saccharomyces cerevisia. During this fermentation process the yeast also produces other metabolites, resulting in a change in the chemical composition, when going from must to wine. Lactic acid bacteria (LAB) can also play a role in the wine production and thereby also affect the chemical composition, mainly through the malolactic fermentation (malic acid to lactic acid).

Wine is a complex microbial matrix, where it is crucial to have the "right" kind of yeast and bacteria present.

The first couple of steps in the winemaking process are vulnerable when it comes to microbial spoilage, due to high sugar concentrations and no/low ethanol concentration, making it a favorable media for various microorganisms (lactic acid bacteria, acetic acid bacteria, yeast and molds). Microbial contamination of the wine during production can have negative consequences for the winemaker, as they may have to discard the wine due to a downgrade in quality. Therefore, sulphur dioxide (S0 ₂ ) has been used in winemaking for centuries and it is considered a fundamental additive in modern winemaking (Ribereau-Gayon et al. 2006). In winemaking S0 ₂ is used both as an antimicrobial and antioxidant compound (Ribereau-Gayon et al . 2006). S0 ₂ inhibits growth of mold, yeast, lactic acid bacteria and to lesser extent acetic acid bacteria in must and wine (Santos et al. 2012).

Spoilage mold, yeast and bacteria can produce unwanted chemical compounds with a negative sensory character as well as allergens and toxins. Acetic acid bacteria are mainly unwanted in wine production, due to their production of excessive amounts of acetic acid, which results in a vinegar smell and contributes to the overall volatile acid (VA) in a wine. Acetic acid bacteria can live of both sugars and ethanol, meaning that they can be found both in the must and in the wine (Bartowsky & Henschke 2008). Furthermore, acetic acid bacteria are tolerant to low pH and also fairly tolerant to S0 ₂ (Bartowsky & Henschke 2008).

Molds are mainly a problem on the grapes where a mold infection, of e.g. Aspergillus or PeniciHium, can lead to a reduced yield as well as toxins in the wine, such as Ochratoxin A, which is a carcinogen (Nielsen et al. 2009; Mogensen 2010; Vega et al . 2012). Toxigenic and spoilage fungi are responsible for numerous diseases (Oliveria et al 2004). Furthermore, a mold infection can result in further microbial infection, due to grapes bursting, making them more susceptible for further microbial contamination. S0 ₂ is toxic as well as an allergenic compound, creating a health risk for consumers as well as the people working in the wineries (Valley et al. 2009; Comuzzo & Zironi 2013). Sulphite-sensitive individuals can experience a range of symptoms including dermatitis, urticarial, flushing, hypotension, abdominal pain, diarrhea, anaphylactic and asthmatic reactions (Valley et al . 2009; Santos et al. 2012). Due to these health issues associated with S0 ₂ , there are regulations regarding S0 ₂ addition in wine. In EU wines with more than 10 mg/L S0 ₂ need to be labeled and the maximum S0 ₂ concentration of conventional wine is 150 mg/L for dry red wines and 200 mg/L for dry white wine, and higher for sweet wines (Regulation (EC) 607/2009; Regulation (EC) 606/2009; Regulation (EC) 203/2012). Due to the recent labeling criteria for S0 ₂ in wine, the consumers have become more aware of the S0 ₂ issues in wine and the negative health effect S0 ₂ may have. This has led to a S0 ₂ reducing trend in winemaking as well as an increased customer demand for low and no S0 ₂ wines (Comuzzo & Zironi 2013). Therefore, replacement of S0 ₂ is currently the most critical winemaking issue in Europe and a challenge for winemakers worldwide (Santos et al. 2012).

Various alternative antimicrobial methods have been tested in wine, in the hope of finding a replacement for S0 ₂ . Physical methods, such as pulsed electric fields (PEF), ultrasound, ultraviolet radiation and high pressure (Santos et al. 2012) have been successful to some extent. However, they require costly equipment which is not normally found in a winery.

