EA042154B1 - LACTOBACILLUS RHAMNOSUS WITH INCREASED DIACETYL PRODUCTION - Google Patents

Description

Technical field

The present invention relates to a composition suitable for the preparation of a dairy product containing a strain of Lactobacillus rhamnosus capable of imparting an enhanced creaminess to a dairy product through significant production of diacetyl without adversely affecting the rheology of the dairy product. The composition according to the invention can be used together with a starter culture or in its composition to obtain a dairy product. In addition, the present invention relates to processes for producing a dairy product such as low fat yogurt, cheese and sour cream, which has a high content of diacetyl. A strain of Lactobacillus rhamnosus useful for making such dairy products is also part of the present invention.

Prior Art

WO2012/136830 discloses an antifungal strain of Lactobacillus rhamnosus for the production of a dairy product.

In the dairy industry, low-fat or no-fat products are experiencing a growing demand from consumers. However, such reduced fat dairy products often exhibit a lack of creaminess.

Diacetyl is a valuable product and is used in the dairy industry as a butter flavoring compound added to products such as margarines and vegetable oil based butter products.

The heterolactic acid bacteria form diacetyl/acetoin as a by-product along with lactate as the main product. Cells form active acetaldehyde from pyruvate and thiamine pyrophosphate via pyruvate oxidase. Active acetaldehyde condenses with another molecule of pyruvate and forms alpha-acetolactate synthase. Diacetyl formation in Lactobacillus rhamnosus is not well understood - it has been suggested that Lactococcus lactis subsp. lactis biovar. diacetylactis alpha-acetolactate is oxidized to diacetyl by alpha-acetolactate oxidase (Jyoti et al 2003). Acetoin is formed directly by decarboxylation of alpha-acetolactate. The formation of acetoin can also occur irreversibly from diacetyl to acetoin via diacetyl reductase.

Lactobacillus rhamnosus is a heterolactic acid bacterium that can be used to produce flavor compounds like diacetyl and acetoin (Jyoti et al. 2003). The level of diacetyl produced depends on the strain as well as the substrate on which it is grown.

US4867992 and US5236833 relate to processes for producing diacetyl by fermenting a coffee substrate and a pectin substrate, respectively, with lactic acid producing bacteria.

The preparation, concentration and addition of diacetyl and/or acetoin to foodstuffs is costly.

US 4,678,673 is directed to oilseed products fermented with a strain of Lactobacillus rhamnosu that produces diacetyl and acetoin. Fermented oilseed products have a butter or milk-like taste. There is no mention of the use of Lactobacillus rhamnosus in dairy products.

WO2012/136832 discloses a diacetyl-producing strain of Lactobacillus rhamnosus for the production of a dairy product.

There is a need for lactic acid bacteria with increased production of diacetyl for the manufacture of dairy products.

Brief summary of the invention

The object of the present invention is to provide a composition and method for producing a dairy product enhanced by the enhanced creaminess imparted by the presence of a strain of Lactobacillus rhamnosus.

Yet another object of the present invention is to provide a new strain of Lactobacillus rhamnosus with improved properties in terms of the ability to impart enhanced creaminess to a dairy product such as yogurt or cheese.

Additional objects will become apparent later in this document, and all other objects will be obvious to a person skilled in the art to which the invention belongs.

As can be seen in the current examples in this document, the disclosed strain of Lactobacillus rhamnosus CHCC15871, which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM32666, produces diacetyl and acetoin, thereby imparting enhanced creaminess to a dairy product without significant negative effects on rheology and subsequent acylation of the dairy product.

Also, surprisingly, it was found that the strain of Lactobacillus rhamnosus CHCC15871 according to the invention, in addition to its high activity of producing diacetyl, has a high antifungal activity. In particular, the strain of Lactobacillus rhamnosus CHCC15871 according to the invention has an increased activity against the growth of yeast compared to conventional antifungal strains of Lactobacillus rhamnosus.

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Thus, the first aspect of the present invention relates to a composition containing a strain of Lactobacillus rhamnosus CHCC15871, which was deposited in the German collection of microorganisms and cell cultures (DSMZ) with accession number DSM32666, or a mutant strain, where the mutant strain is obtained by using the deposited strain in as the mother strain, and wherein the mutant strain has the same or higher diacetyl production as compared to the mother strain.

The second aspect of the present invention relates to the use of the composition according to the first aspect of the present invention for the production of a dairy product.

The third aspect of the present invention is directed to a method for preparing a dairy product, where the method includes the steps:

a) inoculating the milk substrate with a composition according to the first aspect of the present invention;

b) fermentation of the milk substrate;

c) possibly adding additional microorganisms and/or additives to the milk substrate;

d) possibly post-treatment of the milk substrate; And

e) possibly the packaging of a dairy product.

The fourth aspect of the invention relates to a dairy product obtained by the method according to the third aspect of the invention.

The fifth aspect of the present invention relates to a strain of Lactobacillus rhamnosus CHCC15871, which has been deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM32666, or a mutant strain thereof, where the mutant strain is obtained by using the deposited strain as a mother strain, and where the mutant the strain has the same or higher diacetyl production as compared to the parent strain.

Brief description of graphic materials

In FIG. 1 shows diacetyl levels after storage at 7±1° C. for 14 days in fermented dairy products fermented with starter culture alone (control) or starter cultures in combination with Lb. rhamnosus CHCC12483 or together with Lb. rhamnosus CHCC15871. LOD: Limit of detection. LOQ: Limit of quantification.

In FIG. 2 shows acetoin levels after storage at 7±1° C. for 14 days in fermented dairy products fermented with starter culture alone (control) or starter cultures in combination with Lb. rhamnosus CHCC12483 or together with Lb. rhamnosus CHCC15871. LOD: Limit of detection. LOQ: Limit of quantification.

In FIG. 3 shows yeast growth on plates of milk fermented with only one starter culture (control, first plate), together with Lb. rhamnosus CHCC12483 (second plate) or together with Lb. rhamnosus CHCC15871 (third plate) for 4 different yeast contaminants A, B, C and D and for three yeast concentrations (top, middle and bottom row).

In FIG. 4 shows yeast growth on plates of milk fermented with only one starter culture (control, first plate), together with Lb. rhamnosus CHCC12483 (second plate), along with a reference strain Lb. rhamnosus (third cup) or together with Lb. rhamnosus CHCC15871 (fourth plate) for one yeast contaminant and for three yeast concentrations (top, middle, and bottom row).

In FIG. 5 shows mold growth on plates of milk fermented with only one starter culture (control, first plate) or together with Lb. rhamnosus CHCC12483 (second plate) or together with Lb. rhamnosus CHCC15871 (third plate) for 3 different mold contaminants A, B and C and for 2 different temperatures (top and bottom row of plates).

In FIG. 6 shows the acidification profile of fermented dairy products during fermentation at 43°C. The products are fermented with a single starter culture, control, or starter culture in combination with Lb strains. rhamnosus CHCC12483 or Lb. rhamnosus CHCC15871.

