Lactic acid bacterial strain with improved texturizing properties

Abstract

The present invention relates to novel Streptococcus thermophilus strains having improved texturizing properties, compositions comprising said strains and fermented products manufactured using said strains.

Description

Technical Field

[0001] This invention relates to a mutant strain of *Streptococcus thermophilus*, which has been found to have improved textural properties while retaining the growth characteristics of its parent strain. Furthermore, this invention relates to compositions comprising this mutant strain, such as starter cultures, and fermentation products prepared using this mutant strain. Background Technology

[0002] The food industry uses large numbers of bacteria, especially lactic acid bacteria, to improve the taste and texture of food, and also to extend its shelf life. In the dairy industry, lactic acid bacteria are used extensively to acidify milk (through fermentation) and to adjust the texture of products in which they are incorporated.

[0003] Among the lactic acid bacteria used in the food industry, the main genera are Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, and Bifidobacterium. Streptococcus thermophilus lactic acid bacteria are widely used alone or in combination with other bacteria such as Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) in food production, especially fermented products. They are particularly used in formulating starters for the production of fermented milk (e.g., yogurt). Some of them play a dominant role in the textural development of fermented products. This characteristic is closely related to the production of extracellular polymers secreted by lactic acid bacteria into the surrounding environment.

[0004] The current trend in yogurt is towards mild flavor and excellent texture. This is currently achieved by using cultures that produce a mild flavor and adding thickeners or proteins to achieve the desired consistency. Yogurt producers want to be able to produce yogurt with these properties without adding thickeners. This would help them reduce costs and offer simpler labeling. A very attractive way to achieve this is to have starter cultures that produce a high level of texture.

[0005] Many strains of *Streptococcus thermophilus* synthesize extracellular polysaccharides (EPS). These molecules can be produced as tightly associated vesicles within cells, or they can be released into the culture medium as a loose mucus (i.e., “viscous” polysaccharides). Although the presence of EPS does not provide any significant advantage to the growth or survival of *Streptococcus thermophilus* in dairy products, in situ production by this species or other dairy lactic acid bacteria often imparts the desired “viscous” or sticky texture to fermented dairy products. Studies have also shown that *Streptococcus thermophilus* that produce EPS can enhance the functional properties of mozzarella cheese. For more details, see the review article by Broadbent et al. (J. Dairy Sci. 86:407-423).

[0006] To meet industrial requirements, it has become essential to provide novel textured strains of lactic acid bacteria (especially Streptococcus thermophilus) for food texturing. In particular, there is a need for novel textured strains of Streptococcus thermophilus that can be used in conjunction with textured strains of Lactobacillus delbrueckii subsp. bulgaricus. Summary of the Invention

[0007] The problem to be solved by the present invention is to provide novel thermophilic streptococcal strains with improving properties, particularly their ability to improve the texture of fermented dairy products (e.g., yogurt), and which can be used in today's highly industrialized dairy production.

[0008] In particular, this invention discloses a novel thermophilic streptococcal strain, DSM 33677.

[0009] Therefore, one aspect of the present invention relates to a thermophilic streptococcal strain derived from a galactose-negative mother strain having a transposase insertion element in the promoter region of the galactose operon, wherein the thermophilic streptococcal strain becomes galactose-positive due to the lack of a transposase-encoded insertion element.

[0010] In another aspect, the present invention relates to compositions comprising Streptococcus thermophilus strain DSM 33677.

[0011] In another aspect, the present invention relates to a method for producing fermented products, comprising fermenting a substrate with Streptococcus thermophilus strain DSM33677 or a composition containing Streptococcus thermophilus strain DSM 33677.

[0012] In another aspect, the present invention relates to fermented products that can be obtained by the method of the present invention.

[0013] In another aspect, the present invention relates to fermented products comprising Streptococcus thermophilus strain DSM 33677.

[0014] Next, another aspect concerns the use of Streptococcus thermophilus strain DSM 33677 in the manufacture of fermented products. Attached Figure Description

[0015] Figure 1 Milk acidification diagrams of strains DSM 17876 and DSM 33677. Detailed Implementation

[0016] definition

[0017] Before outlining the invention in more detail, let us first define a set of terms and conventions:

[0018] The term "milk" should be understood as the milk secretion obtained by milking any mammal (e.g., cow, sheep, goat, buffalo, or camel). In a preferred embodiment, milk is cow's milk. The term milk also includes protein / fat solutions made partly or entirely of plant material.

[0019] The term "milk substrate" can refer to any raw and / or processed milk material capable of fermentation according to the methods of the present invention. Therefore, useful milk substrates include, but are not limited to, solutions / suspensions of any dairy or dairy product containing proteins, such as whole or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, lactose crystal mother liquor, whey protein concentrate, or cream. Obviously, milk substrates can be derived from any mammal, such as substantially pure mammalian milk, or reconstituted milk powder, or they can be derived from plant material.

[0020] Preferably, at least a portion of the proteins in the milk substrate are (i) proteins naturally occurring in mammalian milk, such as casein or whey protein, or (ii) proteins naturally occurring in plant milk. However, a portion of the proteins may be proteins not naturally occurring in milk.

[0021] Prior to fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.

[0022] As used in this article, "homogenization" means thorough mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, the fats in the milk can be broken down into smaller sizes so that they no longer separate from the milk. This can be achieved by forcing the milk through small pores under high pressure.

[0023] As used herein, "pasteurization" refers to the treatment of a latex substrate to reduce or eliminate the presence of living organisms (e.g., microorganisms). Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. This is typically achieved by heating to the specified temperature. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may then follow.

[0024] Fermentation processes for producing fermented dairy products are well known, and those skilled in the art will know how to select appropriate process conditions, such as temperature, oxygen, the amount and characteristics of microorganisms, and processing time. Clearly, fermentation conditions should be selected to support the implementation of this invention, i.e., to obtain fermented products, such as dairy or non-dairy products (fermented dairy products) in solid or liquid form.

[0025] In this article, the term "starter culture" refers to a culture that is a preparation of one or more bacterial strains (e.g., lactic acid bacteria strains) that contribute to the initiation of the fermentation process in the preparation of fermented products (e.g., various foods, feeds, and beverages).