Other chemical and biotechnological approaches have also been tested, but none of them are able to inhibit both molds and acetic acid bacteria efficiently on the grapes and the must at the same time. Commercial fungicides such as Rally® fungicide (Dow Agrosciences) and Tanos® (DuPont) are very effective against molds on the grapes but are not used on the must as these chemical compounds are unwanted in the final wine. Furthermore, they do not inhibit acetic acid bacteria.

One of the chemical approaches tested involves the addition of dimethyl dicarbonate (DMDC), which is effective against yeast. It can therefore only be used after the alcoholic fermentation (Santos et al. 2012). Furthermore, DMDC does not inhibit bacteria and can therefore not be used as the only antimicrobial agent.

Ozone has also been tested as an antimicrobial agent in wine and it can inhibit growth of both bacteria and yeast (Guzzon et al. 2013). Ozone however, can also only be used after the alcoholic fermentation due to its yeast inhibition.

As a biotechnological approach the enzyme lysozyme can also be used for protection against unwanted bacterial growth, and is currently used in the wine industry (Santos et al. 2012). However, Lysozyme is only active against gram-positive bacteria and thereby lysozyme does not inhibit growth of acetic acid bacteria, as they are gram negative.

Plant material and extracts have also been suggested as S0 ₂ replacement compounds. Salaha et al. (2008) tested a combination of Raphanus niger and acetic acid as a S0 ₂ replacement, but this changed the wines chemical composition and higher levels of VA were also found in the wines.

Antimicrobial plant phenolics have been tested as a replacement for S0 ₂ , but only during storage (Garcia-Ruiz et al. 2013; Gonzales-Rompi et al . 2013). The tested plant phenolics inhibited all bacteria and can therefore not be used in the must, if malolactic fermentation is wanted later in the process, which is common for most red wines. Also, the plant phenolic came from almonds and eucalyptus and had a measurable and sensory detectable impact on the wine aroma (Gonzales-Rompi et al. 2013).

Bacteriocines, such as nisin, Pediocin PA-1 and Lacticine 3147, have been shown to have an antimicrobial effect against wine related bacteria (Rojo-Bezares et al. 2007; Knoll et al. 2008; Diez et al 2012; Garcia-Ruiz et al. 2013B). However, the bacteriocines are mainly active against gram-positive bacteria(Rojo-Bezares et al. 2007; Diez et al 2012; Sonate et al 2012). The research covering the antimicrobial activity of bacteriocin in wine has in general not been successful in using wine-isolated bacteria, producing bacteriocins, as an antimicrobial.

Spoilage microorganisms, such as especially molds and acetic acid bacteria, are detrimental in the process of winemaking, affecting both the quantity as well as the quality of the resulting wine. Despite extensive efforts being employed to control the presence of these unwanted microorganisms, the addition of sulphur dioxide continues to be the most prominent method applied. There is an unmet need for an alternative to this toxic compound which does not possess the disadvantages described above.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an alternative to the antimicrobial agents and methods currently used for reducing both molds on fruits and in the must as well as acetic acid bacteria in the must and in the wine during winemaking.

Surprisingly, the present inventors have identified a Lactobacillus plantarum strain isolated from wine capable of inhibiting both molds as well as acetic acid bacteria on the fruit, in the fermentation medium and in the wine.

The use of the Lactobacillus plantarum strain was shown to inhibit mold growth on whole grapes and in grape juice and significantly reduce the amount of acetic acid bacteria in fermenting wine.

Consequently, the present invention can be used to replace, in part or fully, the use of known physical, chemical, or biotechnological methods, such as the addition of S0 ₂ , during winemaking.

Thus, in the present invention a facultative heterofermentative Lactobacillus plantarum strain isolated from wine is used to protect the fruit, the fruit must and the wine against molds and acetic acid bacteria.

The approach did not shown any negative effect on the yeast performance during alcoholic fermentation.