In FIG. 7 shows the rise in pH in fermented dairy products as a function of time when stored at 7±1° C. for 21 days. The products are fermented with a single starter culture, control, or starter culture in combination with Lb strains. rhamnosus CHCC12483 or Lb. rhamnosus CHCC15871.

In FIG. 8 shows mold growth on plates of milk fermented with only one starter culture (control, first plate) or together with Lb. rhamnosus CHCC15871 (second plate) for 3 different mold contaminants A, B and C.

In FIG. 9 shows the fermentation time (in minutes) to a final pH of 4.50 at 30°C. The products are fermented with only one of five different sour cream starter cultures (SC1-SC5) and in combination with Lb strains. rhamnosus CHCC15871 or Lb. rhamnosus CHCC12697.

In FIG. 10 shows diacetyl levels ( ^ppm , mean plus/minus SD) after storage at 5±1°C for 7 and 21 days in fermented sour cream products fermented with

- 2 042154 using only one of five different starter cultures (SC1-SC5) and in combination with Lb strains. rhamnosus CHCC15871 or Lb. rhamnosus CHCC12697.

In FIG. 11 shows acetoin levels ( ^ppm , mean plus/minus SD) after storage at 5±1°C for 7 and 21 days in fermented sour cream products fermented with only one of five different starter cultures (SC1-SC5 ) and in combination with Lb strains. rhamnosus CHCC15871 or Lb. rhamnosus CHCC12697.

In FIG. 12 shows the measurement of rheological characteristics of fermented sour cream products after storage at 5±1°C for 7 days. Shear stress (Pa, mean plus/minus standard deviation) at a shear rate of 300 (1/s) derived from rheological curve data. Samples fermented with only one of five different starter cultures (SC1-SC5) and in combination with Lb. rhamnosus CHCC15871 or Lb. rhamnosus CHCC12697.

In FIG. 13 shows diacetyl levels ( ^ppm , mean plus/minus SD) after storage at 7±1° C. for 14 days in yoghurt products fermented with only one starter culture and in combination with 8 different Lb strains. rhamnosus (CHCC15871, CHCC12483, strain 1, strain 2, strain 3, strain 4, strain 5 and strain 6).

Detailed description of the invention

Definitions.

As used herein, the term lactic acid bacterium refers to a Gram-positive, microaerophilic, or anaerobic bacterium that ferments sugars to produce acids, including lactic acid as the predominantly produced acid, acetic acid, and propionic acid. The most commercially useful lactic acid bacteria are found in the order Lactobacillales, which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. In addition, lactic acid-producing bacteria belonging to the group of strict anaerobic bacteria, bifidobacteria, namely Bifidobacterium spp., are usually included in the group of lactic acid bacteria. They are often used as food cultures, alone or in combination with other lactic acid bacteria.

Lactic acid bacteria, including Lactobacillus spp. and Streptococcus thermophilus are commonly supplied to the dairy industry as either frozen or freeze-dried cultures for propagating food sourdough or as so-called direct inoculation cultures (DVS) intended for direct inoculation into a fermentation tank or bath to prepare a dairy product such as fermented dairy product or cheese. Such lactic acid bacteria cultures are commonly referred to as starter cultures or starter cultures.

The term mesophile in this document refers to microorganisms that successfully reproduce best at moderate temperatures (15-40°C). The most commercially useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term mesophilic fermentation in this document refers to fermentation at a temperature of from about 12 to about 35°C. The term mesophilic dairy product refers to dairy products obtained by mesophilic fermentation of a mesophilic starter culture and includes dairy products such as buttermilk, curdled milk, sour milk, sour cream, sour heavy cream, and soft cheese such as quark, cottage cheese, and cream cheese.

The term thermophile in this document refers to microorganisms that successfully reproduce best at temperatures above 43°C. The most commercially useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term thermophilic fermentation in this document refers to fermentation at a temperature above about 35°C. The term thermophilic dairy product refers to dairy products obtained by the thermophilic fermentation of a thermophilic starter culture and includes dairy products such as yogurt.

The term milk should be understood as the excretion of milk obtained by milking any mammal such as cows, sheep, goats, buffalo or camels. In a preferred embodiment, the milk is cow's milk. The term milk also includes protein/oil solutions made from vegetable matter, such as soy milk.

The term milk substrate can be any raw and/or processed milk raw material that can be fermented according to the process of the invention. Thus, useful dairy substrates include, but are not limited to, solutions/suspensions of any dairy or dairy-like products containing protein, such as whole or reduced fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, lactose crystallization mother liquor, whey protein concentrate or cream. Obviously, the milk substrate can come from any mammal, for example being mostly pure mammalian milk or reconstituted milk powder.

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Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

Homogenization as used in this document means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is carried out prior to fermentation, it can be carried out in such a way as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This can be done by forcing milk at high pressure through small holes.

Pasteurization as used herein means the treatment of a milk substrate to reduce or eliminate the presence of living organisms such as microorganisms. Preferably, pasteurization is achieved by maintaining a predetermined temperature for a predetermined period of time. The set temperature is usually reached by heating. The temperature and duration may be selected to kill or inactivate specific bacteria, such as harmful bacteria. This can be followed by a rapid cooling step.

Fermentation in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention includes the conversion of lactose to lactic acid.

Fermentation processes for the preparation of dairy products are well known and one skilled in the art will know how to select suitable process conditions such as temperature, oxygen, quantity and characteristics of the microorganism(s), and process duration. Obviously, the fermentation conditions are chosen in such a way as to reveal the achievement of the present invention, i. obtain a dairy product in solid (such as cheese) or liquid form (such as a fermented dairy product).

In this context, the term shear stress determines the viscosity. Viscosity (the unit is Pa s) is defined as the ratio of shear stress (Pa) to shear rate (1/s).

The shear stress value is presented as a standard in this document at a shear rate of 300 1/s. Sensory experiments showed (data not shown) that the best correlation between rheological measurements and sensory viscosity/consistency in the mouth was found when the viscosity measured at a shear rate of 300 1/s was used.

In the context of the present invention, the term shear stress means shear stress, which is measured by the following method.

Seven days after incubation, the fermented milk product was brought to 13° C. and gently stirred by hand using a stick equipped with a perforated disc until the sample was homogeneous. The rheological properties of the sample were evaluated on a rheometer (Anton PaarPhysica Rheometer with ASC, Automatic Sample Changer, Anton Paar® GmbH, Austria) using a cup with a float. The rheometer was adjusted to a constant temperature of 13°C during the measurement time. The settings were as follows.

Holding time (until the original structure is restored to some extent) 5 min without any physical load (vibration or rotation) applied to the sample.

Oscillation stage (to measure the modulus of elasticity and viscosity, G' and G, respectively, thus calculating the total modulus G*).

Strain constant=0.3%, frequency (f)=[0.5...8] Hz 6 measurement points within 60 s (every 10 s).