[0026] In this article, "yogurt starter culture" is a bacterial culture containing at least one strain of *Lactobacillus delbrueckii* subsp. bulgaricus and at least one strain of *Streptococcus thermophilus*. Accordingly, "yogurt" refers to a fermented dairy product obtained by inoculating and fermenting a milk substrate with a composition containing *Lactobacillus delbrueckii* subsp. bulgaricus and *Streptococcus thermophilus* strains.

[0027] In this document, the term "galactose-positive (gal-positive or gal+) Streptococcus thermophilus strain" as defined herein means strains containing at least 10... 4 Cells / ml of M17 were seeded in M17 containing 2% galactose (added galactose as the sole carbohydrate) and incubated at 37°C for 16 hours until the pH decreased by at least 1.0.

[0028] Observations have shown that galactose-positive derivatives typically exhibit a slower acidification curve and a higher final pH after fermentation exceeding 20 hours. Figure 1 ).

[0029] Assay I was disclosed in Example 3, and Assay II was disclosed in Example 4, which discloses the selection of galactose-positive thermophilic streptococci based on galactose release.

[0030] In this invention, "the ability not to secrete galactose" means that the galactose-positive strain cannot secrete a measurable amount of galactose, and in particular, means that no measurable amount of galactose is detected when tested by assay II.

[0031] In this invention, "the ability to secrete galactose but consume the secreted galactose within a maximum of 9 hours" means that the galactose-positive strain is able to secrete galactose but consumes the secreted galactose within a maximum of 9 hours after inoculation (i.e., to a level below measurable), and particularly means that when tested by assay II, the galactose-positive strain secretes galactose but consumes the secreted galactose within a maximum of 9 hours after inoculation (i.e., to a level below measurable).

[0032] In one embodiment, a galactose-positive thermophilic streptococcal strain as defined herein is characterized by its inability to secrete galactose when inoculated at 1% (v / v) into M17 medium supplemented with 0.5% (wt / wol) lactose and incubated at 42°C, particularly when tested by assay II.

[0033] In one embodiment, a galactose-positive thermophilic streptococcal strain as defined herein is characterized by its ability to secrete galactose, but consume the secreted galactose within a maximum of 9 hours, when inoculated at 1% (v / v) into M17 medium supplemented with 0.5% (wt / wol) lactose and incubated at 42°C, particularly when tested by assay II.

[0034] In a particular embodiment, a galactose-positive thermophilic streptococcal strain as defined herein is characterized by its ability to secrete galactose, but consume the secreted galactose within a maximum of 8 hours, when inoculated at 1% (v / v) into M17 medium supplemented with 0.5% (wt / wol) lactose and incubated at 42°C, particularly when tested by assay II.

[0035] In this document, the term "mutant" should be understood as a strain derived from the strain (or parent strain) of the present invention through, for example, genetic engineering, radiation, and / or chemical treatment, or a strain that can be derived from the strain (or parent strain) of the present invention through the above methods. Mutants can also be spontaneously generated mutants. Mutants are preferably functionally equivalent mutants, such as mutants having substantially the same or improved properties as the parent strain (e.g., in terms of viscosity, gel hardness, mouthfeel, flavor, post-acidification, acidification rate, and / or phage robustness). Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenesis treatment (including treatment with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), UV light treatment), or a spontaneously generated mutant. The mutant strain may have undergone several mutagenesis treatments (a single treatment should be understood as a mutagenesis step accompanied by a screening / selection step), but it is currently preferred to perform no more than 20, 10, or 5 treatments (or screening / selection steps). In the currently preferred mutant strains, less than 1%, 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 replaced or deleted by another nucleotide compared to the parent strain.

[0036] In this document, the term "variant" should be understood as a strain that is functionally equivalent to the strains of the present invention, for example, having substantially the same or improved properties, such as in terms of viscosity, gel firmness, mouthfeel, flavor, post-acidification, acidification rate, and / or phage robustness. Such variants, which can be identified using appropriate screening techniques, are part of the present invention.

[0037] Novel thermophilic streptococcal strains and their applications

[0038] The inventors have unexpectedly identified a thermophilic streptococcal strain (i.e., DSM 33677) that meets industrial requirements. Compared to its parent strain, this new strain exhibits improved rheological properties, such as texturing properties, when used alone or as part of a mixed culture in dairy substrates.

[0039] Streptococcus thermophilus strain DSM 33677 can be used, for example, in dairy cultures (such as yogurt cultures) to obtain improved rheological parameters of the final product, such as shear stress (i.e., viscosity) and gel hardness. Rheology is closely related to the sensory quality of a product; therefore, the interaction between rheology and the taste of the final product is crucial.

[0040] Therefore, one aspect of the present invention relates to a thermophilic streptococcal strain derived from a galactose-negative mother strain having a transposase insertion element in the promoter region of the galactose operon, wherein the thermophilic streptococcal strain becomes galactose-positive due to the lack of a transposase-encoded insertion element.

[0041] In one embodiment, the transposase insertion element is located in the promoter region sequence of the galactose operon, corresponding to nucleotides between 133 and 1460 of SEQ ID No:1.

[0042] It is understood that the promoter region sequence of the galactose operon corresponds to nucleotides 105-133 and 1460-1491 of SEQ ID No:1. Similarly, it is understood that the transposase insertion element sequence corresponds to nucleotides 134-1459 of SEQ ID No:1. In yet another embodiment, the parent strain is DSM 17876.

[0043] In yet another embodiment, the present invention relates to Streptococcus thermophilus strain DSM 33677 and its mutants and variants.

[0044] The mutants and variants are expected to exhibit the same or similar shear stress and / or gel hardness characteristics as DSM 33677. In this document, the term "similar shear stress" should be understood as a range from 10% lower than the shear stress characteristic of DSM 33677 to 10% higher than the shear stress characteristic of DSM 33677. This range can also be ±9% of the shear stress characteristic of DSM 33677, for example ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0045] In this document, the term "similar gel hardness" should be understood as a range from 10% lower than the gel hardness characteristic of DSM 33677 to 10% higher than the gel hardness characteristic of DSM 33677. This range can also be ±9% of the gel hardness characteristic of DSM 33677, for example ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0046] The above “characteristics” should be understood in the context of the definition section, which explains how to properly measure shear stress or gel hardness.

