New uses for lactic acid bacteria

Abstract

The present invention relates to the specific use of lactose-positive Streptococcus thermophilus strains in cheese production. The strains used according to the invention can carry one or more mutations in the glcK gene encoding glucokinase (which mutations result in reduced glucokinase activity), a mutation in the ccpA gene, and / or a mutation in at least one gene encoding a protein of the mannose-glucose-specific PTS. The invention further relates to a composition comprising the strains used according to the invention in combination with at least one strain of the genus Lactobacillus and / or a strain of mesophilic lactic acid bacteria, a method for producing cheese, and the cheese obtained by this method.

Description

[Technical Field]

[0001] The present invention relates to the specific use of lactose-positive Streptococcus thermophilus strains in cheese production. The strains used according to the invention can carry one or more mutations in the glcK gene encoding glucokinase (which mutations result in reduced glucokinase activity), a mutation in the ccpA gene encoding catabolite control protein A (CcpA), and / or a mutation in at least one gene encoding a protein of the mannose-glucose specific PTS.

[0002] The present invention further relates to a composition comprising the strain of the invention used according to the invention in combination with at least one strain of the genus Lactobacillus and / or one strain of mesophilic lactic acid bacteria, a method for producing cheese, and cheese obtainable by this method. [Background technology]

[0003] The food industry uses bacteria to improve the taste and texture of food or feed products. In the dairy industry, for example, lactic acid bacteria are commonly used to cause the acidification of milk (by fermenting lactose) and to provide texture to products in which they are incorporated. For example, lactic acid bacteria of the species Streptococcus thermophilus (S. thermophilus) are widely used, alone or in combination with other bacteria, in the production of fresh fermented dairy products such as cheese or yogurt. Summary of the Invention [Problem to be solved by the invention]

[0004] One challenge in the production of semi-hard cheeses is that commonly used starter cultures are primarily 100% mesophilic cultures, such as Lactococcus lactis (Lc. lactis), which only consume a portion of the lactose. As a result, residual lactose present in the cheese promotes the growth of undesirable microorganisms. This, in turn, can lead to undesirable post-acidification, undesirable taste, flavor, and texture issues, and increased proteolysis, reducing shelf life. Existing solutions include adding a curd washing step to the cheese process to limit residual lactose in the cheese, resulting in increased cheesemaking process times and the use of large amounts of water and energy. [Means for solving the problem]

[0005] The present invention has addressed these problems by providing new specific uses for lactose-positive S. thermophilus strains. DETAILED DESCRIPTION OF THE INVENTION

[0006] Starter cultures used in the production of semi-hard cheeses are mainly 100% mesophilic cultures such as Lc. lactis, which only consume a portion of the lactose, and as a result, the residual lactose present in the cheese promotes the growth of undesirable microorganisms, which can lead to undesirable post-acidification, undesirable taste, flavor, texture and the development of proteolysis, reducing shelf life.

[0007] The existing solution consists in adding a curd washing step to the cheese process to limit residual lactose in the cheese, which results in: - The curd washing step adds time to the cheese making process, - The curd washing step uses a lot of water and energy (to heat the water), which is expensive for the cheese maker and bad for the environment.

[0008] The present invention has addressed this problem in the art by providing a solution in terms of the use of specific mutations in Streptococcus thermophilus strains that alter sugar metabolism, particularly modifications that increase lactose consumption.

[0009] Thus, in a broad aspect, the present invention relates to the use of lactose-positive Streptococcus thermophilus strains in the manufacture of cheese, for example continental cheese, such as semi-hard or soft cheese.

[0010] In a first aspect, the present invention relates to the use of a lactose-positive Streptococcus thermophilus strain in the manufacture of cheese, such as semi-hard or soft cheese, wherein the strain is present at a concentration of 1% v / v or about 10% v / v as measured by Test 2 described herein. 9 When grown in raw whole milk inoculated with Streptococcus thermophilus strains at 1 CFU / ml, they have the ability to consume at least approximately 35% of the lactose.

[0011] In a second aspect, the present invention relates to a method for producing a lactic acid bacteria comprising at least one other strain of the genus Lactobacillus, such as Lactobacillus delbrueckii subsp. bulgaricus, and / or a mesophilic lactic acid bacterium, for example a species of the genus Lactococcus, such as a strain of Lactococcus lactis subsp. cremoris, a strain of Lactococcus lactis subsp. hordinae, or a species of Lactococcus lactis, such as Lactococcus lactis subsp. lactis, or a strain of the genus Bifidobacterium, or Leuconostoc mesenteroides. The present invention relates to a composition comprising at least one lactose-positive Streptococcus thermophilus strain as described according to the present invention in combination with a strain of the genus Leuconostoc, such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

[0012] In a third aspect, the present invention relates to a method for producing cheese, such as semi-hard and / or soft cheese, comprising the steps of inoculating a milk substrate with a Streptococcus thermophilus strain as defined herein, or a composition according to the invention, and fermenting the inoculated milk to obtain cheese.

[0013] In a further aspect, the present invention relates to a method for eliminating or reducing the need for a delactosizing step, such as a curd washing step or any other lactose dilution, in a cheese making process, such as semi-hard cheese making and / or soft cheese making, comprising the step of inoculating and fermenting a dairy substrate with a Streptococcus thermophilus strain as defined herein, or a composition according to the invention.

[0014] In a further aspect, the present invention relates to a cheese, such as a semi-hard cheese and / or a soft cheese, comprising at least one Streptococcus thermophilus strain as defined herein, a composition according to the invention or obtainable by a method according to the invention.

[0015] As used herein, "cheese" refers to a dairy product in its ordinary sense, comprising proteins and fats from milk, usually cow's, buffalo, goat's, camel's, or sheep's milk, produced in a wide variety of flavors, textures, and forms by coagulation of the milk protein casein, and produced by a process in which the milk is usually acidified to coagulate the casein, after which the solid curd is separated from the liquid whey and pressed into finished cheese. Some cheeses have aromatic molds on the rind, outer layer, or throughout. Specific examples of fresh or aged cheeses can be any selected from the group consisting of natural cheese, mozzarella, fresh cheese, Camembert, Berg, semi-hard cheeses such as Gouda, Edam, Jarlsberg, Maasdam, Claret, Cheddar, and Tilsit, hard cheeses such as Emmental, Parmesan, and Gruyère, and Appenzeller. Semi-hard cheese refers to cheeses that have a firm, slightly elastic texture but are dense, and are often produced by pressing curds into a solid and draining the remaining whey from the solid during the process. "Soft cheese" is used in its traditional sense to refer to cream cheeses such as Brie and Neuchâtel. As used herein, "soft cheese" also includes semi-soft cheeses such as high-moisture cheeses, including Havarti, Munster, Port Salut, and Butterkäse.

[0016] The "curd washing step" or "curd washing" is a step in the normal conventional process for the production of cheese, such as semi-hard cheese, to reduce the residual lactose content and thereby reduce the potential formation of excessive levels of lactic acid. The curd washing step is necessary to reduce the amount of residual lactose in the curd (also known as "pre-brined cheese") upon fermentation for 6 hours. This step in the cheesemaking process is also called "delactosing" or the delactosizing step. The term "delactosing" can also refer to any step of lactose dilution by adding water during milk standardization or during the cheesemaking process, for example, diafiltration, dilution of concentrated milk, and curd washing.

[0017] Curd washing generally refers to a specific part of the process applied to the production of cheeses such as Dutch-style cheeses, including Gouda, Edam, and Maasdamer, as well as some Swiss / Dutch-style cheeses, such as Samsoe, Havarti, and Dambo. It generally involves removing a portion of the whey (30–45% of the milk volume) after cutting and replacing it with warm water (55–60°C). This is a means of diluting the lactose content in the moisture phase of the cheese curd. The whey is partially pumped out, and the curd is washed by adding water (10–50% of the total volume). This water addition helps remove lactose from the curd. The energy and environmental costs of curd washing are significant for cheesemakers. Indeed, the increased volume of water heated and the volume of whey processed are prohibitively expensive. Avoiding the curd washing step would be beneficial from both an economic and environmental standpoint.

[0018] Some embodiments herein refer to a "variant sequence" of a strain as defined herein that has at least 90%, or at least 95% sequence identity with respect to the particular SEQ ID NO referred to, or with the genomic sequence of the parent strain from which the variant sequence is derived, with or without a particular mutation or insertion of the strain, and in particular has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98%, or at least 99.99% identity with the particular SEQ ID NO referred to, or with the genomic sequence of the parent strain from which the variant is derived. Identity is stated when comparing two sequences over their entire length (global alignment) and can be calculated using any program based on the Needleman-Wunsch algorithm.

[0019] In some embodiments, the present invention relates to a method for producing a mannose-glucose specific PTS comprising the steps of: 1) one or more mutations in the glcK gene encoding glucokinase; 2) one or more mutations in the ccpA gene encoding catabolite control protein A (CcpA), the mutations being selected from nonsense mutations or mutations resulting in a frameshift in the open reading frame of the ccpA gene; 3) at least one of the genes encoding proteins of the mannose-glucose specific PTS, such as the manL gene, the manM gene, and the manN gene (the mutations are selected from the group consisting of the IIAB gene, ... man , IIC man or IID man1) one or more mutations in the glcK gene encoding glucokinase and at least one gene encoding a protein of the mannose-glucose specific PTS; 2) one or more mutations in the glcK gene encoding glucokinase and at least one gene encoding a protein of the mannose-glucose specific PTS; 3) any combination thereof, for example, one or more mutations in the glcK gene encoding glucokinase and at least one gene encoding a protein of the mannose-glucose specific PTS; 4) any combination thereof, for example, one or more mutations in the glcK gene encoding glucokinase and at least one gene encoding a protein of the mannose-glucose specific PTS; 5) one or more mutations in the ccpA gene encoding catabolite control protein A (CcpA) and at least one gene encoding a protein of the mannose-glucose specific PTS; 6) one or more mutations in the glcK gene encoding glucokinase and the ccpA gene; and 7) one or more mutations in the glcK gene encoding glucokinase and a gene encoding a protein of the mannose-glucose specific PTS and the ccpA gene encoding catabolite control protein A (CcpA).