Rotation stage (to measure shear stress at 300 1/s).

Two stages have been developed:

1) shear rate=[0.3-300] 1/s and

2) shear rate=[275-0.3] 1/s.

Each stage included 21 measurement points for 210 s (every 10 s).

The shear stress at 300 1/s was chosen for further analysis as it correlates with mouth texture when swallowing a fermented dairy product.

In this context, the term mutant should be understood as a strain obtained from the strain of the invention by, for example, genetic engineering, irradiation, UV light and/or chemical treatment, and/or methods that induce changes in the genome. Preferably, the mutant is a functionally equivalent mutant, e.g., a mutant that has substantially the same or improved properties as the parent strain (e.g., diacetyl production, viscosity, gel hardness, mouth coating, taste, postacylation, acylation rate and/or phage resistance). Such a mutant is part of the present invention. The term mutant generally refers to a strain obtained by subjecting a strain of the invention to any conventional mutagenization treatment, including treatment with a chemical mutagen such as ethyl methanesulfonate (EMS) or K-methyl-K'-nitro-K-nitroguanidine (NTG) , UV radiation, or to a spontaneously occurring mutant. In particular, the mutant is a natural mutant, i. e. a mutant obtained, for example, by subjecting the mother strain to a mutagenization treatment involving growing the mother strain in a medium devoid of one

- 4 042154 or more commonly used growth factors, also called selection pressure. In this regard, the term natural mutant means a mutant that has not been obtained through the use of genetic engineering, irradiation, UV radiation and/or chemical processing. The mutant may be subjected to several mutagenization treatments (a single treatment should be understood as containing one mutagenization step followed by a screening/selection step), but in this case it is preferred that no more than 20, or no more than 10, or no more than 5 treatments be performed ( or screening/selection steps). In this case, in the preferred mutant, less than 5%, or less than 1%, or even less than 0.1% of the nucleotides in the bacterial genome have been replaced with another nucleotide or deleted compared to the parent strain. In a specific embodiment, the mutant contains no more than 20, in particular no more than 10, in particular no more than 5, in particular no more than 4, in particular no more than 3, in particular no more than 2 and in particular no more than 1 nucleotide mutation of the bacterial genome compared with the parent strain, where the mutation is a substitution, insertion or deletion of one nucleotide.

The use of terms and similar references in the context of the description of the invention (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated in this document or is not clearly refuted by the context. The terms containing, having, including, and including are to be construed as non-limiting terms (ie, meaning including, but not limited to), unless otherwise indicated. The description of ranges of values in this document is intended to serve only as a shorthand referring individually to each individual value falling within the range, unless otherwise indicated in this document, and each individual value is included in the description of the invention as if it were individually described in this document. document. All of the methods described in this document may be performed in any suitable order, unless otherwise indicated in this document or otherwise clear from the context. The use of any and all examples, or introductory words (eg, such as) offered in this document is intended only to better illuminate the invention and is not a limitation on the scope of the invention, unless otherwise stated. No word in the description of the invention should be construed as indicating any unclaimed element as essential to the practice of the invention.

Composition according to the invention.

The inventors of the present invention have surprisingly found that by inoculating and fermenting a dairy substrate with a strain of Lactobacillus rhamnosus, in addition to the starter culture, it is possible to impart a pleasant creamy taste to the resulting dairy product without negatively affecting the consistency of the dairy product, fermentation time and postacylation.

Enhanced creaminess has been found in dairy products prepared by both mesophilic and thermophilic fermentation processes in the presence of Lactobacillus rhamnosus CHCC15871, which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM32666.

By the term enhanced creaminess is meant that the diacetyl and/or acetoin content of the product is increased and/or that the creamy taste of the product, as measured by the sensory scale, is enhanced compared to a product that does not contain the Lactobacillus rhamnosus strain of the present invention.

Without being limited by theory, it can be assumed that the enhanced creaminess imparted to the dairy product by the strain of Lactobacillus rhamnosus of the present invention is due to the increased production of diacetyl and/or acetoin by the strain of Lactobacillus rhamnosus.

The dairy product in preferred embodiments is a reduced/fat-free fermented dairy product or cheese that is substantially lacking in creaminess when the Lactobacillus rhamnosus strain of the present invention was not used in the fermentation.

In a specific embodiment of the invention, the composition contains at least one additional lactic acid bacterium.

In a particular embodiment of the composition of the invention, the additional lactic acid bacterium is at least one antifungal lactic acid bacterium selected from the group consisting of a Lactobacillus rhamnosus strain, a Lactobacillus fermentum strain, a Lactobacillus paracasei strain, a Lactobacillus plantarum strain, a Propionibacterium freudenreichii strain, e.g. Propionibacterium freudenreichii subsp. shermanii, Propionibacterium thoenii strain, Propionibacterium jensenii strain, Pediococcus acidilactici strain, Enterococcus faecium strain, Lactococcus lactis strain and Lactobacillus amylovorus. In this embodiment, the composition is suitable for use in addition, together with a starter culture, to a dairy substrate during the preparation of a dairy product, such as a fermented dairy product, such as yogurt or sour cream, or cheese, at the start of fermentation to increase the diacetyl content and/or to obtain antifungal effect in fermented product or cheese. Alternatively, the composition may be added during the fermentation process, i. after the start and before the end of fermentation.

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In a specific embodiment of the composition of the invention, at least one antifungal lactic acid bacterium is a strain of Lactobacillus rhamnosus CHCC15860, which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number: 32092.

In a specific embodiment of the invention, the composition contains a starter culture for the preparation of a fermented dairy product containing one or more strains selected from the group consisting of the genera Lactococcus, Streptococcus and Lactobacillus.

Typically, such a composition contains bacteria in concentrated form, including frozen, dried or lyophilized concentrates, typically having a viable cell concentration that is in the range of 10 ⁴ to 10 ¹² cfu (colony forming units) per gram of the composition, comprising at least 10 ⁴ cfu per grams of the composition, such as at least 105 cfu/g, such as at least 106 cfu/g, such as at least 10 ⁷ cfu/g, such as at least 10 ⁸ cfu/g, such as at least 109 cfu /g, for example at least 10 ¹⁰ CFU/g, such as at least 10 ¹¹ CFU/g. Thus, the antimicrobial composition of the invention is preferably present in frozen, dry or lyophilized form, for example as a direct application culture (DVS). However, as used herein, the antimicrobial composition may also be a liquid that is obtained after suspending frozen, dry, or lyophilized cell concentrates in a liquid medium such as water or PBS buffer. When the antimicrobial composition of the invention is a suspension, the viable cell concentration is in the range of 10 ⁴ to 10 ¹² cfu (colony forming units) per ml of composition, including at least 10 ⁴ cfu per ml of composition, such as at least 105 cfu/ml , such as at least 106 cfu/ml, such as at least 10 ⁷ cfu/ml, such as at least 10 ⁸ cfu/ml, such as at least 10 ⁷ cfu/ml, such as at least 10 ¹⁰ cfu/ ml, such as at least 10 ¹¹ cfu/ml.