[0047] Methods for determining the texture of fermented products (such as dairy products) include measuring the shear stress (viscosity) or gel hardness of the fermented product, and are readily available and known in the art, and have been illustrated herein.

[0048] In a preferred embodiment, the shear stress generated by the thermophilic streptococcal strain DSM 33677 is increased by at least 1% compared to its parent strain, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0049] In a preferred embodiment, the complex modulus produced by the thermophilic streptococcal strain DSM 33677 is increased by at least 1% compared to its parent strain, and by, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%.

[0050] In a preferred embodiment, when at least 10 per ml of dairy product 7 When inoculated at a cell volume, the shear stress (measured at 300 1 / s (Pa)) generated by Streptococcus thermophilus strain DSM 33677 after growth at 43°C in skim milk (0.5% fat) for 16 hours was at least 1% higher than that of its parent strain, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than that of its parent strain.

[0051] In a preferred embodiment, when at least 10 per ml of dairy product 7When inoculated at a quantity of 1,000 cells, the complex modulus (measured by 1.52 Hz oscillation) of Streptococcus thermophilus strain DSM 33677 after growth at 43°C in skim milk (0.5% fat) for 16 hours was at least 1% higher than that of its parent strain, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than that of its parent strain.

[0052] "Texture" or "taste" refers to the physical and chemical interactions of a product in the mouth.

[0053] In one embodiment of the invention, DSM 33677 is galactose-positive.

[0054] In addition to being galactose-positive, Streptococcus thermophilus strain DSM 33677 has been found to be protease-negative (Prt-). Furthermore, it has been found that Streptococcus thermophilus strain DSM 33677 can be stimulated by the addition of peptides (e.g., but not limited to casein hydrolysate).

[0055] Composition

[0056] Another aspect of the invention relates to compositions comprising or consisting of Streptococcus thermophilus strain DSM 33677.

[0057] The compositions of the present invention can be provided in a variety of forms. They can be in a frozen form, a dried form, a lyophilized form, or a liquid form. Thus, in one embodiment, the composition is in a frozen, dried, lyophilized, or liquid form.

[0058] The compositions of the present invention may further comprise cryoprotectants, lyophilization protectants, antioxidants, nutrients, fillers, flavorings, or mixtures thereof. The compositions preferably comprise one or more of cryoprotectants, lyophilization protectants, antioxidants, and / or nutrients; more preferably, cryoprotectants, lyophilization protectants, and / or antioxidants; and most preferably, cryoprotectants or lyophilization protectants, or both. The use of protectants (e.g., cryoprotectants and lyophilization protectants) is known to those skilled in the art. Suitable cryoprotectants or lyophilization protectants include monosaccharides, disaccharides, trisaccharides, and polysaccharides (e.g., glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch, and gum arabic (acacia), etc.), polyols (e.g., erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol, etc.), amino acids (e.g., proline, glutamic acid), complexes (e.g., skim milk, peptone, gelatin, yeast extract), and inorganic compounds (e.g., sodium tripolyphosphate). In one embodiment, the composition according to the invention may comprise one or more cryoprotectants selected from the group consisting of: inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uracil, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine, and derivatives of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and its salts, gallate, cysteine, sorbitol, mannitol, and maltose. Suitable nutrients include carbohydrates, amino acids, fatty acids, minerals, trace elements, and vitamins (such as B vitamins and vitamin C). The composition may optionally contain other substances, including fillers (such as lactose, maltodextrin) and / or flavoring agents.

[0059] In one embodiment of the invention, the cryoprotectant is a reagent or a mixture of reagents that, in addition to cryoprotection, also has a booster effect.

[0060] The term "promoting effect" is used to describe situations where, when a cryoprotectant is inoculated into a culture medium to be fermented or transformed, the cryoprotectant imparts enhanced metabolic activity (promoting effect) to the thawed or reconstituted culture. Viability and metabolic activity are not synonymous. Commercially available frozen or lyophilized cultures may retain their viability, although they may have lost a significant portion of their metabolic activity; for example, even with short storage times, cultures may lose their acid-producing (acidifying) activity. Therefore, viability and the promoting effect must be evaluated using different assays. Viability is assessed by viability assays (such as measuring colony-forming units), while the promoting effect is assessed by quantifying the relevant metabolic activity of the thawed or reconstituted culture relative to the culture's viability. The term "metabolic activity" refers to the oxygen removal activity of a culture, its acid-producing activity (i.e., the production of, for example, lactic acid, acetic acid, formic acid, and / or propionic acid), or its activity in producing metabolites (e.g., the production of aromatic compounds such as acetaldehyde, α-acetolactone, acetoin, diacetyl, and 2,3-butanediol).

[0061] In one embodiment, the composition of the present invention contains or comprises a mixture of 0.2% to 20% of a cryoprotectant or reagent, measured as % w / w of the material. However, it is preferred to add a mixture of cryoprotectants or reagents in amounts ranging from 0.2% to 15%, 0.2% to 10%, 0.5% to 7%, and 1% to 6% by weight, including a mixture of cryoprotectants or reagents ranging from 2% to 5% by weight, measured as % w / w of the frozen material. In a preferred embodiment, the culture contains about 3% of a mixture of cryoprotectants or reagents, measured as % w / w of the material by weight. The amount of about 3% cryoprotectant corresponds to a concentration in the range of 100 mM. It should be understood that for each aspect of the embodiments of the present invention, the range may be incremental.

[0062] In another aspect, the compositions of the present invention contain or comprise ammonium salts (e.g., ammonium salts of organic acids (e.g., ammonium formate and ammonium citrate) or ammonium salts of inorganic acids) as promoters (e.g., growth promoters or acidification promoters) for bacterial cells (e.g., cells belonging to the thermophilic streptococcus species, such as (essentially) urease-negative bacterial cells). The terms "ammonium salt," "ammonium formate," etc., should be understood as a source of salt or a combination of ions. For example, the term "source" of "ammonium formate" or "ammonium salt" refers to a compound or mixture of compounds that provides ammonium formate or an ammonium salt when added to a cell culture. In some embodiments, the ammonium source releases ammonium into the growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, the ammonium is not provided by the emulsion substrate. It should be understood, of course, that ammonia can be added instead of the ammonium salt. Therefore, the term ammonium salt includes ammonia (NH3), NH4OH, NH4+ wait.