[0020] In some embodiments, the lactose-positive Streptococcus thermophilus strain carries a mutation in one gene encoding a protein of the mannose-glucose-specific PTS. In one embodiment, the gene encoding a protein of the mannose-glucose-specific PTS is selected from the group consisting of the manL gene, the manM gene, the manN gene, and the manO gene. In one embodiment, the gene encoding a protein of the mannose-glucose-specific PTS is selected from the group consisting of the manL gene, the manM gene, and the manN gene. In one embodiment, the lactose-positive Streptococcus thermophilus strain carries a mutation in one gene selected from the group consisting of the manL gene, the manM gene, and the manN gene. In one embodiment, the lactose-positive Streptococcus thermophilus strain carries mutations in two genes selected from the group consisting of the manL gene, the manM gene, and the manN gene. In one embodiment, the lactose-positive Streptococcus thermophilus strain carries mutations in the manL, manM, and manN genes. In some embodiments, the lactose-positive Streptococcus thermophilus strain carries a mutation in only one gene encoding a protein of the mannose-glucose-specific PTS, the manM gene, as described in the Examples of WO 2019122365, WO 2019197051, and EP 22159955.8.

[0021] Lactose overconsumption assay: To assess the effect of a particular mutation on lactose overconsumption, the following assay can be used, in which the engineered strain and an appropriate control strain are used to ferment milk as described in Test 2 of Example 2.

[0022] Therefore, the amount of lactose can be measured by HPLC (high-performance liquid chromatography) on a Vanquish system provided by Thermo Fisher Scientific, equipped with a refractive index detector. Five grams of inoculated milk before incubation (TO sample) and five grams of inoculated milk after incubation (sample T24h) or five grams of curd / cheese are diluted in 25 mL of 0.025N sulfuric acid solution (or, in the case of cheese samples, ground at 13,500 rpm with an IKA Ultra-Turrax). After homogenization, the sample is centrifuged at 4,600 rpm for 10 minutes, and the supernatant is filtered through a nylon 0.2 μm filter into an HPLC vial. Five microliters of sample are injected onto an H+ ion-exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm × 7.8 mm). Elution is performed in isocratic mode with 0.025N sulfuric acid at 0.7 mL / min. The molecules are separated in 20 minutes at 40°C. Carbohydrates are detected by refractometry at 35°C.

[0023] In some embodiments, the lactose-positive Streptococcus thermophilus strain used in accordance with the present invention does not carry any mutations in a gene selected from a gene encoding a protein of the mannose-glucose specific PTS and the ccpA gene encoding catabolite control protein A (CcpA).

[0024] For the avoidance of doubt, the species Streptococcus thermophilus should be understood as the strain Streptococcus salivarius subsp. thermophilus.

[0025] The term "lactose positive" refers to a Streptococcus thermophilus strain that can grow on lactose as the sole carbohydrate source, particularly on M17 medium supplemented with 3% lactose. In one embodiment, "lactose positive" refers to the phenotype of a Streptococcus thermophilus strain that, when assayed by inoculating an overnight culture of the S. thermophilus strain being tested at 1% into M17 broth containing 3% lactose and incubating at 37°C for 20 or 24 hours, achieves a pH of 5.5 or less at the end of incubation.

[0026] The term "galactose-negative" refers to a Streptococcus thermophilus strain that cannot grow on galactose as the sole carbohydrate source, specifically on M17 medium supplemented with 2% galactose. In a specific embodiment, the "galactose-negative" phenotype is assayed by inoculating M17 broth containing 2% galactose with 1% of an overnight culture of the S. thermophilus strain being tested and incubating at 37°C for 20 hours; a pH of 6 or higher at the end of incubation indicates a galactose-negative phenotype.

[0027] The expression "derivative" (e.g., a DSM28255 derivative) in reference to a parent strain means a strain obtained from the original strain (e.g., from the DSM28255 strain) by replacing one of its genes (e.g., glcK, manM, ...) with another allele of the same gene (in particular an allele carrying some specific mutation). In one embodiment, a derivative is obtained by replacing a gene and its promoter of the original strain with another allele of that gene and promoter. In one embodiment, a derivative is obtained by replacing all or part of the coding sequence of a gene of the original strain with another allele of that gene or part thereof.

[0028] For embodiments involving glcK sequencing, transferring the glcK allele of a suitable strain into the genome of other S. thermophilus strains, screening and selecting Streptococcus thermophilus strains for related traits, identifying relevant mutations in the glcK gene, identifying and using additional glcK mutations such as the non-conservative amino acid difference G144S, or identifying and using specific mutations in the glcK gene such as the non-conservative amino acid difference E275K in the GlcK sequence, see co-pending international patent applications WO 2019122365 and WO 2019197051, and European Patent Application No. 22159955.8.

[0029] A derivative of strain DSM28255 was engineered in which the glcK gene encodes glucokinase and the glutamic acid (E) at position 275 was replaced with the amino acid lysine (K). This derivative (DGCC12534) was deposited at DSMZ on August 15, 2017, under accession number DSM32587 (also referred to herein as ST21-G). The sequence of the GlcK protein is as defined in SEQ ID NO: 22.

[0030] Lactic acidification performance The acidification properties of the S. thermophilus strains used according to the invention can be assessed by recording the pH over time during milk fermentation as described in the Milk Acidification Assay as follows: Raw whole milk, previously pasteurized at 74°C for 1 minute, is inoculated with 1% (v / v, approximately 10%) of a culture of the S. thermophilus strain to be assayed (an overnight culture grown in semi-skimmed milk 10% BBA inoculated at 1% from a frozen vial and heated at 120°C for 20 minutes). 9 The inoculated milk flasks were inoculated with 10 ...

[0031] In some embodiments, the strain used in accordance with the present invention comprises a mutation in a gene encoding a protein of the mannose-glucose-specific PTS that, alone or in combination with mutations in other genes, confers on the bacterium the ability to overconsume lactose from the medium. In some embodiments, the mutant glcK gene encodes glucokinase, and glucokinase activity in the strain is significantly reduced, but not zero. In some embodiments, the lactose-positive Streptococcus thermophilus strain possesses a mutation in a gene encoding a protein of the mannose-glucose-specific PTS that confers on the bacterium the ability to overconsume lactose from the medium, and possesses a mutation in the glcK gene encoding glucokinase such that glucokinase activity in the strain is significantly reduced, but not zero. In any of these embodiments, the gene encoding a protein of the mannose-glucose-specific PTS may be selected from the group consisting of the manL gene, the manM gene, and the manN gene.

[0032] In some embodiments, a mutation in a gene encoding a protein of a mannose-glucose-specific PTS, alone or in combination with mutations in other genes, results in the ability to overconsume lactose from the medium. In some embodiments, the mutated glcK gene encodes glucokinase, and glucokinase activity in the strain is significantly reduced, but not zero, in the strain. In some embodiments, the lactose-positive Streptococcus thermophilus strain harboring a mutation in the glcK gene also harbors a mutation in the ccpA gene.

[0033] The following sections describe mutations in the glcK gene, mutations in genes encoding proteins of the mannose-glucose specific PTS (such as mutations in the manL, manM and manN genes), and mutations in the ccpA gene, respectively.

[0034] Although these mutations are disclosed separately herein (for clarity), any embodiment of one moiety can be combined with any embodiment of another moiety or with any embodiment of two other moieties to design lactose-positive Streptococcus thermophilus strains as defined herein.

[0035] For the avoidance of doubt, in some embodiments the present invention relates to the use of a lactose-positive Streptococcus thermophilus strain as defined herein, said strain being 1) one or more mutations in the glcK gene encoding glucokinase that result in a decrease in glucokinase activity; or 2) one or more mutations in the glcK gene encoding glucokinase, which mutations lead to a decrease in glucokinase activity, and a mutation in at least one, particularly one, gene encoding a protein of the mannose-glucose specific PTS; or 3) one or more mutations in the glcK gene encoding glucokinase, which mutations lead to a decrease in glucokinase activity, and at least one, in particular one, gene encoding a protein of the mannose-glucose specific PTS, and a mutation in its ccpA gene; or 4) one or more mutations in the glcK gene encoding glucokinase, which mutations lead to a decrease in glucokinase activity, and a mutation in its ccpA gene; 5) one or more mutations in at least one, in particular one, gene encoding a protein of the mannose-glucose specific PTS and a mutation in its ccpA gene; 6) one or more mutations in at least one, in particular one, gene encoding a protein of the mannose-glucose-specific PTS; 7) one or more mutations in its ccpA gene; and each strain carrying such a mutation gains the ability to overconsume lactose.

[0036] Mutation in the glcK gene This section describes mutations in the glcK gene affecting the glucose transporter that can be used in the context of lactose-positive Streptococcus thermophilus strains used according to the present invention, either alone, in combination with one or more mutations in genes encoding proteins of the mannose-glucose specific PTS, in combination with one or more mutations in the ccpA gene defined herein, or in combination with both one or more mutations in genes encoding proteins of the mannose-glucose specific PTS defined herein and one or more mutations in the ccpA gene defined herein.

[0037] In some embodiments, the mutant glcK gene of the strain used in accordance with the invention encodes glucokinase, and the glucokinase activity in the strain is significantly reduced, but not zero. Indeed, the inventors have provided evidence that several mutant alleles of the glcK gene encode glucokinase (GlcK), and that the glucokinase activity is significantly reduced, but not zero, when the mutant glcK gene is present in lactose-positive Streptococcus thermophilus strains.