DETAILED DISCLOSURE OF THE INVENTION

The inventors of the present invention surprisingly found that whole grapes, grape must and wine, when inoculated with a lactic acid bacterium derived from wine, a Lactobacillus plantarum strain isolated from grape juice from the Western Cape region in South Africa, both the growth of molds and unwanted acetic acid bacteria (gram-negative bacteria) were reduced with comparison to whole grapes, grape must and wine, which had not been inoculated with the Lactobacillus plantarum strain.

Thus, one aspect of the present invention relates to use of a Lactobacillus plantarum strain for inhibiting growth of mold and/or gram-negative bacteria during winemaking. In a preferred embodiment the gram-negative bacteria are acetic acid bacteria. As used herein, the term "acetic acid bacterium" designates a gram-negative, aerobic bacterium of the family Acetobacteraceae, which oxidizes ethanol to acetic acid during fermentation in neutral or acidic media (Krieg et al. 1984) . Genera of acetic acid bacteria include Acetobacter and Gluconobacter which can be found in wine (Bartowsky and Henschke 2008).

In another aspect the present invention relates to use of a Lactobacillus plantarum strain for inhibiting gram-negative bacteria.

In a much preferred embodiment the gram-negative bacteria are acetic acid bacteria. In another preferred embodiment the inhibition of gram-negative bacteria is in fruit must or in wine, preferably in grape must or wine, preferably in wine.

As used herein, the term "must" designates the unfermented or fermenting juice expressed from grapes or other fruits, such as apples and pears.

The term "wine", as used herein, refers to a must, in which the alcohol quantity produced by alcoholic fermentation is at least 4% (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.

Another aspect relates to use of a Lactobacillus plantarum strain for inhibiting growth of mold and/or gram-negative bacteria on fruit or in fruit must.

In a preferred embodiment the inhibition of growth of mold and/or gram-negative bacteria is on grapes or in grape must. Preferably, the gram-negative bacteria are acetic acid bacteria.

An even further aspect relates to use of a Lactobacillus plantarum strain for reducing growth of mold on fruit or in fruit must.

In a preferred embodiment the reduction of growth of mold is on grapes or in grape must.

Growth of mold and/or gram-negative bacteria on fruit can be reduced by dipping or submerging the fruit completely in an aqueous solution comprising at least one viable Lactobacillus plantarum strain or spraying the aqueous solution onto the fruit.

It was found herein that at a relatively low concentration of viable Lactobacillus plantarum cells growth of mold on fruit, such as grapes, could be inhibited. Thus, one further aspect relates to an aqueous solution comprising at least one

Lactobacillus plantarum strain in an amount of at the most lxlO ⁸ CFU/ml, such as at the most 5xl0 ⁷ CFU/ml, such as at the most lxlO ⁷ CFU/ml, such as at the most 5xl0 ⁵ CFU/ml.

Preferably, the aqueous solution has a pH of at least 3.2, such as at least 3.3, such as at least 3.4, such as at least 3.5.

In preferred embodiments of the four above-mentioned aspects the Lactobacillus plantarum strain is selected from the group consisting of the Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM 27565 and a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material, and wherein the mutant has retained or further improved the anti-fungal effect and/or the anti-bacterial effect that characterize DSM 27565. In a further preferred embodiment the fruit must is inoculated and fermented with at least two or more Lactobacillus plantarum strains, such as 2, 3, 4, 5 or more Lactobacillus plantarum strains.

In the present context, the term "mutant strain" should be understood as a strain derived from a strain of the invention by means of e.g. genetic engineering, radiation and/or chemical treatment, and/or selection, adaptation, screening, etc. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. antifungal effect and/or antibacterial effect) as the mother strain. Such a mutant is a part of the present invention. Especially, the term "mutant strain" refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N- nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred mutant, less than 1%, or less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the mother strain.

The present invention in a further aspect is directed to a Lactobacillus plantarum strain selected from the group consisting of the Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM 27565 and a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material, and wherein the mutant has retained or further improved the anti-fungal effect and/or the antibacterial effect that characterize DSM 27565.