[0063] In one embodiment, the composition of the present invention may comprise a thickener and / or a stabilizer, such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soybean fiber / soybean polymer, starch, modified starch, carrageenan, alginate, and guar gum.

[0064] In one embodiment, in which microorganisms produce polysaccharides (such as EPS) that give the acidified dairy product a high-texture / viscous texture, the acidified dairy product is substantially free of or completely free of any thickeners and / or stabilizers, such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soybean fiber / soybean polymer, starch, modified starch, carrageenan, alginate, and guar gum. "Substantially free" should be understood to mean that the product contains 0% to 20% (w / w) (e.g., 0% to 10%, 0% to 5%, 0% to 2%, or 0% to 1%) of thickeners and / or stabilizers.

[0065] The composition may be in the form of a mixture or a kit-of-parts kit, comprising:

[0066] i) Streptococcus thermophilus strain DSM 33677, and

[0067] ii) Strains belonging to the Lactobacillus delbrueckii subspecies bulgaricus.

[0068] To obtain the optimal combination of acidity, flavor, and texture in products (such as dairy products like yogurt), a combination of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is typically used.

[0069] In one embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 22935 and Lactobacillus delbrueckii subsp. bulgaricus strain DSM 22586.

[0070] In one embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 24655 and Lactobacillus delbrueckii subsp. bulgaricus strain DSM 33571.

[0071] In another embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 24655 and Lactobacillus delbrueckii subsp. bulgaricus strain DSM 24074.

[0072] Example 3 shows a mixed culture containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0073] probiotic strains

[0074] The term "probiotics" refers to live bacteria administered to consumers in adequate quantities to achieve health-promoting effects. After ingestion, probiotics can survive in gastrointestinal conditions and colonize the consumer's intestines.

[0075] It should be understood that the classification of Lactobacillus species was updated in 2020. Zheng et al. disclosed a new classification in 2020, and unless otherwise notified, this document will use that classification. For the purposes of this invention, Table 1 lists the old and new names of some Lactobacillus species relevant to this invention.

[0076]

[0077]

[0078] Table 1. Old and new names of some Lactobacillus species related to this invention

[0079] In specific embodiments of the present invention, the probiotic strains according to the present invention are selected from the group consisting of the following bacteria: *Lactobacillus*, such as *Lactobacillus acidophilus*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Lactobacillus casei*, *Lactobacillus delbrueckii*, *Lactobacillus lactis*, *Lactobacillus plantarum*, *Limosilactobacillus reuteri*, and *Lactobacillus johnsonii*; *Bifidobacterium*, such as *Bifidobacterium longum*, *Bifidobacterium adolescentis*, *Bifidobacterium bifidum*, and *Bifidobacterium breve*. Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudo-small chain, and Bifidobacterium infantis, etc.

[0080] In a specific embodiment of the present invention, the probiotic lactobacillus strain is selected from the group consisting of: Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus johnsonii.

[0081] In a specific embodiment of the present invention, the probiotic Lactobacillus strain is selected from the group consisting of Lactobacillus rhamnosus and Lactobacillus paracasei.

[0082] In a specific embodiment of the present invention, the probiotic strain is *Lactobacillus rhamnosus* strain preserved as ATCC53103.

[0083] In a specific embodiment of the present invention, the probiotic strain is Lactobacillus paracasei strain CRL 431, preserved as ATCC 55544.

[0084] In a specific embodiment of the present invention, the probiotic Bifidobacterium strain is selected from the group consisting of: Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentatum, Bifidobacterium chain, Bifidobacterium angularis, Bifidobacterium macrocarpa, Bifidobacterium pseudosmata and Bifidobacterium infantis.

[0085] In a specific embodiment of the present invention, the probiotic Bifidobacterium strain is Bifidobacterium animalis subsp. BB-12, preserved as DSM15954.

[0086] The above mixtures or kits can be further combined with other lactic acid bacteria (e.g., but not limited to probiotics). In one embodiment, at least one lactic acid bacteria is selected from the group consisting of: Bifidobacterium, such as Bifidobacterium animalis subsp. lactis (e.g. Lactobacillus acidophilus Lactobacillus rhamnosus (e.g.) (and their combinations). The choice of which Bifidobacterium, Lactobacillus acidophilus, and / or Lactobacillus rhamnosus to use depends on their application and the food to be produced.

[0087] In one embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 22935, Lactobacillus delbrueckii subsp. bulgaricus strain DSM 22586 and at least one lactic acid bacteria selected from the group consisting of Bifidobacterium, Lactobacillus acidophilus, Lactobacillus rhamnosus and combinations thereof.

[0088] In one embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 24655, Lactobacillus delbrueckii subsp. bulgaricus strain DSM 33571 and at least one lactic acid bacteria selected from the group consisting of Bifidobacterium, Lactobacillus acidophilus, Lactobacillus rhamnosus and combinations thereof.

[0089] In another embodiment, the mixture or kit may include a combination of Streptococcus thermophilus strain DSM 33677 with Streptococcus thermophilus strain DSM 24655, Lactobacillus delbrueckii subsp. bulgaricus strain DSM 24074 and at least one lactic acid bacteria selected from the group consisting of Bifidobacterium, Lactobacillus acidophilus, Lactobacillus rhamnosus and combinations thereof.

[0090] The mixtures or kits described above can be further combined with other galactose-positive strains. For example, these strains could be DSM32823 and / or DSM32587.

[0091] The term "mixture" refers to the physical mixing of one or more strains of Streptococcus thermophilus and one or more strains of Lactobacillus delbrueckii subsp. bulgaricus. In one embodiment, one or more strains of Streptococcus thermophilus and one or more strains of Lactobacillus delbrueckii subsp. bulgaricus are contained in the same box or bag.

[0092] Conversely, the description of a "kit containing one or more Streptococcus thermophilus strains and one or more Lactobacillus delbrueckii subsp. bulgaricus strains" means that cultures of one or more Streptococcus thermophilus strains and cultures of one or more Lactobacillus delbrueckii subsp. bulgaricus strains are physically separated but intended to be used together. Therefore, cultures of one or more Streptococcus thermophilus strains and cultures of one or more Lactobacillus delbrueckii subsp. bulgaricus strains are in separate boxes or pouches. In one embodiment, cultures of one or more Streptococcus thermophilus strains and cultures of one or more Lactobacillus delbrueckii subsp. bulgaricus strains are in the same form, i.e., both are in frozen form, granule or frozen granule form, or powder form (e.g., dry powder or lyophilized powder).