[0038] The phrase "glcK gene encoding glucokinase" refers to any DNA sequence of a Streptococcus thermophilus strain that encodes the glucokinase enzyme, which catalyzes the conversion of glucose and ATP to glucose-6-phosphate (G6P) and ADP. Non-limiting examples of Streptococcus thermophilus glucokinase sequences are disclosed as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.

[0039] In some embodiments within the present invention, glucokinase activity in a Streptococcus thermophilus strain is significantly reduced, but not zero, as a result of a mutation in the glcK gene thereof, i.e., the allele of the glcK gene carried by the strain is such that glucokinase activity in the strain is significantly reduced, but not zero.

[0040] The phrase "glucokinase activity is reduced, but not zero" or "glucokinase activity in said strain is significantly reduced, but not zero" refers to a strain in which the glucokinase activity is both: - the glucokinase activity is significantly reduced in said strain, in particular compared to a strain carrying a non-mutated glcK gene; and - non-zero, i.e. activity is detectable by the glucokinase activity assay as defined herein.

[0041] According to embodiments of the present invention, the characteristic that "the glucokinase activity in the strain is significantly reduced, but not zero," can be determined by methods well known in the art. Accordingly, methods for measuring glucokinase activity in Streptococcus thermophilus strains are known and include enzyme assays using commercially available reagents. Reference is made herein to paragraph 2.4 of Pool et al. (2006. Metabolic Engineering 8(5); 456-464), incorporated herein by reference. The method may be as disclosed in Pool et al. (2006.5:456-464, incorporated herein by reference). In certain embodiments, glucokinase activity in Streptococcus thermophilus strains used in accordance with the present invention is assayed by a glucokinase activity assay (i.e., a glucokinase activity assay is performed using the Streptococcus thermophilus strain used in accordance with the present invention).

[0042] Glucokinase activity assay: A fresh overnight culture of the Streptococcus thermophilus strain to be assayed in M17 containing 30 g / L lactose was obtained and used at 1% (vol / vol) to inoculate 10 ml of fresh M17 30 g / L lactose. Cells were harvested by centrifugation (6000 g, 10 min, 4°C) at an optical density at 600 nm (OD600) of 0.8 ± 0.2, washed with 5 ml of cold GLCK buffer (5 mM MgCl2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]), and resuspended in 500 μl of cold GLCK buffer. EDTA-free protease inhibitor "cOmplete™" (Roche, supplier reference 04693132001) was added to the GLCK buffer as described by the supplier. Cells were disrupted by adding 100 mg of glass beads (150–212 μm, Sigma G1145) to 200 μl of resuspended cells and shaking for 6 min at 30 cycles / s in an MM200 vibration mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14,000 g, 15 min, 4°C), and the supernatant was transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content was determined using the FLUKA Protein Quantification Kit-Rapid (ref. 51254). Glucokinase activity in cell extracts was determined spectrophotometrically by the glucose-6-phosphate dehydrogenase (G-6PDH, EC 1.1.1.49):NADPH binding assay (Porter et al., 1982), essentially as described by Pool et al. (2006). Each sample (5, 10, and 20 μL) was added to a final volume of 250 μL of assay buffer (10 mM KHPO / KHPO [pH 7.2], 5 mM MgCl, 1 mM ATP, 20 mM glucose, 1 mM NADP, 1 U G-6PDH), and the mixture was left at 30°C for 5 min. The optical density at 340 nm was measured for 5 min using a Synergy HT multidetection microplate reader (BIO-TEK).One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 μmole of D-glucose to D-glucose 6-phosphate per minute under assay conditions. Glucokinase activity is calculated as follows: Glucokinase activity (U / g of total protein extract) = dOD × V / [dt × l × ε × Qprot] (In the formula, - dOD is the variation of optical density (OD) at 340 nm - V is the volume of the reaction (here 250 μL) dt = measurement time (min) l = optical path length (here 0.73 cm) ε = molar extinction coefficient of NADPH; H + (Here, 6220 cm2 / μmol) Qprot = amount of protein in the cuvette (g) Measurements were performed in triplicate for each sample, and the glucokinase specific activity values ​​presented herein under Glucokinase Activity Assay are the average of three independent experiments.

[0043] In a first particular embodiment of the feature "the glucokinase activity in said strain is significantly reduced but not zero," the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is 200-1500 U / g of total protein extract, as assayed by a glucokinase activity assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is 300-1200 U / g of total protein extract, as assayed by a glucokinase activity assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is 400-1000 U / g of total protein extract, as assayed by a glucokinase activity assay. In certain embodiments, the glucokinase activity of a Streptococcus thermophilus strain used in accordance with the present invention, as assayed by a glucokinase activity assay, is between a minimum value selected from the group consisting of 200, 300, and 400 U / g of total protein extract and a maximum value selected from the group consisting of 1000, 1200, and 1500 U / g of total protein extract. As noted for the glucokinase activity assay, it is worth noting that the glucokinase activity values ​​disclosed herein are the average of three independent experiments (triplicates).

[0044] In a second specific embodiment of the feature "the glucokinase activity in the strain is significantly reduced but not zero," the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is 5 to 60% of the glucokinase activity of strain DGCC7710, deposited with the DSMZ on January 14, 2014, under accession number DSM28255. "Glucokinase activity of strain DSM28255" refers to the activity of glucokinase (i.e., SEQ ID NO: 2) in strain DSM28255, as assayed by a glucokinase activity assay in strain DSM28255 [i.e., the glucokinase activity assay is performed using strain DSM28255]. The percentage value is calculated based on the glucokinase activity in the strain used according to the invention and the glucokinase activity of strain DSM28255, both assayed by the glucokinase activity assay. In a specific embodiment, the glucokinase activity of the Streptococcus thermophilus strain used in accordance with the invention is 10 to 50% of the glucokinase activity of strain DSM 28255. In a specific embodiment, the glucokinase activity of the Streptococcus thermophilus strain used in accordance with the invention is 15 to 40% of the glucokinase activity of strain DSM 28255. In a specific embodiment, the glucokinase activity of the Streptococcus thermophilus strain used in accordance with the invention is between a minimum percentage selected from the group consisting of 5, 10, and 15% of the glucokinase activity of strain DSM 28255 and a maximum percentage selected from the group consisting of 40, 50, and 60% of the glucokinase activity of strain DSM 28255. Regardless of the particular embodiment and percentage range, the activity of glucokinase activity is assayed by a glucokinase activity assay as described herein.It is worth noting that the percentage values ​​disclosed herein are calculated based on glucokinase activity values ​​that are the average of three independent experiments (triplicates) assayed by glucokinase activity assay.

[0045] In the first and second particular embodiments, the following strains can be used as controls for the glucokinase activity assay: As a positive control (i.e. a Streptococcus thermophilus strain representative of strains carrying a non-mutated glcK gene): strain DGCC7710 deposited at the DSMZ on 14 January 2014 under the accession number DSM28255.

[0046] The feature "the glucokinase activity in the strain is significantly reduced, but not zero" can also be characterized by the maximum process velocity of glucokinase (referred to herein as Vmax, defined as the rate of conversion of glucose + ATP to G6P + ADP) or the reciprocal of the affinity of glucokinase for one or two of its substrates, i.e., glucose and ATP (referred to as Km). In some embodiments, the feature "the glucokinase activity in the strain is significantly reduced, but not zero" for a strain used according to the present invention is further characterized by the maximum process velocity (Vmax) of that glucokinase in the strain.

[0047] Thus, in combination with the first or second particular embodiment of the feature "the glucokinase activity in said strain is significantly reduced, but not zero" defined herein, the maximum forward velocity (Vmax) of glucokinase in lactose-positive Streptococcus thermophilus strains used according to the invention is significantly reduced, but not zero. The feature "the glucokinase Vmax of said strain is significantly reduced, but not zero" can be defined by one or two of these parameters: - When assayed by glucokinase Vmax assay, Vmax is 200-1500 U / g of total protein extract. - When both are assayed by the glucokinase Vmax assay, the Vmax is 5-60% of the Vmax of glucokinase of strain DSM28255 deposited at DSMZ on January 14, 2014 under accession number DSM28255.

[0048] In a particular embodiment, the mutated glcK gene of the lactose-positive Streptococcus thermophilus strain used according to the invention encodes glucokinase, and the glucokinase activity (as defined herein) in said strain is significantly reduced, but not zero, and the maximum processive velocity (Vmax) of that glucokinase in said strain is significantly reduced, but not zero, as defined by one or two of the following parameters: - When assayed by glucokinase Vmax assay, Vmax is 200-1500 U / g of total protein extract. - When both are assayed by the glucokinase Vmax assay, the Vmax is 5-60% of the Vmax of glucokinase of strain DSM28255 deposited at DSMZ on January 14, 2014 under accession number DSM28255.

[0049] The glucokinase maximum forward velocity (Vmax) in the Streptococcus thermophilus strains described herein is assayed by a glucokinase Vmax assay (performed using the Streptococcus thermophilus strains used in accordance with the present invention).

[0050] Glucokinase Vmax Assay: The maximum forward velocity (Vmax) was determined by using various concentrations of glucose (0, 5, 10, 15, and 20 mM) on crude extracts prepared as described in the glucokinase activity assay. Measurements were performed in triplicate for each sample, and the Vmax values ​​obtained under the glucokinase Vmax assay are the average of three independent experiments. A linear regression of the inverse of the specific velocity as a function of the inverse of the glucose concentration gives the inverse of the maximum forward velocity at the point of intersection with the Y-axis of the graph.