In yet a further aspect the present invention relates to a method for preparing a wine comprising the steps of:

a) inoculating fruit, used for preparing a must, or the fruit must with a Lactobacillus plantarum strain; and

b) fermenting the fruit must with a yeast strain to obtain the wine.

Step b) is carried out simultaneously or after step a). In a preferred embodiment sulphur dioxide is added in a concentration of at the most 20 mg/L, such as at the most 15 mg/L, such as at the most 10 mg/L, such as at the most 5 mg/L.

In another preferred embodiment no sulphur dioxide is added. In a preferred embodiment the pH of the fruit must is at least 3.2, such as at least 3.3, such as at least 3.5, such as at least 3.6, such as at least 3.7, such as at least 3.8. Preferably, the Lactobacillus plantarum strain is selected from the group consisting of the Lactobacillus plantarum strain CHCC12399 that was deposited with the German

Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM 27565 and a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material, and wherein the mutant has retained or further improved the anti-fungal effect and/or the antibacterial effect that characterize DSM 27565.

In a preferred embodiment the Lactobacillus plantarum strain is inoculated in an amount of at least lxlO ⁴ CFU/ml, such as at least 5xl0 ⁴ CFU/ml, such as lxlO ⁵ CFU/ml, such as 5xl0 ⁵ CFU/ml, such as at least lxlO ⁵ CFU/ml, such as at least 5xl0 ⁵ CFU/ml, such as lxlO ⁷ CFU/ml, such as at least 5xl0 ⁷ CFU/ml, such as lxlO ⁸ CFU/ml, such as 5xl0 ⁸ CFU/ml. 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 2, 3, 4, 5 or more yeast strains.

The fruit must may also be fermented by the means of the spontaneous growth of an indigenous flora of yeast.

In another further aspect the present invention relates to a wine obtainable by the method according to the previous aspect.

An even further aspect relates to a kit comprising a Lactobacillus plantarum strain and a yeast strain. In a preferred embodiment, the Lactobacillus plantarum strain is selected from the group consisting of the Lactobacillus plantarum strain CHCC12399 that was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession No. DSM 27565 and a mutant strain thereof, wherein the mutant strain is obtained by using the deposited strain as starting material, and wherein the mutant has retained or further improved the anti-fungal effect and/or the antibacterial effect that characterize DSM 27565. In a preferred embodiment the kit further comprises instructions on how to use the kit to prepare a wine with reduced growth of molds and/or gram-negative bacteria during winemaking.

The term "reduced growth of molds and/or gram-negative bacteria" in a wine relates to a lesser extent of growth of molds on the fruit and the must and a lower amount of acetic acid bacteria in the must and in the wine as compared to fruit, must and wine prepared under identical conditions but without the addition of the Lactobacillus plantarum strain.

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. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Any combination of the above-described elements, aspects and embodiments in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1 depicts Merlot grape juice at pH 3.5 and pH 3.8 inoculated with Lactobacillus plantarum strain CHCC12399 and left for 16 days at 20°C.

FIGURE 2 shows whole grapes treated (left) and not treated (right) with Lactobacillus plantarum strain CHCC12399 after 3 days at ambient temperature.

FIGURE 3 depicts YGC plates plated with samples from the control tank (left) and the test tank (right) diluted 1 : 10000 in duplicates on day 4. FIGURE 4 illustrates the mold inhibition measurement technic.

FIGURE 5 shows acetic acid bacteria counts (CFU/mL) in the four tanks at winery (1) on day 7. FIGURE 6 shows the ethanol concentration over time in the four tanks (A, B, C and D) at winery (1) during alcoholic fermentation.

FIGURE 7 shows acetic acid bacteria counts (CFU/mL) in two tanks at winery (2). The test tank was sampled on day 0 (before inoculation of Lactobacillus plantarum strain CHCC12399), day 3 and day 4, whereas the control tank was only sampled on day 1.