[0093] In a specific embodiment of the present invention, the composition comprises 10 4 Up to 10 12 thermophilic streptococcal strains with CFU (colony forming units) / g, for example, 10 5 Up to 10 11 CFU / g, for example 10 6 Up to 10 10 CFU / g, or for example 10 7 Up to 10 9 Thermophilic Streptococcus strains with CFU / g.

[0094] In a specific embodiment, the composition further comprises 10 4 Up to 10 12 CFU / g of Lactobacillus delbrueckii subsp. bulgaricus strain, for example 10 5 Up to 10 11 CFU / g, for example 10 6Up to 10 10 CFU / g, or for example 10 7 Up to 10 9 CFU / g of Lactobacillus delbrueckii subsp. bulgaricus.

[0095] Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and other lactic acid bacteria are commonly used as starter cultures for technical purposes in the production of various foods, such as in the dairy industry, for example, for fermented dairy products. Therefore, in another preferred embodiment, the composition is suitable as a starter culture.

[0096] The composition can be a starter culture, such as a yogurt starter culture.

[0097] The composition and / or starter culture may be in the form of freeze-dried, spray-dried, lyophilized, vacuum-dried, air-dried, tray-dried, or liquid. Generally, the storage stability of food can be extended by formulating articles with low water activity. By controlling water activity (Aw), the effect of moisture migration on the product can be predicted and adjusted. Therefore, the water activity (Aw) of the dried compositions described herein is preferably in the range of 0.01-0.8, more preferably in the range of 0.05-0.4.

[0098] Another aspect of the present invention relates to a method for producing fermented products, comprising fermenting a substrate with Streptococcus thermophilus strain DSM33677 or a composition according to the present invention.

[0099] Depending on the product to be produced, the substrate can be a milk substrate. A milk substrate is particularly preferred when the final product is a fermented dairy product (such as yogurt, buttermilk, or kefir).

[0100] The base product can be an animal or plant-based product. Therefore, in one embodiment, the fermented product is a dairy product. Dairy products can be selected from the group consisting of fermented dairy products (e.g., but not limited to yogurt, buttermilk, and kefir) and cheeses (e.g., but not limited to fresh cheese or pasta filata).

[0101] Even though fermented products and / or dairy products themselves contain acids and flavors produced during fermentation, it is desirable that fermented products and / or dairy products contain ingredients selected from the group consisting of: fruit concentrates, syrups, probiotic cultures, colorings, thickeners, flavorings, preservatives, and combinations thereof.

[0102] Similarly, enzymes can be added to the substrate (e.g., milk substrate) before, during, and / or after fermentation, for example, by means of enzymes selected from the group consisting of: enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, chymosin, rennet, and combinations thereof.

[0103] In one implementation, the fermented product may be in the form of a stirred product, a set-type product, or a drinkable product.

[0104] Obviously, another aspect of the present invention relates to fermented products obtainable by the method of the present invention. Therefore, another aspect of the present invention is a fermented product comprising Streptococcus thermophilus strain DSM 33677. This fermented product may be a dairy product.

[0105] The final aspect of this invention relates to the use of Streptococcus thermophilus strain DSM 33677 in the manufacture of fermented products. Again, the fermented product can be a dairy product.

[0106] Unless otherwise stated herein or clearly contradicted by the context, nouns without quantifiers in the context of describing the invention (particularly in the context of the following claims) shall be construed as encompassing both singular and plural nouns. Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” shall be construed as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise stated herein, the enumeration of numerical ranges herein is intended only as a shorthand for each individual value falling within that range, and each individual value is incorporated into the specification as if it were individually enumerated herein. Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise stated, the use of any and all instances or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0107] The enumeration or discussion of obviously prior published documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common general knowledge.

[0108] Unless the context otherwise requires, preferred, alternative, and embodiments of a given aspect, feature, or parameter of the invention should be considered as having disclosed any and all preferred, alternative, and embodiments thereof with all other aspects, features, and parameters of the invention. This is especially true for the description of microencapsulated microbial cultures and all their characteristics (which are readily included as part of the final composition obtained by the methods described herein). Embodiments and features of the invention are also outlined in the following items and illustrated by the following non-limiting examples.

[0109] Preservation and expert solutions

[0110] The thermophilic streptococcus strain was deposited by Chr. Hansen of Helsholm, Denmark on October 29, 2020, in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, 7B Inhofenstrasse, D-38124).

[0111] The applicant requires that the preserved microbial samples described below be provided only to experts until the date of patent grant.

[0112] Streptococcus thermophilus DSM 33677 October 29, 2020

[0113] Table 2. Deposits made at depositary institutions that have obtained international depositary status, in accordance with the Budapest Treaty on the international recognition of the preservation of microorganisms for patent procedures: Leibniz Institute DSMZ - German Center for the Preservation of Microorganisms and Cell Cultures, Braunschweig, 7B Inhofenstrasse, 38124, Germany.

[0114] project

[0115] X1. A thermophilic streptococcal strain derived from a galactose-negative mother strain having a transposase insertion element in the promoter region of the galactose operon, wherein the thermophilic streptococcal strain becomes galactose-positive due to the lack of the transposase insertion element.

[0116] X2. The thermophilic streptococcal strain according to item 1, wherein the transposase insertion element is located in the promoter region sequence of the galactosidon, corresponding to nucleotides between 133 and 1460 of SEQ ID No:1.

[0117] X3. The thermophilic streptococcal strain according to Project 2, wherein the promoter region sequence of the galactosooperon corresponds to nucleotides 105-133 and 1460-1491 of SEQ ID No:1.

[0118] X4. The Streptococcus thermophilus strain according to any one of items 1-3, wherein the transposase insertion element sequence corresponds to nucleotides 134-1459 of SEQ ID No:1.

[0119] X5. The thermophilic streptococcal strain according to any one of items X1-X4, wherein the parent strain is DSM17876.

[0120] X6. The thermophilic streptococcal strain according to any one of items X1-X5, wherein the thermophilic streptococcal strain is DSM 33677 and its mutants and variants.