[0051] In certain embodiments, the maximum processive velocity of glucokinase in a Streptococcus thermophilus strain used according to the invention, as assayed by a glucokinase Vmax assay, is between 200 and 1500 U / g of total protein extract. In certain embodiments, the Vmax is between 300 and 1200 U / g of total protein extract. In certain embodiments, the Vmax is between 400 U / g and 1000 U / g of total protein extract. In certain embodiments, the Vmax of glucokinase in a Streptococcus thermophilus strain used according to the invention, as assayed by a glucokinase Vmax assay, is between a minimum value selected from the group consisting of 200, 300, and 400 U / g of total protein extract and a maximum value selected from the group consisting of 1000, 1200, and 1500 U / g of total protein extract.

[0052] In a specific embodiment of the maximum processive velocity of glucokinase in a Streptococcus thermophilus strain used in accordance with the present invention, the Vmax is 5-60% of the Vmax of glucokinase in strain DSM28255. "Vmax of glucokinase in strain DSM28255" refers to the Vmax of glucokinase in strain DSM28255 (i.e., SEQ ID NO: 2) as assayed by a glucokinase Vmax assay in strain DSM28255 [i.e., the glucokinase Vmax assay is performed using strain DSM28255]. The percentage value is calculated based on the Vmax of glucokinase in the strain used in accordance with the present invention and the Vmax of strain DSM28255, both assayed by a glucokinase Vmax assay. In certain embodiments, the glucokinase Vmax of a Streptococcus thermophilus strain used in accordance with the invention is 10-50% of the Vmax of the glucokinase of strain DSM 28255, when both are assayed by a glucokinase Vmax assay. In certain embodiments, the glucokinase Vmax of a Streptococcus thermophilus strain used in accordance with the invention is 15-40% of the Vmax of the glucokinase of strain DSM 28255. In certain embodiments, the glucokinase Vmax of a Streptococcus thermophilus strain used in accordance with the invention is between a minimum percentage selected from the group consisting of 5, 10, and 15% of the Vmax of the glucokinase activity of strain DSM 28255 and a maximum percentage selected from the group consisting of 40, 50, and 60% of the Vmax of the glucokinase activity of strain DSM 28255.

[0053] A lactose-positive Streptococcus thermophilus strain used in accordance with the present invention can carry one or more mutations in the glcK gene encoding glucokinase, such that glucokinase activity in said strain is significantly reduced, but not zero, as defined herein, and optionally the maximum processive velocity of glucokinase in said strain is significantly reduced, but not zero, as defined herein.

[0054] Within the present invention, a "mutation in the glcK gene" refers to any nucleotide mutation within the glcK gene, which mutation at the nucleotide level results in a significantly reduced, but non-zero glucokinase activity, as defined herein, in a strain carrying this mutant glcK gene (as the only glcK gene), and optionally results in a significantly reduced, but non-zero maximum processive velocity of glucokinase, as defined herein, in said strain. In a particular embodiment, within the present invention, a "mutation in the glcK gene" refers to any nucleotide mutation within the open reading frame of the glcK gene, which mutation at the nucleotide level results in a significantly reduced, but non-zero glucokinase activity, as defined herein, in a strain carrying this mutant glcK gene (as the only glcK gene), and optionally results in a significantly reduced, but non-zero maximum processive velocity of glucokinase, as defined herein, in said strain.

[0055] Thus, two Streptococcus thermophilus strains may differ by the sequence of their respective glcK genes, but this does not necessarily mean that one of these two glcK genes is mutated within the meaning of the present invention. Indeed, the following are not considered mutations within the scope of the present invention: - mutations at the nucleotide level that do not result in any changes at the protein level (silent mutations) and do not affect the translation of glcK RNA, and - a mutation at the nucleotide level, as defined herein, that results in a change at the protein level but that does not affect the glucokinase activity of the resulting GlcK protein and, optionally, the maximum processive velocity of the resulting GlcK protein. Indeed, such mutations can be observed at the level of the glcK gene of Streptococcus thermophilus described herein without affecting the extent of protection.

[0056] Non-limiting examples of glcK genes that are not considered mutated for use in accordance with the present invention are: a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 2, in particular as defined in SEQ ID NO: 1. This GlcK type is one of the strains DGCC7710 deposited at the DSMZ on January 14, 2014 under the accession number DSM28255, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 4, in particular as defined in SEQ ID NO: 3, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 6, in particular as defined in SEQ ID NO: 5, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 8, in particular as defined in SEQ ID NO: 7, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 10, in particular as defined in SEQ ID NO: 9, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 12, in particular as defined in SEQ ID NO: 11; a polynucleotide encoding glucokinase as defined in SEQ ID NO: 14, in particular as defined in SEQ ID NO: 13, a polynucleotide encoding glucokinase as defined in SEQ ID NO: 16, in particular as defined in SEQ ID NO: 15; a polynucleotide encoding glucokinase as defined in SEQ ID NO: 18, in particular as defined in SEQ ID NO: 17; a polynucleotide encoding glucokinase as defined in SEQ ID NO: 20, in particular as defined in SEQ ID NO: 19.

[0057] Furthermore, some nucleotide mutations in the glcK gene are not considered suitable for the present invention because they result in a glucokinase whose activity, when assayed by a glucokinase activity assay, is zero or less than the minimum activity defined herein. In some embodiments, the Streptococcus thermophilus described herein does not possess a mutation selected from the group consisting of a mutation that results in a knockout of the glcK gene and a large deletion in the glcK gene.

[0058] In some embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention carry a mutation in the open reading frame of the glcK gene that results in a substitution of an amino acid in the GlcK protein, such that glucokinase activity is significantly reduced (as defined herein) in said strains carrying the mutant glcK gene, but not zero, and optionally, the maximum processive velocity of glucokinase in said strains is significantly reduced (as defined herein) but not zero. In particular embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention carry a mutation in the glcK gene that results in a substitution of an amino acid in the GlcK protein, such that glucokinase activity is significantly reduced (as defined herein) in said strains carrying the mutant glcK gene, but not zero, and optionally, the maximum processive velocity of glucokinase in said strains is significantly reduced (as defined herein) but not zero. In certain embodiments, a Streptococcus thermophilus strain used in accordance with the invention carries a mutation in the glcK gene such that the GlcK protein is 322 amino acids in length, and glucokinase activity in said strain is significantly reduced, as defined herein, but not zero, and optionally the maximum processive velocity of glucokinase in said strain is significantly reduced, as defined herein, but not zero.

[0059] As described above, several DNA modifications that do not affect the glucokinase activity of the strain can be observed at the level of the glcK gene of Streptococcus thermophilus described herein. Based on the glucokinase activity assays defined herein, along with the control strains defined herein, one skilled in the art would know how to identify 1) a glcK gene encoding a glucokinase in which glucokinase activity (as defined herein) is significantly reduced, but not zero, in a strain carrying the glcK gene, and optionally, the maximum processive velocity of glucokinase (as defined herein) in a strain carrying the mutant glcK gene is significantly reduced, but not zero, in a strain carrying the modified glcK gene; 2) a glcK gene with a modification that does not affect glucokinase activity in a strain carrying the modification; or 3) a glucokinase-encoding glcK gene in which glucokinase activity (as defined herein) is zero in a strain carrying the modified glcK gene.

[0060] The inventors have identified two positions within glucokinase where the nature of the amino acids has been shown to affect the activity of glucokinase such that the glucokinase activity is significantly reduced, but not zero, as defined herein, and where the Vmax of glucokinase has been shown to be significantly reduced, but not zero, as defined herein: positions 144 and 275 of glucokinase (i.e., codons 144 and 275 of the glcK gene). It is worth noting that, based on the glucokinase activity assays and glucokinase Vmax assays defined herein, along with control strains, one skilled in the art would know how to identify other positions and suitable amino acids within glucokinase to obtain significantly reduced, but not zero, glucokinase activity (as defined herein) and possibly a significantly reduced, but not zero, maximum processive velocity, and therefore the corresponding glcK gene.

[0061] In some embodiments, the amino acid at position 275 of glucokinase (encoded by the glcK gene of a Streptococcus thermophilus strain used in accordance with the invention) is not glutamic acid (i.e., any amino acid other than glutamic acid). Thus, in some embodiments, codon 275 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is not GAA or GAG. In particular embodiments, the amino acid at position 275 of glucokinase is not an acidic amino acid (i.e., any amino acid other than an acidic amino acid). Thus, in some embodiments, codon 275 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is a codon encoding a non-acidic amino acid. In particular embodiments, the amino acid at position 275 of glucokinase is selected from the group consisting of lysine and any of its conservative amino acids. Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention is a codon encoding an amino acid selected from the group consisting of lysine and its conserved amino acids. In particular embodiments, the amino acid at position 275 of glucokinase is lysine. Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention is either AAA or AAG. In particular embodiments, nucleotides 823-825 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention are AAA or AAG.

[0062] In a particular embodiment, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 25, in which the amino acid at position 275 is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine; and b) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In a particular embodiment, the GlcK variant sequence is 322 amino acids in length.

[0063] In another embodiment, the amino acid at position 144 of glucokinase (encoded by the glcK gene of a Streptococcus thermophilus strain used in accordance with the invention) is not glycine (i.e., it is any amino acid except glycine). Thus, in some embodiments, codon 144 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is not GGT, GGC, GGA, or GGG. In particular embodiments, the amino acid at position 144 of glucokinase is not an aliphatic amino acid (i.e., it is any amino acid except an aliphatic amino acid). In particular embodiments, the amino acid at position 144 of glucokinase is selected from the group consisting of serine and any of its conserved amino acids. Thus, in some embodiments, codon 144 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is a codon encoding an amino acid selected from the group consisting of serine and any of its conserved amino acids. In certain embodiments, the amino acid at position 144 of glucokinase is serine. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention is AGT, AGC, TCT, TCC, TCA, or TCG. In certain embodiments, nucleotides 430-432 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention are AGT, AGC, TCT, TCC, TCA, or TCG.