FIGURE 8 depicts total Lactobacillus counts (CFU/ml) in the three trial tanks (T9 with Lactobacillus plantarum strain CHCC12399, Ti l Control and T12 Control) on the first four days of wine fermentation.

FIGURE 9 depicts the decrease in acetic acid bacteria from day 0 to day 4 in the three trial tanks (T9 with Lactobacillus plantarum strain CHCC12399, Til Control and T12 Control) shown as log(CFU/ml).

FIGURE 10 shows the total acetobacteraceae (acetic acid bacteria) 16S rRNA reads normalized with the grape material DNA reads of the three trial tanks (A9 - Lb refers to T9 with Lactobacillus plantarum strain CHCC12399, All - conl is Til Control and A12 - con2 refers to T12 Control).

EXAMPLES

EXAMPLE 1 : Mold inhibition in grape juice by Lactobacillus plantarum strain CHCC12399

The pH of two batches of Merlot grape juice was adjusted to pH 3.5 and pH 3.8, respectively, with 2M NaOH. The parameters of the juice can be seen in Table 1.

T le 1 : Parameters of Merlot grape juice.

Two plastic bottles (250mL) were filled with pH 3.5 Merlot juice and two plastic bottles (250mL) were filled with pH 3.8 Merlot.

All four bottles were inoculated with Lactobacillus plantarum strain CHCC12399 (approx. 1x10 ^s CFU/mL) and incubated at 20°C for a total of 16 days.

The viability of Lactobacillus plantarum viability was analyzed, by anaerobic plating on grape juice agar (GJ5; 77.5 g/L grape juice concentrate (K V Saft Vallo), 22.4 g/L yeast extract (Bio Springer), 0.6 g/L Tween® 80 (Sigma-Aldrich), 0.1 g/L MnS0 ₄ , H ₂ 0 (Merck), 15 g/L agar (SO-BI-GEL) in tap water) with Natamycin (0,075g/L; Delvocide from DSM Food Specialities B.V.), on the day of inoculation (day 0) and on day 3. The plates were incubated for 3 days at 30°C and then counted; only plates with between 25-250 colonies were found valid.

Due to the harsh juice conditions and the low pH the Lactobacillus plantarum bacteria died off in the pH 3.5 grape juice, whereas growth of the Lactobacillus plantarum bacteria was observed in the pH 3.8 grape juice (Table 2). After 16 days, mold was visible on the grape juice with pH 3.5 whereas an inhibition of mold growth was detected on the grape juice of pH 3.8, were Lactobacillus plantarum strain CHCC12399 was active (See Figure 1). Table 2: Viability of Lactobacillus plantarum strain CHCC12399 (CFU/mL) in the four bottles illustrated in Figure 1, on day 0 (after inoculation) and day 3.

This result was surprising, as molds generally grow better at higher pH values.

Therefore, one would have expected the opposite (mold growth in the high pH). This clearly indicates that active Lactobacillus plantarum strain CHCC12399 inhibits mold growth on grape juice. The juice was not added any S0 ₂ .

EXAMPLE 2: Mold inhibition on whole grapes by Lactobacillus plantarum strain CHCC12399

Organic grapes were used for the experiment.

One grape bunch was dipped in a Lactobacillus plantarum strain CHCC12399 solution (lxlO ⁷ CFU/mL of water) and another was not treated in any way.

Both bunches were left on the laboratory bench at ambient temperature for 3 days.

The mold inhibition activity of Lactobacillus plantarum strain CHCC12399 was also observed on the whole grapes (Figure 2). After 3 days, the untreated grape bunch is showing mold growth, whereas nothing is seen on the grape bunch that was inoculated with Lactobacillus plantarum strain CHCC12399 (Figure 2). This is clearly confirming a mold inhibition effect by Lactobacillus plantarum strain CHCC12399.

EXAMPLE 3 : Inhibition of molds by Lactobacillus plantarum strain CHCC12399 in full scale wine tanks

The trials were performed on Merlot in the South of France in 2012.