The composition may further contain, as additional components, cryoprotectants and/or conventional additives, including nutrients such as yeast extracts, sugars and vitamins, for example vitamin A, C, D, K or vitamins of the vitamin B family. Suitable cryoprotectants that can be added to the compositions according to the invention are components that improve the cold resistance of microorganisms, such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerin, raffinose, maltodextrin, erythritol, treitol, trehalose, glucose and fructose. Other additives may include carbohydrates, flavorings, minerals, enzymes (eg rennet, lactase and/or phospholipase).

Since it is normal in lactic acid bacteria fermentation processes to use a mixed culture as a starter culture, the composition in some embodiments will contain multiple strains either from the same species or from different species. A typical example of such a useful combination of lactic acid bacteria in a thermophilic starter culture is a mixture of a strain of Lactobacillus bulgaricus and a strain of Streptococcus thermophilus. A typical example of such a useful combination of lactic acid bacteria in a mesophilic starter culture is a mixture of a strain of Lactococcus lactis in combination with a strain selected from the group consisting of a strain of Leuconostoc, a strain of Lactococcus lactis subsp. lactis biovar. diacetylactis and a strain of Streptococcus thermophilus.

In a preferred embodiment of the present invention, the starter culture is a thermophilic starter culture and the composition is suitable for thermophilic fermentation.

In another preferred embodiment, the starter culture is selected from the group consisting of the genera Streptococcus and Lactobacillus. The starter culture in a preferred embodiment contains at least one strain of Lactococcus lactis. The starter culture may contain any strain of Lactococcus lactis known in the art, such as strains from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae or Lactococcus lactis subsp. lactis. In another preferred embodiment, the starter culture contains a strain of Lactococcus lactis subsp. cremoris and a strain of Lactococcus lactis subsp. lactis.

The composition can be used to obtain a dairy product with an enhanced creamy taste.

In a preferred embodiment, the dairy product is a fermented dairy product such as yogurt. In yet another preferred embodiment, the dairy product is cheese.

In a preferred embodiment, the dairy product contains at least 1.0 ^ppm diacetyl, specifically at least 3.0 ^ppm diacetyl, more specifically at least 5.0 ^ppm diacetyl, more specifically at least 7.0 ^ppm diacetyl, more specifically at least 9.0 ^ppm diacetyl, more specifically at least 11.0 ^ppm diacetyl, more specifically at least 13.0 ^ppm diacetyl, more specifically at least 15 .0 ^ppm diacetyl, more specifically at least 17.0 ^ppm diacetyl, and most specifically at least 19.0 ^ppm diacetyl.

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The person skilled in the art is aware of numerous methods for determining the content of diacetyl in dairy products according to the invention. For example, the content can be determined by suitable chromatographic methods such as static headspace gas chromatography (HSGC). Diacetyl recovery can be determined using various assays known in the art, but is preferably determined in an assay comprising:

1) obtaining a fermented dairy product by:

a) inoculating the milk with Lactobacillus rhamnosus at a concentration of at least 5x10 cfu/g and with a starter culture,

b) fermentation until the target pH is reached, eg pH 4.6; And

2) storage of the fermented milk product at 7±1°C for 14 days;

3) adding 200 µl of 4 N H ₂ SO ₄ to 1 gram of fermented dairy product and determining the diacetyl concentration by static headspace gas chromatography.

Antifungal composition according to the invention.

In a specific embodiment, the composition of the invention is an antifungal composition comprising Lactobacillus rhamnosus CHCC15871 of the invention and at least one additional antifungal lactic acid bacterium.

In a preferred embodiment, the compositions of the invention are used against fungi such as yeasts and molds. This means that the compositions are used to suppress and/or prevent the growth of fungi that cause contamination in dairy processes, in particular in milk fermentation processes. The compositions of the present invention can be used, for example, to suppress and/or prevent the growth of yeasts such as yeasts of the genera Klyveromyces (e.g. K. marxianus, K. lactis), Pichia (e.g. P. fermentans), Yarrowia (e.g. Y. lipolytica), Candida (eg C. sake), Debaryomyces (eg D. hansenii) and the like; or moulds, such as molds of the genera Penicillium (e.g. P. nalgiovense, P. commune, P. crustosum, P. brevicompactum, P. glabrum), Mucor spp., Cladiosporium ssp., Aspergillus (e.g. A. versicolor) and similar. It is particularly preferred to use the compositions according to the invention to inhibit and/or prevent the growth of Klyveromyces marxianus, Yarrowia lipolytica, Penicillium nalgiovense, Cladiosporium ssp., Penicillium commune, Mucor ssp., Penicillium brevicompactum, Aspergillus versicolor, Penicillium crustosum, Kluyveromyces lactis and/or Debaryo myceshansenii species .

In a specific embodiment of the invention, the antifungal activity of the strain Lactobacillus rhamnosus CHCC15871 can be defined as the ability to inhibit the growth of the fungus Penicillium solitum deposited with accession number DSM32093 or the growth of the fungus Penicillium brevicompactum deposited with accession number DSM32094 or the growth of Penicillium crutosum.

In the context of the present invention, the ability to inhibit the growth of the fungus Penicillium solitum deposited with accession number DSM32093 or the growth of the fungus Penicillium brevicompactum deposited with accession number DSM32094 is preferably determined using an assay comprising:

1) obtaining a fermented dairy product by:

a) inoculating the milk with Lactobacillus rhamnosus CHCC15871 at a concentration of at least 5x106 cfu/g and with a starter culture,

b) fermentation until a target pH is reached, e.g. pH 4.6, and

c) solidifying the fermented milk by adding agar;

2) obtaining at least one spot of P. solitum or P. brevicompactum on agar-hardened fermented milk at a concentration of 500 spores/spot and incubating it for 7 days at 25°C;

3) determining the percentage of suppression by determining the maximum diameter of a colony formed as a result of growth of P. solitum or P. brevicompactum, and expressing the diameter as a percentage of the maximum diameter formed under the same conditions, but in the absence of a strain of Lactobacillus rhamnosus.

In a specific embodiment of the invention, the antifungal activity of a strain of Lactobacillus rhamnosus CHCC15871 is defined as the ability to inhibit the growth of one or more yeasts selected from the group consisting of Torulaspora delbrueckii, Cryptococcus fragicola, Debaryomyces hansenii, Yarrowia lipolytica, Rhodotorula mucilaginosa, Pichia quilermondii and Candida spp, preferably one or more yeast selected from the group consisting of Torulaspora delbrueckii, Cryptococcus fragicola, Debaryomyces hansenii, Yarrowia lipolytica and Rhodotorula mucilaginosa.