[0064] In a particular embodiment, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine; and b) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid in glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. In a particular embodiment, the GlcK variant sequence is 322 amino acids in length.

[0065] For the definition of a GlcK variant having at least 90% similarity or identity to SEQ ID NO:25, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portions of the sequences). Position 275, as defined in SEQ ID NO:25, is not considered for the purpose of calculating similarity or identity. In certain embodiments, the GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% similarity or identity to SEQ ID NO:25, and the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO: 25, and the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO: 25, and the amino acid corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine.

[0066] In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by 1 to 30 amino acid substitutions, and the amino acid at position 275 of the GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, particularly lysine (position 275 is not considered for calculating the number of substitutions). In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by 1 to 20 amino acid substitutions, and the amino acid at position 275 of the GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, particularly lysine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by 1 to 15 amino acid substitutions, and the amino acid at position 275 of the GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, particularly lysine. In certain embodiments, the GlcK variant sequence differs from SEQ ID NO: 25 by 1 to 10 amino acid substitutions, and the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In certain embodiments, the GlcK variant sequence differs from SEQ ID NO: 25 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, and the amino acid at position 275 of the GlcK variant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine.

[0067] For the definition of a GlcK variant having at least 90% similarity or identity to SEQ ID NO: 46, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portions of the sequences). Position 144, as defined in SEQ ID NO: 46, is not considered for the purpose of calculating similarity or identity. In certain embodiments, a GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% similarity or identity to SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine.

[0068] In certain embodiments, a GlcK mutant sequence differs from SEQ ID NO: 46 by 1 to 30 amino acid substitutions, and the amino acid at position 144 of the GlcK mutant is any amino acid except glycine, particularly any amino acid except aliphatic amino acids, particularly serine (position 144 is not considered for calculating the number of substitutions). In certain embodiments, a GlcK mutant sequence differs from SEQ ID NO: 46 by 1 to 20 amino acid substitutions, and the amino acid at position 144 of the GlcK mutant is any amino acid except glycine, particularly any amino acid except aliphatic amino acids, particularly serine. In certain embodiments, a GlcK mutant sequence differs from SEQ ID NO: 46 by 1 to 15 amino acid substitutions, and the amino acid at position 144 of the GlcK mutant is any amino acid except glycine, particularly any amino acid except aliphatic amino acids, particularly serine. In certain embodiments, the GlcK variant sequence differs from SEQ ID NO: 46 by 1 to 10 amino acid substitutions, and the amino acid at position 144 of the GlcK variant is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. In certain embodiments, the GlcK variant sequence differs from SEQ ID NO: 46 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, and the amino acid at position 144 of the GlcK variant is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine.

[0069] In some embodiments, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, and the amino acid at position 275 of the variant is any amino acid except glutamic acid, particularly any amino acid except an acidic amino acid, and particularly lysine. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34, and the amino acid at position 275 of glucokinase is not glutamic acid, particularly not an acidic amino acid, and particularly lysine, respectively.

[0070] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase), as in SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (particularly SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34), is not glutamic acid. Thus, in some embodiments, codon 275 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is not GAA or GAG. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used according to the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34), wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not glutamic acid.

[0071] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase), as in SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (particularly SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34), is not an acidic amino acid. Thus, in some embodiments, codon 275 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is a codon that does not encode an acidic amino acid. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used according to the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34), wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not an acidic amino acid.

[0072] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) as set forth in SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (particularly SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34) is selected from the group consisting of lysine and any of its conserved amino acids. Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the present invention is a codon encoding an amino acid selected from the group consisting of lysine and any of its conserved amino acids. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used according to the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33, or 34), wherein the amino acid in glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is either lysine or a conserved amino acid thereof.

[0073] In particular embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) as set forth in SEQ ID NO: 25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25 defined herein (particularly SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, or 34) is lysine. Thus, in some embodiments, codon 275 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is a codon encoding lysine, particularly AAA or AAG, respectively. Thus, in particular, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is selected from the group consisting of SEQ ID NOs: 22, 35, 36, 37, 38, 39, 40, 41, 42, and 43. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 22, 35, 36, 37, 38, 39, 40, 41, 42, and 43. In a particular embodiment, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is as defined in SEQ ID NO: 21.

[0074] In another embodiment, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55, and the amino acid at position 144 of said variant is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55, and the amino acid at position 144 of glucokinase is not glycine, particularly not an aliphatic amino acid, and particularly serine.

[0075] In certain embodiments, the amino acid in glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase), as in SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (particularly SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55), is not glycine. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention is not GGT, GGC, GGA, or GGG. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used according to the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55), wherein the amino acid in glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is not glycine.

[0076] In certain embodiments, the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase), as either SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (particularly SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55), is not an aliphatic amino acid. Thus, in some embodiments, codon 144 of the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is a codon that does not encode an aliphatic amino acid. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used according to the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55), wherein the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is not an aliphatic amino acid.

[0077] In certain embodiments, the amino acid at position 144 of glucokinase corresponding to SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is selected from the group consisting of serine and its conserved amino acids, as in SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (particularly SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55). Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the present invention is a codon encoding an amino acid selected from the group consisting of serine and its conserved amino acids. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55), wherein the amino acid in glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is either serine or a conserved amino acid thereof.

[0078] In particular embodiments, the amino acid in glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is serine, as in SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (particularly SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, or 55). Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain used in accordance with the invention is a codon encoding serine, particularly AAA or AAG. Thus, in particular, the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is selected from the group consisting of SEQ ID NOs: 45, 56, 57, 58, 59, 60, 61, 62, 63, and 64. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 45, 56, 57, 58, 59, 60, 61, 62, 63, and 64. In a particular embodiment, the glcK gene carried by a Streptococcus thermophilus strain used in accordance with the invention is as defined in SEQ ID NO: 44.

[0079] When the sequence of the GlcK protein of a lactose-positive Streptococcus thermophilus strain used in accordance with the present invention is defined, the present teachings provide that the glucokinase activity in a strain expressing this GlcK protein is significantly reduced, as defined herein, but not zero, and optionally the Vmax of glucokinase in this strain is significantly reduced, as defined herein, but not zero.

[0080] Mutations in genes encoding proteins of the mannose-glucose specific PTS, in particular mutations in the manL, manM and manN genes. This section describes mutations in genes encoding proteins of the mannose-glucose-specific PTS, in particular mutations in the manL, manM and manN genes, that may be used in combination with a mutation in the glcK gene defined herein, or in combination with a mutation in the ccpA gene defined herein, or in combination with both a mutation in the glcK gene and a mutation in the ccpA gene defined herein, in the context of lactose-positive Streptococcus thermophilus strains used according to the invention.

[0081] In lactose-positive Streptococcus thermophilus strains, any mutation in a gene encoding a protein of the mannose-glucose-specific PTS is suitable and can be combined with a mutant glcK gene as defined herein, or with a mutation in the ccpA gene as defined herein, or with both a mutant glcK gene and a mutant ccpA gene as defined herein.

[0082] The present inventors have shown that mutations in genes encoding proteins of the mannose-glucose specific PTS, in particular the mutant manL gene, the mutant manM gene, or the mutant manN gene, that confer the ability to overconsume lactose from the medium in lactose-positive Streptococcus thermophilus strains, are particularly advantageous in the present invention.

[0083] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, is a mutation that results in the knockout (ie, complete disruption) of the gene.

[0084] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, particularly the manL gene, manM gene or manN gene, is a mutation in the promoter of the gene, particularly a mutation in the promoter of the gene that reduces or inhibits transcription of the gene.

[0085] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, manM gene or manN gene, is a mutation introduced into the coding sequence of the gene, in particular a mutation that results in the ability to overconsume lactose.

[0086] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, results in a truncated protein, in particular the truncated IIAB Man Protein, truncated IIC Man Protein or truncated IID Man Proteins, particularly truncated proteins (e.g., truncated IIAB) that confer the ability to overconsume lactose Man Protein, truncated IIC Man Protein or truncated IID Man In some embodiments, the mutation introduced into the gene is a nucleotide substitution that results in a stop codon, or a deletion, insertion, or deletion / insertion that results in a frameshift of the open reading frame and a premature stop codon. In some embodiments, the mutation introduced into the gene is a nucleotide substitution that results in a stop codon. In some embodiments, the mutation introduced into the gene is a deletion, insertion, or deletion / insertion that results in a frameshift of the open reading frame and a premature stop codon.

[0087] Two Streptococcus thermophilus strains may differ by the sequence of their respective manL, manM, or manN genes, but this does not necessarily mean that one of these genes is mutated within the meaning of the present invention. Indeed, what is not considered a mutation in a manL, manM, or manN gene relevant to the present invention is the following: - mutations at the nucleotide level that do not result in any changes at the protein level (silent mutations) and do not affect the translation of manL, manM or manN RNA, and - a mutation at the nucleotide level that results in a change at the protein level in the lactose-positive phenotype of a Streptococcus thermophilus strain, but which does not result in excessive lactose consumption when combined with a mutant glcK gene as defined herein, or when combined with a mutation in the ccpA gene as defined herein, or when combined with both a mutant glcK gene and a mutant ccpA gene.