[0121] X7. The thermophilic streptococcal strain DSM 33677 according to any one of items X1-X6, wherein the mutant strain and variant strain exhibit the same or similar shear stress and / or gel hardness characteristics as DSM 33677.

[0122] X8. The thermophilic streptococcal strain DSM 33677 according to any one of items X1-X7, wherein the strain is galactose-positive.

[0123] Y1. A composition comprising Streptococcus thermophilus strain DSM33677 according to any one of items X1-X8.

[0124] Y2. The composition according to item Y1, which comprises, in the form of a mixture or kit, i) Streptococcus thermophilus strain DSM 33677 according to any one of items X1-X8; and ii) a strain belonging to Lactobacillus delbrueckii subsp. bulgaricus.

[0125] Y3. The composition according to any one of items Y1-Y2, wherein the composition is a starter culture.

[0126] Y4. The composition according to any one of items Y1-Y2, wherein the composition and / or the starter culture is in the form of freeze-dried, spray-dried, lyophilized, vacuum-dried, air-dried, tray-dried, or liquid.

[0127] Z1. A method for producing dairy products, comprising fermenting a substrate with a composition of any one of the Streptococcus thermophilus strain DSM 33677 according to any one of items X1-X8 or any one of items Y1-Y4.

[0128] Z2. The method described in project Z1, wherein the substrate is an emulsion substrate.

[0129] Z3. The method according to any one of items Z1-Z2, wherein the emulsion substrate is a product of animal or plant origin.

[0130] Z4. The method according to any one of items Z1-Z3, wherein the fermented product is a dairy product.

[0131] Z5. The method according to item Z4, wherein the dairy product is selected from the group consisting of: fermented dairy products (e.g., yogurt, buttermilk, or kefir) or cheese (e.g., fresh cheese or pastoral cheese).

[0132] Z6. The method according to any one of items Z1-Z5, wherein the fermented product further comprises an ingredient selected from the group consisting of: fruit concentrate, syrup, probiotic strains or cultures, coloring agents, thickeners, flavoring agents, preservatives, and mixtures thereof.

[0133] Z7. The method according to any one of items Z1-Z5, wherein an enzyme is added to the substrate before, during and / or after fermentation, said enzyme being selected from the group consisting of: enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, rennet, chymotrypsin and combinations thereof.

[0134] Z8. The method according to any one of items Z1-Z7, wherein the fermented product is in the form of a stirred product, a fixed product, or a drinkable product.

[0135] Q1. A fermented product that can be obtained by any one of items Z1-Z8.

[0136] Q2. The fermented product according to item Q1, wherein the fermented product is a dairy product.

[0137] P1. A fermented product comprising Streptococcus thermophilus strain DSM33677 according to any one of items X1-X8.

[0138] P2. Fermented products according to item P1, wherein the fermented products are dairy products.

[0139] W1. Use of any one of items X1-X8, Streptococcus thermophilus strain DSM 33677, in the preparation of fermented products.

[0140] W1. The use according to claim W1, wherein the fermented product is a dairy product.

[0141] sequence list

[0142] SEQ ID NO 1: DNA sequence from DSM 17876, encompassing galK initiation, gal operon promoter (including the inserted sequence), and galR initiation.

[0143]

[0144] galK gene initiation nucleotide (negative strand): 67

[0145] The transposase encoding the insertion sequence includes nucleotides 134-1459.

[0146] galR gene initiation nucleotides (positive strand): 1526

[0147] The starter sub-regions on both sides of the inserted element (IS element is located in the middle):

[0148] Part 1: 105-133

[0149] Part 2: 1460-1491.

[0150] Example

[0151] Example 1: Selecting a galactose-positive mutant strain of Streptococcus thermophilus strain DSM 17876

[0152] Isolation of galactose fermentation strains

[0153] A galactose-fermenting mutant of Streptococcus thermophilus strain DSM 17876 was isolated. DSM 17876 cells were not mutagenized with mutagenic compounds or UV light prior to the mutant isolation step. Therefore, the isolated strain resembled a spontaneous galactose-positive mutant of DSM 17876.

[0154] Before isolating the mutants, DSM 17876 was streaked on M17 agar plates (M17-gal plates) containing 2% galactose. DSM 17876 did not grow when galactose was the sole carbohydrate source.

[0155] Typically, galactose-fermenting mutant strains are isolated by spreading cultures of *Streptococcus thermophilus* strains onto M17-2% gal plates and then selecting colonies that appear after two days of growth at 37°C. This method is not suitable for isolating the galactose-fermenting mutant strain of DSM 17876. Even inoculation in liquid M17+2% galactose did not result in any significant growth.

[0156] Streptococcus thermophilus DSM 17876 grows on M17 medium containing lactose. To produce a large number of growing cells, the strain was inoculated into M17 medium containing 0.2% lac + 2% gal. Lactose promotes a large number of cell growths. Once the lactose is depleted, only naturally galactose-positive cells in the cell pool continue to grow. After 24 hours, a denser culture was obtained. 100 μl of this culture was inoculated into fresh M17 medium containing 2% galactose. The culture dilution was spread onto M17-gal plates, and after 24 hours, some colonies were selected and streaked three times on M17-gal plates to purify the strain. Finally, one clone was selected for further analysis. DNA was isolated and the genome was sequenced. The new strain was deposited as DSM 33677. Compared to DSM 17876, DSM 33677 is an acidified B-milk (made by reconstituted skim milk powder in distilled water at a dry matter level of 9.5% (w / v) and pasteurized at 99°C for 30 minutes, then cooled to 30°C). Figure 1 ).

[0157] Genome sequencing of DSM 17876 and DSM33677 revealed why it was more difficult to select galactose-fermenting mutants from DSM 17876. DSM 17876 is galactose-positive because the promoter upstream of the galactose operon contains a transposase insertion element in that region, which disables transcription. In DSM 33677, this insertion element jumps out, leaving the promoter open for transcription. This results in transcription of the gal operon, and the strain becomes galactose-positive.

[0158] Example 2: Production of Plain Stirred Yogurt using a blend containing DSM 33677

[0159] Materials and methods

[0160] Milk base: 4.0% protein, 1.5% fat. Fresh milk containing Arla skimmed milk powder (SMP).