In the context of the present invention, the ability to inhibit the growth of one or more yeasts selected from the group consisting of Torulaspora delbrueckii, Cryptococcus fragicola, Debaryomyces hansenii, Yarrowia lipolytica and Rhodotorula mucilaginosa is preferably determined using an assay comprising:

1) obtaining a fermented dairy product by:

a) inoculating the milk with Lactobacillus rhamnosus CHCC15871 at a concentration of at least 5x106 cfu/g and with a starter culture,

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b) fermentation until a target pH is reached, e.g. pH 4.6, and

c) solidifying the fermented milk by adding agar;

2) obtaining at least one spot from four yeasts Torulaspora delbrueckii, Cryptococcus fragicola, Debaryomyces hansenii and Yarrowia lipolytica on fermented milk hardened from agar at concentrations of 103, ¹⁰² and 101 CFU/spot and incubating them for 27 days at 7°C C;

3) determining the percentage of suppression by determining the number of 12 spots where growth occurs (from 0 to 12 spots) for a plate containing Lactobacillus rhamnosus CHCC15871 with starter culture and expressing the amount as a percentage of the number of spots when growth occurs for a plate containing only the starter culture.

In an alternative embodiment, step (3) is performed as a qualitative determination by visual inspection and comparison of the plates and an assessment of the level of suppression by comparing the plates, taking into account 1) the number of spots with no growth and 2) the level of growth for spots with growth (diameter, density and thickness of the spot ).

Use according to the invention.

As stated above, the invention further relates to the use of the composition of the invention for the production of a dairy product.

In a specific embodiment, the dairy product is a fermented dairy product. In a specific embodiment, the fermented dairy product is yogurt. In yet another specific embodiment, the dairy product is cheese.

In a particular embodiment, the dairy product contains at least 0.75 ^ppm diacetyl, in particular at least 1.5 ^ppm diacetyl.

The present invention further relates to the use of the composition of the invention for the production of diacetyl in a dairy product manufacturing process.

The present invention further relates to the use of the composition of the invention for inhibiting the growth of fungi in a dairy product manufacturing process. The present invention further relates to the use of the composition of the invention for inhibiting the growth of yeast in a dairy product manufacturing process.

Method according to the invention.

As stated above, one aspect of the invention relates to a method for making a dairy product with a creamy taste, including:

a) inoculation of the milk substrate with the composition according to the first aspect of the invention;

b) fermentation of the milk substrate;

c) possibly adding additional microorganisms and/or additives to the milk substrate;

d) possibly post-treatment of the milk substrate; And

e) possibly the packaging of a dairy product.

As described above, the milk substrate for use in step (a) can be any raw and/or processed milk raw material that can be fermented according to the process of the invention.

The milk substrate may be inoculated with the above composition by any suitable method. For example, the milk substrate can be inoculated by direct inoculation into a fermentation tank.

In one preferred embodiment, step (b) comprises fermenting the milk substrate at a temperature above about 37°C, preferably at a temperature of from about 38 to about 45°C, and more preferably from about 39 to about 42°C. In yet another preferred embodiment, step (b) comprises fermenting the milk substrate at a temperature of from about 22 to about 34°C, more preferably from about 24 to about 32°C, and more preferably from about 26 to about 30°C.

Fermentation processes used in the production of dairy products are well known and one skilled in the art will understand how to select appropriate process conditions such as temperature, oxygen, amount and characteristics of the microorganism(s) and duration of the process. Obviously, the fermentation conditions are chosen in such a way as to obtain a fermented dairy product suitable for obtaining a dairy product with improved taste and special texture.

Additional microorganisms and/or additives may be added to the milk substrate before, during or after the fermentation of the milk substrate in step (b). Microorganisms that can be added to dairy substrates will enrich the properties of the dairy product in a favorable manner. For example, the microorganism can improve or maintain diacetyl production, gel viscosity, gel hardness, mouth coating, flavor, post-acylation, and/or acylation rate in a dairy product. Possibly, other ingredients can be added to the milk substrate, such as colorants, stabilizers, eg pectin, starch, modified starch, CMC (carboxymethyl cellulose), and so on; or polyunsaturated fatty acids, such as ome

- 8 042154 ha-3 fatty acids. Such ingredients may be added at any time during the manufacturing process, such as before or after fermentation.

The milk substrate may subsequently be further processed in any manner necessary to produce the desired dairy product. For example, additional components may be added to the dairy substrate, such as conventional supplements, including nutrients such as sugars and vitamins. In addition, the milk substrate may, for example, be homogenized or heat treated, i. e. pasteurized

The dairy product may be packaged in any suitable manner known in the art. For example, the dairy product may be packaged in a sealed container having a volume in the range of, for example, 25 to 1500 ml. The product can be packaged at any time during the production process, for example packaged following the inoculation step and then fermented in the package.

Dairy products obtained by the process include, as typical examples, products such as yogurt, sour cream, cheese and buttermilk.

The dairy product obtained by the above method is also part of the present invention.

In a preferred embodiment, the dairy product is a fermented dairy product. A fermented dairy product as used herein includes, but is not limited to, products such as thermophilic fermented dairy products such as yogurt, mesophilic fermented dairy products such as sour cream and buttermilk, and fermented whey.

The term thermophilic fermented dairy product refers to fermented dairy products obtained by the thermophilic fermentation of a thermophilic starter culture and includes such fermented dairy products as thermostatted yogurt, tank yogurt, and drinking yogurt, such as Yakult.

The term mesophilic fermented dairy product refers to fermented dairy products obtained by the mesophilic fermentation of a mesophilic starter culture and includes fermented dairy products such as buttermilk, curdled milk, thick curdled milk, sour cream, sour heavy cream, and soft cheese such as quark, cottage cheese, and buttermilk. cheese.

In yet another preferred embodiment, the dairy product is a cheese such as cottage cheese, feta, cheddar, parmesan, mozzarella, emmental, danbo, gouda, edam, feta cheese, blue cheese, pickled cheese, camembert or brie.

In a preferred embodiment, the dairy product contains at least 0.75 ^ppm diacetyl, in particular at least 1.0 ^ppm diacetyl, in particular at least 1.5 ^ppm diacetyl.

The strain CHCC15871 according to the invention.

A fifth aspect of the invention relates to a strain of Lactobacillus rhamnosus CHCC15871 which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM 32666. they were obtained by using the deposited strain CHCC15871 as the mother strain. The mutant strain can be obtained from CHCC15871, for example, through genetic engineering, irradiation, UV radiation, chemical treatment and/or methods that induce changes in the genome. Essentially, the mutant according to the invention will have the same characteristics as the mother strain, in terms of production levels of acetate, acetaldehyde, diacetyl and/or acetoin. Preferably, the mutant produces at least 80% or more, at least 90% or more, at least 95% or more, or at least 100% or more of acetate, acetaldehyde, diacetyl and/or acetoin. compared to its parent strain.

In this context, the expression the same or higher production of diacetyl compared to the mother strain means at least 100% production of diacetyl compared to the mother strain.