[0088] The manL, manM and manN genes (IIAB, respectively) are not considered to be mutated as appropriate according to the present invention. Man Protein, IIC Man Proteins and IIDs Man Non-limiting examples of genes encoding proteins are: IIAB as defined in SEQ ID NO: 78 Man A polynucleotide encoding a protein, in particular the polynucleotide defined in SEQ ID NO: 77. Man The type is that of strain DSM28255. IIAB as defined in SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110 ManPolynucleotides encoding proteins, in particular polynucleotides defined in SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109. IIAB defined in SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110. Man The protein sequence is 98.4 to 99.6% identical to SEQ ID NO: 78. - IIC as defined in SEQ ID NO: 130 Man A polynucleotide encoding a protein, in particular the polynucleotide defined in SEQ ID NO: 129. Man The type is DSM28255. IICs as defined by SEQ ID NOs: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156 Man Polynucleotides encoding proteins, in particular polynucleotides defined in SEQ ID NOs: 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 and 155. IICs defined in SEQ ID NOs: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156. Man The protein sequence is 98.5-99.6% identical to SEQ ID NO: 130. - IID defined in sequence number 167 Man A polynucleotide encoding a protein, in particular the polynucleotide defined in SEQ ID NO: 166. Man The type is that of strain DSM28255. - IIDs defined by SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205 ManPolynucleotides encoding proteins, in particular polynucleotides defined in SEQ ID NOs: 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204. IIDs defined in SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205. Man The protein sequence is 97.3-99.6% identical to SEQ ID NO:167.

[0089] The present inventors have identified at least one mutation in the manL gene that, when inserted into the manL gene of an original lactose-positive Streptococcus thermophilus strain (mutated in the glcK gene, the ccpA gene, or both the glcK and ccpA genes as defined herein), enables the strain to overconsume lactose compared to the original strain when assayed by Test 2 in Example 2.

[0090] In some embodiments, the mutation in the manL gene is IIAB at position 305 Man In some embodiments, the mutation in the manL gene is a substitution of nucleotide G for nucleotide T at position 916 (resulting in a stop codon at position 306). Man The protein is referred to herein as IIAB Man 305 It is called.

[0091] In some embodiments, the IIAB Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 112, and b) IIAB, which has at least about 90%, for example at least 95%, similarity or identity to SEQ ID NO: 112, and is particularly 305 amino acids in length Man Mutant sequences.

[0092] IIAB having at least about 90%, e.g., at least 95%, similarity or identity to SEQ ID NO: 112 Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences). Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:112.

[0093] In certain embodiments, IIAB Man The variant sequence differs from SEQ ID NO: 112 by 1 to 30 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 112 by 1 to 20 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 112 by 1 to 15 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 112 by 1 to 10 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 112 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the IIAB sequence of the lactose-positive Streptococcus thermophilus strain used in accordance with the present invention Man The protein sequence is selected from the group consisting of SEQ ID NOs: 112-128.

[0094] In some embodiments, the manL gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is SEQ ID NO: 112 and any IIAB gene whose sequence has at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 112 as defined herein. Man IIAB selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 113 to 128) Man In some embodiments, the manL gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is as set forth in SEQ ID NO:111.

[0095] The present inventors have identified at least two mutations in the manM gene that, when each inserted into the manM gene of an original lactose-positive Streptococcus thermophilus strain (mutated in the glcK gene, the ccpA gene, or both the glcK and ccpA genes as defined herein), enable the strain to overconsume lactose compared to the original strain when assayed by Test 2 in Example 2.

[0096] In some embodiments, the mutation in the manM gene is IIC at position 208 Man In some embodiments, the mutation in the manM gene is a substitution of the nucleotide G for the nucleotide T at position 625 (resulting in a stop codon at position 209). Man Proteins are referred to herein as IIC Man208 It is called.

[0097] In some embodiments, the IIC truncated at position 208 Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 158, and b) IIC having at least 90% similarity or identity with SEQ ID NO: 158, in particular an IIC having a length of 208 amino acids Man Mutant sequences.

[0098] IIC with at least 90% similarity or identity to SEQ ID NO: 158 Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences). In certain embodiments, IIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:158.

[0099] In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 158 by 1 to 30 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 158 by 1 to 20 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 158 by 1 to 15 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 158 by 1 to 10 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 158 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the IIC of lactose-positive Streptococcus thermophilus strains used in accordance with the invention Man The protein sequence is selected from the group consisting of SEQ ID NOs: 158-165.

[0100] In some embodiments, the manM gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is SEQ ID NO: 158 and any IIC whose sequence has at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 158 as defined herein. Man IIC selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 159 to 165) Man In some embodiments, the manM gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is as set forth in SEQ ID NO:157.

[0101] In some embodiments, the mutation in the manM gene is the IIC at position 179 or 180 of SEQ ID NO: 130. Man This results in protein truncation.

[0102] In some embodiments, the mutation in the manM gene is an insertion of a single nucleotide C at any one of positions 438, 439, or 440 of SEQ ID NO: 129, which creates a frameshift in manM (SEQ ID NO: 212), resulting in a stop codon at position 180 of SEQ ID NO: 130 (manM 12997 The resulting Streptococcus thermophilus IIC truncated at position 179 Man Proteins are referred to herein as IIC Man179 (SEQ ID NO: 213).

[0103] In some embodiments, the IIC truncated at position 179 Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 213, and b) IIC having at least 90% similarity or identity to SEQ ID NO: 213, in particular an IIC having a length of 179 amino acids Man Mutant sequences.

[0104] IIC with at least 90% similarity or identity to SEQ ID NO: 213 Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences). In certain embodiments, IIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:213.

[0105] In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 213 by 1 to 30 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 213 by 1 to 20 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 213 by 1 to 15 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 213 by 1 to 10 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 213 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the IIC of lactose-positive Streptococcus thermophilus strains used in accordance with the invention Man The sequence of the protein is selected from the group consisting of SEQ ID NO:213.

[0106] In some embodiments, the manM gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is a gene encoding the manM gene of any IIC whose sequence has at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 213 and to SEQ ID NO: 213 as defined herein. Man IIC selected from the group consisting of mutant sequences Man Encodes proteins.

[0107] The present inventors have identified at least one mutation in the manN gene that, when inserted into the manN gene of an original lactose-positive Streptococcus thermophilus strain (mutated in the glcK gene, the ccpA gene, or both the glcK and ccpA genes as defined herein), enables the strain to overconsume lactose compared to the original strain when assayed by Test 2 in Example 2.

[0108] In some embodiments, the mutation in the manN gene is the IID at position 28 of SEQ ID NO: 167. Man In some embodiments, the mutation in the manN gene is an insertion of a nucleotide A in a stretch of five nucleotide A at positions 37-41 (a stretch of six nucleotide A, a frameshift of the open reading frame, and an IID at position 28 of SEQ ID NO: 167). Man This Streptococcus thermophilus IID truncated at position 28 of SEQ ID NO: 167 Man Proteins are referred to herein as IIDs. Man 28 It is called.

[0109] In some embodiments, the IID is truncated at position 28. Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 207, and b) an IID having at least 90% similarity or identity to SEQ ID NO: 207, in particular an IID that is 28 amino acids in length Man Mutant sequences.

[0110] IIDs with at least 90% similarity or identity to SEQ ID NO: 207 ManFor the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment (i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences). In certain embodiments, IIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:207.

[0111] In certain embodiments, an IID Man The variant sequence differs from SEQ ID NO: 207 by 1 to 10 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 207 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the IID of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is Man The protein sequence is selected from the group consisting of SEQ ID NOs: 207-211.

[0112] In some embodiments, the manN gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is selected from the group consisting of SEQ ID NO: 207 and any IID whose sequence has at least 90% similarity or identity to SEQ ID NO: 207 as defined herein. Man IID selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 208 to 211) Man In some embodiments, the manN gene carried by a Streptococcus thermophilus strain used in accordance with the present invention is as set forth in SEQ ID NO:206.

[0113] At least one gene encoding a protein of the mannose-glucose-specific PTS is mutated as defined herein, whether the glcK gene mutation as defined herein and the ccpA gene mutation as defined herein (either alone or in combination) are present in a lactose-positive Streptococcus thermophilus strain used in accordance with the invention. Regardless of the embodiment, the invention encompasses lactose-positive Streptococcus thermophilus strains harboring mutations in one, two, three, or more genes selected from the group consisting of the manL gene, the manM gene, and the manN gene. In some embodiments, a lactose-positive Streptococcus thermophilus strain used in accordance with the invention harbors a mutation in manL. In some embodiments, a lactose-positive Streptococcus thermophilus strain used in accordance with the invention harbors a mutation in manM. In some embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention possess a mutation in manN. In some embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention possess a mutation in manL and a mutation in manM. In some embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention possess a mutation in manL and a mutation in manN. In some embodiments, lactose-positive Streptococcus thermophilus strains used in accordance with the invention possess a mutation in manM and a mutation in manN.In some embodiments, a lactose-positive Streptococcus thermophilus strain used in accordance with the present invention harbors a mutation in manL, a mutation in manM, and a mutation in manN.

[0114] Any method can be used to identify mutations in genes encoding proteins of the mannose-glucose specific PTS, in particular the manL gene, manM gene or manN gene, suitable for lactose-positive Streptococcus thermophilus strains used according to the invention.

[0115] By way of example, to identify suitable mutations in the manL, manM or manN genes, one skilled in the art can proceed by the following method: a) providing a parent strain, for example any one of the strains DSM32587, DSM34909, DSM34910 and DSM34911 (mutated in their glcK gene); b) performing mutagenesis, for example by random or directed mutagenesis, on the manL, manM or manN gene of the strain of a) to obtain a manL, manM or manN gene whose sequence differs from the sequence of the manL, manM or manN gene of DSM 32587 or another original strain, to obtain a man mutant strain, for example a man mutant DSM 32587 strain; c) determining whether a phenotype has been achieved that allows the strain to overconsume lactose compared to the original strain; and d) pH, if necessary STOP To determine whether the phenotype was achieved.