Two tanks of 200 hL of Merlot grape must were used, one was inoculated with

Lactobacillus plantarum strain CHCC12399 (1x10 ^s CFU/mL) on day 0 and the control tank was not inoculated.

On day 1 both tanks were inoculated with a Saccharomyces sp. strain. Lactobacillus plantarum inoculation was successful (Table 3). However, the CFU of the Lactobacillus plantarum had decreased on day 4, due to the alcohol concentration, which was 6% on day 4 in the test tank.

Table 3 : Viability of Lactobacillus plantarum strain CHCC12399 (CFU/mL) in the test and control tank in the 2012 Merlot, South of France. The results are averages of two plates.

On day 4 standard YGC (yeast extract, glucose, chloramphenicol) agar plates it was surprisingly seen that the control tank had mold growth and the test tank did not have any mold (Figure 3).

The plates in Figure 3 clearly show a mold inhibition activity by Lactobacillus plantarum (the plates to the right).

EXAMPLE 4: Inhibition of Aspergillus steynii, Aspergillus niger, Penicillium verrucosum and Penicillum brevicompactum on malt extract agar by Lactobacillus plantarum strain CHCC12399

Lactobacillus plantarum strain CHCC12399 was tested in its ability to inhibit growth of the four common vinerelated molds Aspergillus steynii, Aspergillus niger, Penicillium verrucosum and Penicillium brevicompactum.

Plates of standard malt extract agar (MEA) were prepared and surface plated with the Lactobacillus plantarum strain CHCC12399. Some plates were not inoculated with bacteria, these where the control plates.

The plates were then spot-inoculated with one of the above mentioned molds and the plates were inoculated at 30°C for 7days.

The area of the molds were measured and compared with the area of the mold on the control plate - as shown in Figure 4. The reduction in the area is calculated into percentage - % growth reduction.

The laboratory analysis showed that Lactobacillus plantarum strain CHCC12399 has a high inhibitory effect of 66% inhibition against Aspergillus steynii, 28% inhibition against Aspergillus niger, 48% against Penicillium verrucsum and 47% against Penicillium brevicompactum .

EXAMPLE 5: Inhibition of acetic acid bacteria by Lactobacillus plantarum strain

CHCC12399

Field trials were carried out at two different wineries in Bordeaux:

Winery (1) :

Merlot must was divided into 4 steel tanks of 3 hL. The tanks were placed in a temperature controlled room of 25°C.

The tanks were name A, B, C and D.

C and D were inoculated with Lactobacillus plantarum strain CHCC12399 (lxlO ⁷ CFU/mL) on day 0.

A and B were not inoculated with anything on day 0.

On day 1 all four tanks were inoculated with a Saccharomyces cerevisiae yeast.

The viability of Lactobacillus plantarum strain CHCC12399 was analyzed by anaerobic pure plating on GJ5 agar, with Natamycin (0.075 g/L). The plates were incubated at

25°C and counted after 2 days.

The viability of acetic acid bacteria was analyzed by aerobic plating on GJ5 agar, with

Natamycin (0.075g/L) and Penicillin (12.5 mg/L; from Sigma). The plates were incubated at 25°C and counted after 7 days.

Winery (2) :

Two tanks (80 hL) of Merlot were used for the trial.

One tank was inoculated with Lactobacillus plantarum strain CHCC12399 on day 0. The other (control) was not inoculated with anything on day 0.

The temperature of the tank at inoculation was approx. 10°C. The temperature of the tank was set to 7°C. For the cold soak maceration, prior to the alcoholic fermentation. On day two the test tank was inoculated with a Torulaspora delbrueckii and the Control tank was inoculated with a Saccharomyces cerevisiae. The temperature was kept at 7°C in both tanks

The test tank was inoculated with Saccharomyces cerevisiae on day 4.

The temperature of both tanks was increased to 20°C on day 5to initiate the alcoholic fermentation.