The skilled artisan will appreciate that by using the deposited strain as the parent strain, the skilled artisan can, through conventional mutagenesis or reisolation techniques according to current practice, obtain additional mutants or derivatives thereof that retain the significant features and benefits described herein. Thus, the term mutant thereof in the first aspect refers to mutant strains obtained by using the deposited strain as the mother strain.

Embodiments of the present invention are described below using non-limiting examples.

Examples

Example 1 Semi-quantitative analysis of diacetyl production and Lb inhibitory effect. rhamnosus CHCC15871 against various yeast and mold contaminants.

For semi-quantitative analysis of the inhibitory effect of Lb. rhamnosus CHCC15871 used an agar assay similar to the manufacturing process and production of yogurt.

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Homogenized reduced fat milk (1.5% w/v) was heat treated at 90±1° C. for 20 minutes and immediately cooled. A commercially available yogurt starter culture (F-DVSYoFlex Mild 2.0) was inoculated at a concentration of 0.02% (v/w) and the inoculated milk was dispensed into 200 ml bottles. One bottle was inoculated with Lb. rhamnosus CHCC15871 at a total concentration of 1x10 ⁷ CFU/g, one bottle was inoculated with Lb. rhamnosus CHCC12483 with a commercially available bioprotective strain (obtained from Holdbac® YM-B) at a total concentration of 1×10 ⁷ CFU/g, one bottle was inoculated with the reference strain Lb. rhamnosus with antifungal activity and one bottle was used as control and inoculated with starter culture only. All bottles were incubated in a water bath at 43±1°C and fermented under these conditions until a pH of 4.60±0.1 was reached. After fermentation, the bottles were shaken vigorously to break the curd and cooled on ice. Then the fermented milk was heated to a temperature of 40°C and added 40 ml of 5% sterile agar solution, which was melted and cooled to 60°C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the dishes were dried on a table in a laminar flow hood for 30 minutes.

A spore suspension of three different mold species Penicillium brevicompactum (DSM32094), P. crustosum, P. solitum (DSM32093) was spotted at a concentration of 500 spores/spot. Three types of mold fungus were spotted on the same cup. The dishes were incubated at 25±1°C and periodically examined for mold growth. Five yeast species including Torulaspora delbrueckii, Cryptococcus fragicola, Debaryomyces hansenii, Yarrowia lipolytica and Rhodotorula mucilaginosa were spotted at concentrations of 103, ¹⁰² and 101 cfu/spot. Cups were incubated at 7±1°C and periodically examined for the presence of yeast growth.

On day 14, samples were analyzed for the presence of diacetyl by static headspace gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a static headspace feeder connected to a gas chromatograph with a flame ionization detector (FID). For this purpose, the following equipment was used.

HS autosampler: HS40XI, TurboMatrix 1110, Perkin Elmer.

HS-software: HSControl v.2.00, Perkin Elmer.

GC: Autosystem XL, Perkin Elmer.

GC software: Turbochrom navigator, Perkin Elmer.

Column: HP-FFAP 25 m x 0.20 mm x 0.33 µm, Agilent Technologies.

Known concentration standards were used to determine the response factors (calibration), controls were used to ensure that the response factors used were stable across assay runs as well as intermediate runs and over time (months). The concentration of volatiles ( ^ppm ) in the samples and controls was determined using the response factors, based on the standards. Samples were prepared by adding 200 µl 4N H2SO4 to 1 g yoghurt sample and immediately analyzed by HSGC.

The effect on diacetyl production is illustrated in FIG. 1, showing that when adding Lb. rhamnosus CHCC15871 produces significantly higher levels of diacetyl during milk fermentation compared to the commercially available strain Lb. rhamnosus.

The effect on acetoin production is illustrated in FIG. 2, showing that when adding Lb. rhamnosus CHCC15871 produces significantly higher levels of acetoin during milk fermentation compared to the commercially available strain Lb. rhamnosus.

In FIG. 3 shows yeast growth on prepared plates with milk fermented with only one starter culture (control, first plate), together with Lb. rhamnosus CHCC12483 (second plate) or together with Lb. rhamnosus CHCC15871 (third plate). Cups were incubated at 7±1°C for 27 days. Target contaminants were added at concentrations of 1 x 10 ³ cfu/spot (top row), 1 x 10 ² cfu/spot (middle row) and 1 x 10 ¹ cfu/spot (bottom row): (A) Torulaspora delbrueckii, (B) Cryptococcus fragicola, (C ) Debaryomyces hansenii and (D) Yarrowia lipolytica.

In FIG. 4 shows yeast growth on prepared plates with milk fermented with only one starter culture (control, first plate), together with Lb. rhamnosus CHCC12483 (second plate), along with a reference strain Lb. rhamnosus (third cup) or together with Lb. rhamnosus CHCC15871 (fourth plate). Cups were incubated at 7±1°C for 26 days. The target contaminant, Rhodotorula mucilaginosa, was added at concentrations of 1 x 10 ³ cfu/spot (top row), 1 x 10 ² cfu/spot (middle row) and 1 x 10 ¹ cfu/spot (bottom row).

As is apparent from the results of the yeast agar assay shown in FIG. 3 and 4, the test yeast grew very well on agar plates made with milk fermented with starter culture only (control). However, when Lb was present during milk fermentation. rhamnosus CHCC15871, all yeasts tested were inhibited in the plates obtained. The level of suppression was even higher than the suppression observed for the commercially available bioprotective strain, Lb. rhamnosus CHCC12483, and for the comparative strain Lb.

In FIG. 5 shows the growth of molds on prepared cups of milk, fermented

- 10 042154 with only one starter culture (control, first plate) or together with Lb. rhamnosus CHCC12483 (second plate) or together with Lb. rhamnosus CHCC15871 (third plate). The plates were incubated at 25±1°C for 12 days (top row) or 7±1°C for 27 days (bottom row). Target contaminants were added at concentrations of 500 spores/spot: (A) Penicillium brevicompactum, (B) Penicillium crustosum, and (C) Penicillium solitum.

In FIG. 5 shows that all molds tested were able to grow on agar plates made with milk fermented with starter culture only (control). When Lb was present during milk fermentation. rhamnosus CHCC12483, mold growth was suppressed. When Lb was present during milk fermentation. rhamnosus CHCC15871, mold growth was suppressed even more than for CHCC12483.

Example 2 Influence of strain Lb. rhamnosus CHCC15871 for postacylation.

Strain Lb. rhamnosus (CHCC15871) was tested for acylation rate effects and post-acylation effects.

Homogenized reduced fat milk (1.5% w/v) was heat treated at 90±1° C. for 20 minutes and immediately cooled.

A commercially available yogurt starter culture (F-DVS YoFlexMild 2.0) was inoculated at 0.02% (v/w) and the inoculated milk was dispensed into 200 ml bottles. One bottle was inoculated with Lb. rhamnosus CHCC15871 at a total concentration of 1x10 CFU/g, one bottle was inoculated with Lb. rhamnosus CHCC12483 with a commercially available bioprotective strain (obtained from Holdbac® YM-B) at a total concentration of 1x107 cfu/g and one bottle was used as a control and inoculated with starter culture only. All bottles were incubated in a water bath at 43±1°C and fermented under these conditions until a pH of 4.60±0.1 was reached. After fermentation, the bottles were shaken vigorously to break the curd and cooled on ice.