[0161] Culture:

[0162] Culture 2 (Lactobacillus delbrueckii subsp. bulgaricus strain DSM 33571 and Streptococcus thermophilus strains DSM 33677 and DSM 24655) and F-DVS YoFlex Premium 1.0 (a proprietary existing culture containing a combination of a Lactobacillus delbrueckii subsp. bulgaricus strain and two different Streptococcus thermophilus strains).

[0163]

[0164] result

[0165] Complex modulus G * - Related to gel hardness

[0166] The complex modulus G was evaluated by oscillation measurement using Anton Paar's ASC DSR502 rheometer. * This method is based on an oscillatory step, where the sample oscillates between two surfaces, with the upper geometry (oscillating) moving while the lower cup remains stationary. Oscillations are performed at 0.5–8 Hz under constant stress. Measurements at 1.52 Hz are extracted for these evaluations. Before measurement, the samples are placed at 13°C for 1 hour. Each sample is gently stirred five times from bottom to top with a small spoon to ensure homogeneity. The rheology cup is filled to the graduations and placed in the sample box. Samples are measured twice using two separate yogurt cups. Measurements are performed on day +7, with the measurement temperature set at 13°C. Samples are stored at 5°C until the day of measurement. Since these results include three products, a total of six data points are available.

[0167]

[0168] Table 3. Complex modulus G measured at 1.52 Hz * The resulting gel hardness was then analyzed. The results are presented as averages, including the standard deviation from three replicates from the ATC, with the sample measured twice. Measurements were taken on day +7, with the temperature adjusted to 13°C.

[0169] Shear stress – related to mouth consistency

[0170] Shear stress was measured using an Anton Paar ASC rheometer, model DSR502. The method utilizes a rotational step, based on the sample's rotational deformation from 10⁻³ s⁻¹ to 300 s⁻¹ and back to 10⁻³ s⁻¹. The corresponding shear stress was measured. For these results, four shear rates (0.3 s⁻¹; 30 s⁻¹; 135 s⁻¹; 300 s⁻¹) were extracted from the flow curves (see Table 4). Before measurement, the samples were placed at 13°C for 1 hour. Each sample was gently stirred five times from bottom to top with a small spoon to ensure homogeneity. Rheology cups were filled to the graduations and placed in the sample cassette. Samples were measured twice, using two separate yogurt cups. Measurements were performed on day +7, with the measurement temperature set at 13°C. Samples were stored at 5°C until the day of measurement. Since these results include three products, a total of six data points were obtained.

[0171] average value

[0172]

[0173] STDev

[0174]

[0175] Table 4. Shear stress results measured by the Anton Paar rheometer. Results are shown as averages, including the standard deviation from three repeated tests from the ATC, with the sample measured twice. Measurements were taken on day +7, with the temperature adjusted to 13°C.

[0176] Example 3: pH-based selection of galactose-positive thermophilic streptococci (Assay I)

[0177] Measurement I is as follows:

[0178] - A thermophilic streptococcal strain characterized by galactose positivity (i.e., described as being able to grow on a medium containing galactose as the sole carbohydrate source) was grown overnight at 37°C in M17 supplemented with 30 g / L sucrose.

[0179] - Wash the culture (v / v) in tryptone-salt solution (tryptone 1 g / L, NaCl 8.5 g / L) as follows: centrifuge the culture at 4000 rpm for 5 minutes; resuspend the precipitate in 10 ml tryptone-salt solution;

[0180] - Inoculate the washed culture at 1% (v / v) into 150 ml of M17 oxoid supplemented with 30 g / L galactose;

[0181] - The inoculated culture medium was incubated at 43°C for 24 hours, and its pH was monitored using a CINAC system (Alliance Instruments, France; Mettler 405DPAS SC pH electrode, Toledo, Spain); pH was measured and recorded every 5 minutes. The following parameters were specifically calculated using CINAC v2.07 software: the time to reach pH 5.2 and the slope (pH / min) between pH 6.4 and pH 5.6 [slope pH 6.4–5.6].

[0182] It is desired that the test strain can grow not only on galactose-containing medium. Furthermore, the strain must be able to reach pH 5.2 in the shortest possible time, i.e., optimally consume the galactose in the medium within an industrially acceptable production time. Therefore, by determination I, it is considered that galactose-positive thermophilic streptococcal strains meeting the following conditions are suitable for this invention:

[0183] - When the I test is passed, pH 5.2 is reached in less than 5 hours (i.e., the pH of the inoculated medium is reduced to 5.2 in less than 5 hours when the I test is passed); and

[0184] - Optionally, when measured by test I, the average acidification rate is at least 0.01 pH / min between pH 6.4 and 5.6.

[0185] Example 4: Selection of galactose-positive thermophilic streptococci based on galactose release (Assay II)

[0186] Measurement II is as follows:

[0187] - A thermophilic streptococcal strain characterized as galactose-positive was grown in M17 supplemented with 0.5% (wt / vol) lactose at 42°C for 12 hours [1% (v / v) inoculation]; this step was repeated a second time under the same conditions;

[0188] - Inoculate the culture at 1% (v / v) into M17 medium supplemented with 0.5% (wt / vol) lactose and incubate the inoculated medium at 42°C for 10 hours;

[0189] - During fermentation, samples were taken every 30 minutes to determine galactose concentration; samples were centrifuged at 14000 x g for 5 minutes and passed through a Phenex nylon 0.45 pm pore size x 15 mm diameter filter. Filter sterilize and store at -20°C until further analysis; 10 ml of each sample was injected onto a 100 HPLC system. Elution was performed using pure H₂O at a constant composition solvent mode at 0.6 ml / min. Within 40 minutes, Pb... 2+ Ion exchange column (SP0810 Shodex) TM Separate the sugar on a 300mm x 8mm x 7pm microscope. Determine the concentration of galactose (if present) in g / L. Galactose concentrations below 0.05 g / L are considered unmeasurable.