The viability of Lactobacillus plantarum strain CHCC12399 was analyzed by anaerobic pure plating on GJ5 agar, with Natamycin (0.075g/L). The plates were incubated at 25°C and counted after 2 days. The viability of acetic acid bacteria was analysed by aerobic plating on GJ5 agar, with Natamycin (0.075g/L) and Penicillin (12.5 mg/L). The plates were incubated at 25°C and counted after 7 days. Surprisingly, inhibition of acetic acid bacteria by Lactobacillus plantarum strain CHCC12399 was observed at both wineries in Bordeaux.

At winery (1) acetic acid bacteria were detected on day 7, in the two tanks without Lactobacillus plantarum CHCC12399 (the control tanks A and B) (Figure 5). Whereas, no acetic acid bacteria were detected on day 7, in the two tanks inoculated with Lactobacillus plantarum strain CHCC12399 (Figure 5).

Unfortunately the initial acetic acid bacteria CFU is not known. However, it would be expected to be higher in the initial must than at approx. 9% v/v ethanol, which was the ethanol level on day 7 (Figure 6). As the acetic acid bacteria decrease during alcoholic fermentation and then may increase again after the alcoholic fermentation (Du Toit & Lambrechts 2002).

The acetic acid bacteria inhibition seen in Figure 6, was obtained despite the fact that Lactobacillus plantarum strain CHCC12399 had died off at day 7, in the two inoculated tanks (Table 4).

Table 4: Viability of Lactobacillus plantarum strain CHCC12399 (CFU/mL) in the inoculated tanks at winery (1).

*Days after the grape must was inoculated with Lactobacillus plantarum strain CHCC12399.

At winery (2) a decrease in acetic acid bacteria was seen during the cold soak maceration, in the tank with Lactobacillus plantarum strain CHCC12399 (Figure 7). This was an unexpected result as acetic acid bacteria would be expected to increase during a cold soak maceration step (Drysdale and Fleet 1988; Du toit & Lambrechts 2002). In the control tank, more than double the amount of acetic acid bacteria where found on day 1, when compared to the test tank with Lactobacillus plantarum on day 0 (Figure 7).

The growth inhibition of acetic acid bacteria was obtained even with a relatively low survival rate of Lactobacillus plantarum strain CHCC12399 in the test tank (Table 5). Table 5: Viability of Lactobacillus plantarum CHCC12399 (CFU/mL) in the inoculated test tank at winery (2).

*Days after the grape must was inoculated with Lactobacillus plantarum strain CHCC12399.

EXAMPLE 6: Inhibition of acetic acid bacteria by Lactobacillus plantarum strain CHCC12399 in Spanish white wine

The results are derived from Spanish field trials in La Mancha. The volumes of the tanks were 500hL grape must and the grape variety was Airen, the pH 3.6 and the total S0 ₂ 30 ppm. Three tanks were used for the trial; one tank was inoculated with approximately lxlO ⁷ CFU/mL Lactobacillus plantarum strain CHCC12399 (day 0) and two control tanks were not inoculated with any bacteria.

After 24 hours (on day 1) the three tanks were all inoculated with Saccharomyces cerevisiae yeast (approximately lxlO ⁵ CFU/mL).

Tank 9: Lactobacillus plantarum strain CHCC12399

Tank 11 : Control

Tank 12: Control

Apart from the Lactobacillus plantarum inoculation in one tank (tank 9) the three tanks were treated similarly.

To verify the viability and the survival of the inoculated Lactobacillus plantarum strain CHCC12399 in the fermenting wine, total Lactobacillus colony forming units (CFU) per mL was determined on day 0, 2 and 4. To determine the viability of acetic acid bacteria in the fermenting wine, these were also followed by CFU per mL counts on the same days (0, 2 and 4).