To observe the effect on postacylation, three fermented milk samples (sourdough only, Lb. rhamnosus CHCC12483 and Lb. rhamnosus CHCC15871) were stored at 7±1°C for 21 days and pH was measured on days 1, 7, 14 and 21.

In FIG. 6 shows the acylation profile of fermented dairy products during fermentation at 43°C. The products are fermented with starter culture alone, control or starter culture in combination with Lb strains. rhamnosus CHCC12483 or Lb. rhamnosus CHCC15871.

As is evident from FIG. 6, none of the two strains of Lb. rhamnosus did not change the acylation profile of the fermentation when added.

In FIG. 7 shows the pH increase (postacylation) in fermented dairy products as a function of time when stored at 7±1°C for 21 days. The products are fermented with starter culture alone, control or starter culture in combination with Lb strains. rhamnosus CHCC12483 or Lb. rhamnosus CHCC15871.

As is evident from FIG. 7, none of the two strains of Lb. rhamnosus, when added, did not alter the postacylation properties of the fermented dairy products to any significant extent.

Example 3 Semi-quantitative analysis of the inhibitory effect of Lb. rhamnosus CHCC15871 against various mold contaminants.

For semi-quantitative analysis of the inhibitory effect of Lb. rhamnosus CHCC15871, an agar assay similar to the manufacturing process and production of sour cream was used.

Homogenized standardized milk (9% fat w/v) was heat treated at 90±1° C. for 20 minutes and immediately cooled. A commercially available sour cream starter culture (F-DVS XT-314) was inoculated at a concentration of 0.02% (v/w) and the inoculated milk was dispensed into 200 ml bottles. One bottle was inoculated with Lb. rhamnosus CHCC15871 at a total concentration of 5x10 cfu/g and one bottle was used as a control and inoculated with starter culture alone. Both bottles were incubated in a water bath at 26±1°C and fermented under these conditions until a pH of 4.60±0.1 was reached. After fermentation, the bottles were shaken vigorously to break the curd and cooled on ice. Then the fermented milk was heated to a temperature of 40°C and added 40 ml of 5% sterile agar solution, which was melted and cooled to 60°C. This solution of fermented milk and agar was then poured into sterile Petri dishes and the dishes were dried on a table in a laminar flow hood for 30 minutes.

A spore suspension of three different molds was spotted at a concentration of 500 spores/spot; Penicillium brevicompactum (DSM32094), P. crustosum and P. solitum (DSM32093). Three molds were spotted on the same dish. The dishes were incubated at 25±1°C and periodically examined for mold growth.

In FIG. 8 shows mold growth on prepared plates with milk fermented with only one starter culture (control, first plate) or together with Lb. rhamnosus CHCC15871 (second plate). Cups were incubated at 25±1°C for 12 days. Target contaminants were added at a concentration of 500 spores/spot: (A) Penicillium brevicompactum, (B) Penicillium crustosum, and (C) Penicillium solitum.

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In FIG. 8 shows that all molds tested were able to grow on agar plates made with milk fermented with starter culture only (control). When Lb was present during milk fermentation. rhamnosus CHCC15871, mold growth was strongly suppressed.

Example 4 Influence of strain Lb. rhamnosus CHCC15871 on diacetyl and acetoin production, fermentation time and viscosity.

Strain Lb. rhamnosus (CHCC15871) was tested for effects on diacetyl and acetoin production, fermentation time and viscosity when sour cream was used. The characteristics were compared with conventional Lb. rhamnosus CHCC12697 is known to have high diacetyl production, cf. WO2012/136832, and antifungal activity, cf. WO2012/136830.

The sour cream base (18% fat and 2.7% w/v protein) was homogenized (two stages 200/70 bar (20/7 MPa) at 70°C), heat treated at 85±1°C for 3 min and immediately cooled. In a trial run, the sour cream base was distributed into sterile 200 ml bottles. Five starter cultures (SC1-SC5) consisting of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Streptococcus thermophilus and Leuconostoc were inoculated at a concentration of 0.01% (v/w). Five bottles were inoculated with each starter culture. In addition, two bottles were inoculated with Lb. rhamnosus CHCC15871 at a total concentration of 5x106 CFU/g and the other two bottles with Lb. rhamnosus CHCC12697 (comparison base) at a total concentration of 5x106 CFU/g. One bottle of each starter culture was used as a reference/control. All bottles were incubated in a water bath at 30±1°C and fermented under these conditions until a pH of 4.50±0.1 was reached. At final pH, the coagulum was stirred (20 times with a hand stirrer) to achieve a homogeneous consistency and cooled on ice. After cooling, the bottles were stored at 5±1°C.

The recorded fermentation time is shown in FIG. 9. It is important to note that the combination with Lb. rhamnosus does not cause a significant increase in fermentation time. For SC1, SC2, SC4 and SC5, the fermentation times with starter culture and CHCC15871 mixtures were lower than those for starter culture alone and starter culture and CHCC12697.

On days 7 and 21, samples were analyzed for diacetyl and acetoin by static headspace gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a static headspace feeder connected to a gas chromatograph with a flame ionization detector (FID). For this purpose, the following equipment was used.

HS autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

HS-software: HSControl v.2.00, Perkin Elmer.

GC: Autosystem XL, Perkin Elmer.

GC software: Turbochrom navigator, Perkin Elmer.

Column: HP-FFAP 25 m x 0.20 mm x 0.33 µm, Agilent Technologies.

Known concentration standards were used to determine the response factors (calibration), controls were used to ensure that the response factors used were stable across assay runs as well as intermediate runs and over time (months). The concentration of volatiles ( ^ppm ) in the samples and controls was determined using the response factors, based on the standards.

Samples were prepared by adding 200 μl 4N H ₂ SO ₄ to 1 gram fermented sour cream sample and analyzed by HSGC.

The effect on diacetyl production is shown in FIG. 10, and for acetoin - in Fig. 11. In FIG. 10 shows that the addition of Lb. rhamnosus CHCC15871 during fermentation has a very strong effect on the amount of diacetyl produced, especially after 21 days of storage in the refrigerator, and, surprisingly, the amounts are significantly higher compared to samples with Lb. rhamnosus CHCC12697. In addition, CHCC15871 also produces higher levels of acetoin compared to CHCC12697 as shown in FIG. eleven.

Seven days after production, the fermented sour cream product was brought to 13°C and mixed by hand (20 times) until the sample was homogeneous. The rheological properties of the sample were evaluated on a rheometer (Anton Paar Physica Rheometer with ASC, automatic sampler, Anton Paar!!! GmbH, Austria) using a float cup. The rheometer was adjusted to a constant temperature of 13°C during the measurement time. The settings were as follows.