[0116] Alternatively, one skilled in the art can proceed by the following method: a) a mutated ccpA gene (ccpA Δ1A114-120) herein replaced by DGCC7710-ccpA Δ1A114-120 providing a parent strain such as strain DSM32587 or strain DSM28255, referred to as strain ST21-C (corresponding to strain ST21-C referred to herein); b) performing mutagenesis on the manL, manM or manN gene of the strain of a) by, for example, random or directed mutagenesis, to identify the sequence of DGCC7710-ccpA Δ1A114-120 The manL, manM, or manN gene sequences were obtained that were different from those of the manL, manM, or manN gene sequences of the man mutant DGCC7710-ccpA strain. Δ1A114-120 Obtaining shares, c) determining whether a phenotype has been achieved that allows the strain to overconsume lactose compared to the original strain; and d) pH, if necessary STOP To determine whether the phenotype was achieved.

[0117] Once identified, the mutated manL, manM or manN gene according to the invention can be introduced into a lactose-positive Streptococcus thermophilus strain in place of manL, manM or manN to obtain a lactose-positive Streptococcus thermophilus strain for use according to the invention.

[0118] Mutation in the ccpA gene This section describes mutations in the ccpA gene in the context of lactose-positive Streptococcus thermophilus strains used in accordance with the present invention, which may be used alone, in combination with one or more mutations in genes encoding proteins of the mannose-glucose specific PTS defined herein, in combination with one or more mutations in the glcK gene defined herein, or in combination with both one or more mutations in genes encoding proteins of the mannose-glucose specific PTS defined herein and one or more mutations in the glcK gene defined herein.

[0119] In lactose-positive Streptococcus thermophilus strains, any mutation in the ccpA gene, alone, in combination with one or more mutations in genes encoding proteins of the mannose-glucose-specific PTS as defined herein, in combination with one or more mutations in the glcK gene as defined herein, or in combination with both one or more mutations in genes encoding proteins of the mannose-glucose-specific PTS as defined herein and one or more mutations in the glcK gene as defined herein, is suitable so long as it promotes excessive lactose consumption when grown in milk.

[0120] In some embodiments, the ccpA gene mutation is not a mutation that results in a knockout (ie, complete disruption) of the gene.

[0121] In some embodiments, the ccpA gene mutation is a mutation in the coding sequence of the ccpA gene, particularly in the first 270 nucleotides of the coding sequence of the ccpA gene. In some embodiments, the mutation is a mutation selected from the group consisting of: a) a nonsense mutation located between nucleotide 1 and nucleotide 270 of the coding sequence of the ccpA gene (i.e., resulting in a stop codon), and b) A mutation located in the first quarter of the coding sequence of the ccpA gene (i.e., nucleotide 1 to nucleotide 250) that results in a frameshift in the open reading frame of the ccpA gene.

[0122] In some embodiments, the mutation that results in a frameshift in the open reading frame of the ccpA gene is located in the coding sequence of the ccpA gene from nucleotide 50 to nucleotide 200. In some embodiments, the mutation that results in a frameshift in the open reading frame of the ccpA gene is located in the coding sequence of the ccpA gene from nucleotide 100 to nucleotide 150. Regardless of the location of the mutation that results in the frameshift, the mutation is selected from the group consisting of a deletion, an insertion, or a deletion / insertion (all of which are not a multiple of three).

[0123] Two Streptococcus thermophilus strains may differ by the sequence of their respective ccpA genes, but this does not necessarily mean that one of these two ccpA genes is mutated within the meaning of the present invention. Indeed, what is not considered a mutation in the ccpA gene suitable for the present invention is a mutation at the nucleotide level that results in a change at the protein level.

[0124] Non-limiting examples of ccpA genes that are not considered to be suitably mutated within the meaning of the present invention are: - a polynucleotide defined in SEQ ID NO: 65 (ccpA type ST1), which ccpA type is that of the strain DSM28255; - the polynucleotide defined in SEQ ID NO: 66 (ccpA type ST2), which has 99.8% identity with SEQ ID NO: 65; - a polynucleotide defined in SEQ ID NO: 67 (ccpA type ST3) which has 99.8% identity with SEQ ID NO: 65, this ccpA type being that of strain DSM 33651; - the polynucleotide defined in SEQ ID NO: 68 (ccpA type ST4), which has 99.7% identity with SEQ ID NO: 65; - a polynucleotide defined in SEQ ID NO: 69 (ccpA type ST5), which has 99.8% identity with SEQ ID NO: 65, and - the polynucleotide defined in SEQ ID NO: 70 (ccpA type ST6), which has 99.7% identity with SEQ ID NO: 65.

[0125] In some embodiments, the present invention may relate to the use of a lactose-positive Streptococcus thermophilus strain carrying a mutation in the ccpA gene selected from the group consisting of a nonsense mutation located between nucleotides 1 and 270 of the coding sequence of the ccpA gene, and a mutation located in the first quarter of the coding sequence of the ccpA gene that results in a frameshift in the open reading frame of the ccpA gene.

[0126] In some embodiments, the mutation in the ccpA gene is a deletion of nucleotide A in a stretch of seven nucleotide A's from positions 114 to 120 (resulting in a frameshift in the open reading frame of the ccpA gene). Such a Streptococcus thermophilus mutant ccpA gene is referred to herein as ccpA Δ1A114-120 It is called.

[0127] In some embodiments, the sequence of the ccpA gene having a stop codon at codon 66 is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 71, and b) A ccpA variant sequence having at least 90% identity to SEQ ID NO: 71. A ccpA variant as defined herein carries a mutation as defined above, i.e. selected from the group consisting of a nonsense mutation located from nucleotide 1 to nucleotide 270 of the coding sequence of the ccpA gene and a mutation located in the first quarter of the coding sequence of ccpA, resulting in a frameshift in the open reading frame of the ccpA gene.

[0128] For the definition of a ccpA variant having at least 90% identity to SEQ ID NO:71, identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of identical nucleotides in the aligned portions of the sequences]. In certain embodiments, a ccpA variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity to SEQ ID NO:71. In certain embodiments, a ccpA variant sequence differs from SEQ ID NO:71 by 1 to 30 nucleotide substitutions. In certain embodiments, a ccpA variant sequence differs from SEQ ID NO:71 by 1 to 20 nucleotide substitutions. In certain embodiments, a ccpA variant sequence differs from SEQ ID NO:71 by 1 to 15 nucleotide substitutions. In certain embodiments, a ccpA variant sequence differs from SEQ ID NO:71 by 1 to 10 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions from SEQ ID NO: 71. In some embodiments, the sequence of the ccpA gene of a lactose-positive Streptococcus thermophilus strain used in accordance with the invention is selected from the group consisting of SEQ ID NOs: 71, 72, 73, 74, 75, and 76.

[0129] Those skilled in the art are provided with guidance on how to identify mutations in the ccpA gene other than those specifically disclosed in this section of the application.

[0130] Thus, the skilled person can proceed in the following way: a) the manL gene is a mutated manL gene defined in SEQ ID NO: 111 (IIAB Man 305 The manL gene encoding the ribosomal protein (referred to herein as DGCC7710-IIAB) has been replaced by the manL gene encoding the ribosomal protein. Man 305 providing a parent strain, such as strain DSM28255, referred to as a strain; b) mutagenesis of the ccpA gene of the strain of a) by, for example, random or directed mutagenesis, to identify the sequence DGCC7710-IIAB Man 305 A ccpA mutant strain, e.g., ccpA mutant DGCC7710-IIAB, is obtained by obtaining a ccpA gene sequence different from that of the ccpA gene of the strain. Man 305 Obtaining shares, c) determining whether a phenotype has been achieved that allows the strain to overconsume lactose compared to the original strain; and d) pH, if necessary STOP To determine whether the phenotype was achieved.

[0131] Alternatively, one skilled in the art can proceed by the following method: a) the manM gene is a mutated manM gene (IIC Man 208 The manM gene encoding the .gamma.-protein) is replaced by the DGCC7710-IIC gene, herein DGCC7710-IIC. Man 208 providing a parent strain, such as strain DSM28255, referred to as a strain; b) mutagenesis of the ccpA gene of the strain of a) is carried out, for example by random or directed mutagenesis, to identify the sequence DGCC7710-IIC Man 208 A ccpA mutant strain, e.g., ccpA mutant DGCC7710-IIC, was obtained by obtaining a ccpA gene sequence different from that of the ccpA gene of the strain. Man 208 Obtaining shares, c) determining whether a phenotype has been achieved that allows the strain to overconsume lactose compared to the original strain; and d) pH, if necessary STOP To determine whether the phenotype was achieved.

[0132] A person skilled in the art can also proceed in the following way: a) the manN gene is a mutated manN gene (IID) defined in SEQ ID NO: 206 Man 28 The manN gene encoding the ribosomal protein (referred to herein as DGCC7710-IID) has been replaced by the ribosomal protein Man 28 providing a parent strain, such as strain DSM28255, referred to as a strain; b) mutagenesis of the ccpA gene of the strain of a) is carried out, for example by random or directed mutagenesis, to determine that the sequence is DGCC7710-IID Man 28 A ccpA mutant strain, e.g., ccpA mutant DGCC7710-IID, is obtained by obtaining a ccpA gene sequence different from that of the ccpA gene of the strain. Man 28 Obtaining shares, c) determining whether a phenotype has been achieved that allows the strain to overconsume lactose compared to the original strain; and d) pH, if necessary STOP To determine whether the phenotype was achieved.

[0133] Once identified, the mutant ccpA gene (identified herein) can be introduced into a lactose-positive Streptococcus thermophilus strain in place of the ccpA gene to obtain a lactose-positive Streptococcus thermophilus strain for use in accordance with the present invention.

[0134] Further characterization of the strains of the present invention The inventors have found that strains having one or more mutations in the glcK gene encoding glucokinase, and optionally further having a mutation in a gene encoding a protein of the mannose-glucose specific PTS and / or a mutation in the ccpA gene, when used to ferment milk, 1) Allows for increased lactose consumption in fermented milk, and optionally 2) For strains carrying one or more mutations in the glcK gene in combination with further mutations in genes encoding proteins of the mannose-glucose specific PTS, and optionally also carrying a mutation in the ccpA gene, it is possible to obtain fermented milk that does not undergo post-acidification even when stored at fermentation temperature. It was shown that...