[0190] In addition to the time required to reach pH 5.2, or as an alternative to the time required to reach pH 5.2, among galactose-positive strains, the following strains are expected to provide interesting behavior in terms of galactose catabolism into dairy substrates: either, by assay II, they cannot secrete galactose; or, by assay II, they are able to secrete galactose into the culture medium but are able to completely consume it. Therefore, by assay II, galactose-positive thermophilic streptococcal strains that meet the following conditions are considered suitable for the present invention:

[0191] - No measurable amount of galactose was detected when tested by assay II; or

[0192] - When tested by assay II, the strain secretes galactose but consumes it up to 9 hours after inoculation (i.e., consumes it to a level below measurable). sequence list <110> K.H.S. Ltd. <120> Lactic acid bacteria strains with improved texture properties <130> P7512EP00 <160> 1 <170> BiSSAP 1.3.6 <210> 1 <211> 1642 <212> DNA <213> Streptococcus thermophilus <400> 1 agaaagtatg atctgcttct acaccaaaaa cttctttaaa cttttctctt aactgtgatg 60 tattcataga atgtatctca cttatcttt ttgtttatac tgaaattgta accactttca 120 catggaaaat caatattttt agatactgtc aataattatg tgtaaacact caagtagagt 180 tgaagaatgt taatcaaata agctttcaag tgtgtcagca cattggccaa accctttatg 240 gatgcgttga ttttgcttga aattataatc ttcaaacagg gtaactaaat aacgttccag 300 agcctcctcg ttaggaaaaa gaaccttctt tttcgtttga cgtttgattt ctttgttaag 360 agactcaatg aggtttgtcg aataaatgct atgccaaatc tggtagggaa actgataaaa 420 agttaaaaga ttatccgtat tctccagact ttccatgact ttcctatact ttggtttcca 480 ttcggcgata aagttctcta aagcttgcac tgccatttct aaattttcag cacgataaat 540 cgttttaaat tgctccagaa taaccgctct atctgctcgt ttcactttac tagctagatt 600 tcgactaata tgaattaagc aacgttgttg tttagctaat gggtaagcct gattgataat 660 ctcttcaagc cccttgaagc catcggtcac tacaagaagaa acctgttgga ttccttggtt 720 ttgaagcttg tctaacaggg tggaccaaga agcattgttt tcatttgggg cgatttcata 780 tccaagaaca gccttctgtc cttctggtgt aatgccaagt gcgatatgaa tacattcttt 840 actaacggtt ccacgtctta atggaagata ggttccgtca agaaataaaa cagagtaatt 900 ggcttctaag cttcgctcat gaaaagtagc gacattctcc tgagttgctt ttgagatatt 960 agaaattgtg gcaggactat agtgatgacc atacattcgc tcgatgatat cactaatttc 1020 tcgagtcgtt acaccggtttt gatagagttt gataaccatc tcttccaagt ggtcatctcg 1080 acgtccataa gcgggaagca aagctggact aaagttccca ttacgatctc taggaatact 1140 caactgaaca gtcccatatt tggtttcgaa tttccgtgca tagcttccgt tacgactatt 1200 cccagaatta tagcctaatt tatcgtaagg ttcataccct aaaaaggctg ataactctgc 1260 ttgaagcaga tcattcatag ctgtttcaag agaagtacgg aaaaattcat caatatcttg 1320 cttttgggct aggaagttaa gtagttctgt ggtaaactga gtcataggaa taaatctctt 1380 tctagtaatg ttttgcaact ctactataac ggatttattc cttttgtgt ttacacaact 1440 tattttacac tacctatttt attttttag taaaatatag gtaaaaaata aaagttatgt 1500 tatactgaaa tatgaggagg atacttatggc tacattagca gatatcgcaa aattagcagg 1560 tgtatctatt tcaactgttt cacgtgttct tataaagat gaaactcttt ccgtaacaga 1620 ggatactaga catcggatat ta 1642

Claims

1. A thermophilic streptococcus ( Streptococcus thermophilus The strain is derived from a galactose-negative parent strain having a transposase insertion element in the promoter region of the galactose operon, wherein the thermophilic streptococcal strain becomes galactose-positive due to the lack of the transposase insertion element, wherein the parent strain is DSM 17876, and wherein the transposase insertion element is located in the promoter region sequence of the galactose operon, the transposase insertion element corresponding to nucleotides between 133 and 1460 of SEQ ID No:

1.

2. The thermophilic streptococcal strain according to claim 1, wherein the promoter region sequence of the galactosooperon corresponds to nucleotides 105-133 and 1460-1491 of SEQ ID No:

1.

3. The Streptococcus thermophilus strain according to any one of claims 1-2, wherein the transposase insertion element sequence corresponds to nucleotides 134-1459 of SEQ ID No:

1.

4. The thermophilic streptococcal strain according to any one of claims 1-2, wherein the thermophilic streptococcal strain is DSM33677.

5. The thermophilic streptococcal strain according to claim 4, wherein strain DSM 33677 is galactose-positive.

6. A composition comprising Streptococcus thermophilus strain DSM33677 as defined in any one of claims 4-5.

7. The composition according to claim 6, comprising, in the form of a mixture or kit, i) *Streptococcus thermophilus* strain DSM 33677 as defined in any one of claims 4-5; and ii) *Lactobacillus delbrueckii* subsp. *bulgaricus* (…). Lactobacillus delbrueckii subsp bulgaricus ) strains.

8. The composition of claim 6, comprising, in the form of a mixture or kit, a combination of Streptococcus thermophilus strain DSM 33677 as defined in any one of claims 4-5 with Streptococcus thermophilus strain DSM 22935 and Lactobacillus delbrueckii subsp. bulgaricus strain DSM 22586.

9. The composition of claim 6, comprising, in the form of a mixture or kit, *Streptococcus thermophilus* strain DSM 33677 and *Streptococcus thermophilus* strain DSM 22935, *Lactobacillus delbrueckii* subsp. *bulgaricus* strain DSM 22586, and selected from *Bifidobacterium* (…). Bifidobacterium ), Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus A combination of at least one lactic acid bacteria in a group consisting of ) and combinations thereof.

10. A method for producing dairy products, comprising fermenting a substrate with a Streptococcus thermophilus strain DSM 33677 as defined in any one of claims 4-5 or a composition of any one of claims 6-9.

11. A fermented product that can be obtained by the method according to claim 10.

12. The fermented product according to claim 11, wherein the fermented product is a dairy product.

13. A fermented product comprising Streptococcus thermophilus strain DSM33677 as defined in any one of claims 4-5.

14. Use of the thermophilic streptococcal strain DSM 33677 as defined in any one of claims 4-5 in the preparation of fermented products.

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