Holding time (until the original structure is restored to some extent): 5 min without any physical load (vibration or rotation) applied to the sample.

Rotation stage (to measure shear stress at 300 1/s).

Two stages have been developed:

1) shear rate=[0.3-300] 1/s and

2) shear rate=[275-0.3] 1/s.

Each stage included 21 measurement points for 210 s (every 10 s).

- 12 042154

The shear stress at the maximum point (300 1/s) of the rheological curves was early for further analysis, as this value was shown to correlate well with the consistency felt in the mouth.

Shear stress (Pa) is represented as Viscosity (Pa*s)*Shear rate (1/s).

In FIG. 12 shows the rheological results. Obviously, the addition of Lb. rhamnosus has no negative effect on the properties of the consistency, expressed by shear stress at a shear rate of 300. The shear stress is not reduced in comparison with the control samples (SC1SC5).

Example 5 Comparison of Lb diacetyl production. rhamnosus CHCC15871 with various strains of Lb. Rhamnosus.

Production of diacetyl by strain Lb. rhamnosus CHCC15871 with seven different strains of Lb. rhamnosus.

Homogenized reduced fat milk (1.5% w/v) was heat treated at 90±1° C. for 20 minutes and immediately cooled. A commercially available yogurt starter culture (F-DVS YoFlex Mild 2.0) was inoculated at 0.02% (v/w) and the inoculated milk was dispensed into 200 ml bottles. 8 bottles were inoculated with Lb strains. rhamnosus (CHCC15871, CHCC12483, strain 1, strain 2, strain 3, strain 4, strain 5 and strain 6) at a total concentration of 1x10 CFU/g, one strain per bottle. One bottle was used as a reference and inoculated with starter culture only. All bottles were incubated in a water bath at 43±1°C and fermented under these conditions until a pH of 4.55±0.1 was reached. After fermentation, the bottles were shaken vigorously to break the curd and cooled on ice. After cooling, the samples were stored at 7±1°C.

On day 14, samples were analyzed for diacetyl by static headspace gas chromatography (HSGC), a sensitive method for analyzing volatiles in complex matrices. The setup consisted of a static headspace feeder connected to a gas chromatograph with a flame ionization detector (FID). For this purpose, the following equipment was used.

HS autosampler: HS40XI, TurboMatrix 110, Perkin Elmer.

HS-software: HSControl v.2.00, Perkin Elmer.

GC: Autosystem XL, Perkin Elmer.

GC software: Turbochrom navigator, Perkin Elmer.

Column: HP-FFAP 25 m x 0.20 mm x 0.33 µm, Agilent Technologies.

Known concentration standards were used to determine the response factors (calibration), controls were used to ensure that the response factors used were stable across assay runs as well as intermediate runs and over time (months). The concentration of volatiles ( ^ppm ) in the samples and controls was determined using the response factors, based on the standards. Samples were obtained by adding 200 μl of 4N H ₂ SO ₄ to 1 g of yogurt sample and immediately analyzed by HSGC.

The effect on diacetyl production is shown in FIG. 13, demonstrating that the addition of seven different strains of Lb. Rhamnosus during fermentation resulted in varying levels of diacetyl present in samples after 14 days of storage. Observed levels ranged from 6.3 ^ppm to 20 ^ppm . In comparison, CHCC15871 produced the highest amount of diacetyl.

Deposit and expert solution.

The Applicant requests that a sample of the deposited microorganisms indicated below be made available only to the examiner before the date of grant of the patent.

The strain Lactobacillus rhamnosus CHCC15871 was deposited on October 17, 2017 in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124, Braunschweig, accession number DSM32666.

The strain Lactobacillus rhamnosus CHCC15860 was deposited on July 16, 2015 in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124, Braunschweig, DSM accession number 32092.

The strain Lactobacillus rhamnosus CHCC12697 was deposited on March 1, 2011 in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, with DSM access number 24616.

The strain Penicillium solitum CHCC16948 was deposited on July 16, 2015 in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124, Braunschweig, DSM accession number 32093.

The strain Penicillium brevicompactum CHCC16935 was deposited on July 16, 2015 in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; DSMZ), Inhoffenstr. 7B, D-38124, Braunschweig, DSM accession number 32094.

The deposit data was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

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Links.

Jyoti, B.D., Suresh, A.K., and Venkatesh, K.V. (2003): Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates. World Journal of Microbiology & Biotechnology 19: 509-514.

Claims (18)

CLAIM 1. A composition for the production of a dairy product containing a strain of Lactobacillus rhamnosus CHCC15871 deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM 32666, or a mutant strain thereof, where the mutant strain is obtained by using the deposited strain as the mother strain and where the mutant strain has the same or higher diacetyl production as compared to the parent strain. 2. Composition according to claim 1, containing at least one additional lactic acid bacterium. 3. The composition according to claim 1 or 2, wherein the additional lactic acid bacterium is at least one antifungal lactic acid bacterium selected from the group consisting of a Lactobacillus rhamnosus strain, a Lactobacillus fermentum strain, a Lactobacillus paracasei strain, a Lactobacillus plantarum strain, a Propionibacteria freudenreichii strain , Pediococcus acidilactici strain, Enterococcus faecium strain and Lactococcus lactis strain. 4. The composition according to claim 3, wherein at least one antifungal lactic acid bacterium is a strain of Lactobacillus rhamnosus CHCC15860 deposited in the German collection of microorganisms and cell cultures (DSMZ) with accession number 32092. 5. Composition according to any one of claims 1 to 4, containing a starter culture for the preparation of a fermented dairy product, containing one or more strains selected from the group consisting of the genera Lactococcus, Streptococcus and Lactobacillus. 6. Composition according to any one of claims 1 to 5, which contains bacteria in a form selected from the group consisting of frozen, dried and lyophilized concentrate. 7. The use of a composition according to any one of claims 1 to 6 for the production of a dairy product. 8. Use according to claim 7, wherein the dairy product is a fermented dairy product. 9. Use according to claim 8, wherein the fermented dairy product is yogurt. 10. Use according to claim 7, wherein the dairy product is cheese. 11. Use according to any one of claims 7 to 10, wherein the dairy product contains at least 0.75 ^ppm diacetyl. 12. Use according to claim 11, wherein the dairy product contains at least 1.5 ^ppm diacetyl. 13. A method for obtaining a dairy product, including: a) inoculating the milk substrate with a composition according to any one of claims 1 to 5; And b) fermentation of the milk substrate. 14. The method of claim 13, further comprising: c) adding additional microorganisms and/or additives to the milk substrate. 15. The method according to claim 13 or 14, further comprising: d) subsequent processing of the milk substrate. 16. The method according to any one of claims 13-15, further comprising: e) packaging of a dairy product. 17. The method according to any one of claims 13-16, wherein step (b) comprises fermenting the milk substrate at a temperature above 35°C. 18. Lactobacillus rhamnosus strain CHCC15871 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM 32666.