The method used for counting total Lactobacillus CFU/mL was anaerobic pour plating on grape juice agar (GJ5 agar) with 0.09 g/L Natamycin (Delvocide; which is 50% Natamycin). The incubation temperature was 30°C and the plates were incubated for 2 days before colonies were counted. The method used for counting acetic acid bacteria CFU/mL was aerobic surface plating on grape juice agar (GJ5 agar) 0.09 g/L Natamycin (Delvocide; which is 50% Natamycin) and 12.5 mg/L Penicillium. The incubation temperature was 25°C and the plates were incubated for 7 days before colonies were counted.

5

Results

The CFU results for total Lactobacillus clearly show that the inoculation of Lactobacillus plantarum strain CHCC12399 was successful and that the Lactobacillus plantarum strain CHCC12399 had a fine survival rate during the first four days in the fermenting wine in 10 the inoculated tank (see Figure 8).

Also it can be seen in Figure 8 that the total Lactobacillus CFU is much lower in the two control tanks. From 16S rRNA sequencing of the wine samples (as described in Bergey's Manual of Systematic Bacteriology (2001), Second Edition, Volume 1, eds. Boone and

15 Castenholz) and mapping of the sequences against the SILVA high quality ribosomal RNA databases (LTPsll9, Released November 2014; http://www.arb-silva.de/projects/living- tree/; Pruesse et ai. (2007), Nucleic Acids Res. 35(21) :7188-7196), it was found that approx. 25% of the lactic acid bacteria in the control tanks were Lactobacillus plantarum spp. giving approximately 3xl0 ³ CFU/mL (log(CFU/ml_ = 3.47) of Lactobacillus plantarum

20 initially in the control tanks.

Due to the anaerobic nature of a wine fermentation tank, especially a 500hL tank as used in this trial, it would be expected to see a decrease in acetic acid bacteria, which are aerobic bacteria (Madigan et al. 2002). This decrease is seen in the two control tanks 25 as a decrease of 1.3-1.4 log(CFU/ml_) (Figure 9). However, the tank inoculated with Lactobacillus plantarum strain CHCC12399 decreased considerably more in acetic acid bacteria CFU over the same 4 days. In the Lactobacillus plantarum strain CHCC12399 tank the acetic acid bacteria decreased 2.8 log(CFU/ml_) from day 0 to day 4 (Figure 9).

30 To verify the decrease in acetic acid bacteria, 16S rRNA sequencing was performed on the Airen wine samples (as described in Bergey's Manual of Systematic Bacteriology (2001), Second Edition, Volume 1, eds. Boone and Castenholz) seen in the CFU results plus additional samples from the same tanks. The 16S rRNA sequence data was mapped against the SILVA high quality ribosomal RNA databases (LTPsl l9, Released November

35 2014). The 16S rRNA sequence data are shown in Figure 10 as the amount of DNA in reads, since the amount of reads is affected by the total DNA composition in the samples. All the DNA sequence data is normalised to the amount of Vitis vinifera DNA reads (the grape DNA), since the grape DNA is assumed to be the same throughout the whole fermentation period. The normalization enables the comparison of data across the tanks and bacterial species. The normalized Actobacteraceae (total acetic acid bacteria; Acetobacter spp. and Gluconobacter spp.) can be seen in Figure 10. The results in Figure 10 confirm a drastic decrease in acetic acid bacteria from day 0 to day 1, which is expected due to the environment in the tank (low oxygen and S0 ₂ ). The graph in Figure 10 also confirms on DNA level, that the decrease in acetic acid bacteria is considerably larger in the Lactobacillus plantarum strain CHCC12399 inoculated tank.

It can also be seen in Figure 10 that the amount of total acetic acid bacteria does not increase during the fermentation.

Conclusion

The inoculation of Lactobacillus plantarum strain CHCC12399 decreased the amount of acetic acid bacteria in the wine tank during the first 4 days of the vinification more than can be expected in un-inoculated tanks.

DEPOSIT and EXPERT SOLUTION

The strain of Lactobacillus plantarum CHCC12399 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on 1 August 2013 under the accession number DSM 27565.

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.

The Applicant requests that a sample of the deposited microorganisms should be made available only to an expert approved by the Applicant.

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