[0135] In some embodiments, the lactose-positive Streptococcus thermophilus strain used in accordance with the present invention is characterized by the fact that when used to ferment milk, the strain results in low-lactose fermented milk.

[0136] In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the present invention is characterized by the fact that this strain results in fermented milk that does not undergo post-acidification when stored at fermentation temperatures.

[0137] The expression "not subject to post-acidification" refers to a specific pH value (i.e. pH ) that ensures that the rate of acidification is less than 0.1 mUpH / min when inoculated with the strain used according to the invention and fermented as described in the "Lactose Acidification Performance" section. STOP a dairy product whose pH is reduced to a value below 1000 ppm, STOP The value is between 4.4 and 5.3, and optionally the gradient from pH 6 to pH 5.5 is at least -0.008 UpH / min.

[0138] As used herein, "pH" STOP The term "phenotype" refers to strains of Streptococcus thermophilus that, when used in milk fermentation as described in the "Milk Acidification Performance" section, produce dairy products that are "not subject to post-acidification" as defined herein.

[0139] The absence of post-acidification is therefore characterized by the fact that the pH of the fermented milk stops at 4.4 to 5.3. The pH is considered to have stopped (pH 4.4) when the rate of acidification (ΔpH / Δtime) becomes less than 0.1 mUpH / min (less than 0.0001 mUpH / min). STOP ) is considered to be "certain" STOP This means that after the acidification rate is reached, the acidification rate remains below 0.1 mUpH / min for the remainder of the fermentation as described in the lactate acidification assay (i.e. up to 24 hours at the fermentation temperature).

[0140] In some embodiments, the pH obtained using the strains used in accordance with the present invention STOP In some embodiments, the pH obtained using the strains used in accordance with the present invention is between 4.7 and 5.2. STOP In some embodiments, the pH obtained using the strains used in accordance with the present invention is between 4.8 and 5.1. STOP is between a minimum value selected from the group consisting of 4.4, 4.5, 4.6, 4.7 and 4.8 and a maximum value selected from the group consisting of 5.1, 5.2 and 5.3.

[0141] In some embodiments, the fermented milk that has not undergone post-acidification is also characterized by a slope between pH 6 and pH 5.5. The slope represents the reciprocal of the rate (rate of acidification). In some embodiments, the slope is at least -0.009 UpH / min. In some embodiments, the slope is at least -0.01 UpH / min.

[0142] Compositions, methods and uses using the lactose-positive Streptococcus thermophilus strains of the present invention The present invention also relates to the use of a bacterial composition comprising or consisting of at least one, particularly one, lactose-positive Streptococcus thermophilus strain used according to the invention. In a particular embodiment, the bacterial composition is a pure culture, i.e., comprising or consisting of a single bacterial strain. In another embodiment, the bacterial composition is a mixed culture, i.e., comprising or consisting of a lactose-positive Streptococcus thermophilus strain of the invention and at least one other bacterial strain. "At least" (with respect to strains or bacteria) means one or more, particularly one, two, three, four or five strains.

[0143] Thus, in some embodiments, the bacterial compositions of the invention comprise or consist of at least one lactic acid bacterium of a species selected from the group consisting of lactose-positive Streptococcus thermophilus strains of the invention, as well as Lactococcus species, Streptococcus species, Lactobacillus species, including Lactobacillus acidophilus, Enterococcus species, Pediococcus species, Leuconostoc species, Bifidobacterium species, and Oenococcus species, or any combination thereof. Lactococcus species include Lactobacillus acidophilus and Lactococcus lactis, including Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactis biovar diacetylactis. Bifidobacterium species include Bifidobacterium animalis, particularly Bifidobacterium animalis subsp. lactis.Other lactic acid bacteria species include Leuconostoc species, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus.

[0144] In some embodiments, the bacterial composition comprises or consists of a lactose-positive Streptococcus thermophilus strain of the present invention, and at least one Streptococcus thermophilus strain different from the S. thermophilus strain of the present invention, and / or at least one strain of a Lactobacillus species, and / or any combination thereof. In a particular embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, one or several strains of Lactobacillus delbrueckii sp. subsp. bulgaricus and / or one or several strains of Lactobacillus helveticus sp. and / or any combination thereof, optionally together with at least one Streptococcus thermophilus strain different from the S. thermophilus strain of the invention. In a specific embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, at least one strain of the Streptococcus thermophilus species different from the S. thermophilus strain of the invention, and a strain of Lactobacillus delbrueckii sp. subsp. bulgaricus. In another specific embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention and a strain of Lactobacillus delbrueckii sp. subsp. bulgaricus.

[0145] In some embodiments, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. cremoris.

[0146] In certain embodiments of any bacterial composition defined herein, either as a pure culture or a mixed culture, the bacterial composition further comprises at least one probiotic strain, such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paracasei, or Lactobacillus casei.

[0147] In certain embodiments, the bacterial composition is in a frozen, dried, or lyophilized liquid or solid format, in the form of a pellet or frozen pellet, or in the form of a powder or dry powder, either as a pure culture or a mixed culture as defined above. In certain embodiments, the bacterial composition of the invention is in a frozen format or in the form of a pellet or frozen pellet, particularly contained in one or more boxes or sachets. In another embodiment, the bacterial composition defined herein is in the form of a powder, such as a dry powder or lyophilized powder, particularly contained in one or more boxes or sachets.

[0148] In a particular embodiment, the bacterial composition of the invention, either as a pure culture or a mixed culture as defined above, and in any format (frozen, dried, lyophilized liquid or solid format, pellet or frozen pellet form, or powder or dry powder), contains 10 lactose-positive Streptococcus thermophilus strains of the invention at a concentration of 10 per gram of bacterial composition. 5 ~10 12 In certain embodiments, the concentration of lactose-positive Streptococcus thermophilus strains in the bacterial compositions of the present invention is in the range of 10 cfu (colony forming units) per gram of bacterial composition. 7 ~10 12 cfu, particularly at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 Or at least 10 11 In certain embodiments, when in the form of a frozen concentrate or a dry concentrate, the concentration of lactose-positive Streptococcus thermophilus strains, either as a pure culture or as a mixed culture, in the bacterial composition is in the range of 10 CFU / g of the frozen concentrate or the dry concentrate. 8 ~10 12 cfu / g, more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 Or at least 10 12 cfu / g range.

[0149] The present invention also relates to a method for producing a fermented product, which is cheese, comprising: a) inoculating a substrate with a lactose-positive Streptococcus thermophilus strain of the present invention; and b) fermenting the inoculated substrate to obtain a fermented cheese product. In certain embodiments, the lactose-positive Streptococcus thermophilus strain described herein is inoculated as a bacterial composition, e.g., a pure culture or a mixed culture, as defined herein. In some embodiments, the substrate to which the S. thermophilus strain or bacterial composition described herein is added is a dairy substrate. By "dairy substrate" is meant milk of animal and / or plant origin. In certain embodiments, the dairy substrate is of animal origin, such as cow, goat, sheep, buffalo, zebra, horse, donkey, or camel. The milk can be natural, reconstituted, skim milk, or milk supplemented with compounds essential for bacterial growth or for further processing of the fermented milk. Thus, in certain embodiments, the present invention also provides methods of producing a cheese product, the method comprising: a) inoculating a dairy substrate with a lactose-positive Streptococcus thermophilus strain or bacterial composition described herein; and b) fermenting the inoculated dairy substrate to obtain a fermented cheese product.

[0150] Various features and embodiments of the present invention will now be described by way of non-limiting example.

[0151] Numbered embodiments of the invention: 1. The use of a lactose-positive Streptococcus thermophilus strain in the manufacture of cheese, such as semi-hard or soft cheese, wherein the strain is present at a concentration of 1% v / v or about 10% v / v as measured by Test 2 described herein. 9Use a Streptococcus thermophilus strain that has the ability to consume at least approximately 35% of the lactose when grown in raw whole milk inoculated with 1 CFU / ml.

[0152] 2. Use according to embodiment 1, wherein the use is for eliminating or reducing the need for a delactosing step, such as a curd washing step or any other lactose dilution, in the manufacture of cheese, such as semi-hard cheese production.

[0153] 3. This strain is a) a mutation in the glcK gene encoding glucokinase that leads to a decrease in glucokinase activity, but not to zero; and / or b) mutations in the ccpA gene selected from nonsense mutations or mutations resulting in a frameshift in the open reading frame of the ccpA gene, each mutation being located in the coding sequence of the ccpA gene from nucleotide 1 to nucleotide 270 of SEQ ID NO: 65, and each of these mutations resulting in the expression of a truncated CcpA protein; and / or c) a mutation in at least one gene encoding a protein of the mannose-glucose specific PTS, for example, one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene, each of which is IIAB Man , IIC Man or IID Man Mutations that result in the expression of a truncated form of any one of the proteins 3. The use according to embodiment 1 or 2, comprising one or more mutations selected from:

[0154] 4. The strain comprises a mutation in one or more genes encoding proteins of a mannose-glucose specific PTS, for example, a manL gene, a manM gene, and a manN gene, each of which is a IIAB Man , IIC Man or IIDMan and a mutation resulting in expression of a truncated form of any one of the following: 305 ), a mutation resulting from a substitution of nucleotide G with T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ); e.g., IIC at position 179 of SEQ ID NO: 130 Man IIC by the insertion of nucleotides at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene, resulting in a truncation of the protein Man Mutations that result in a frameshift in the open reading frame of a protein-coding gene (manM 12997 ); and / or the IID at position 28 of SEQ ID NO: 167 Man A mutation by insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41 of SEQ ID NO: 166, which generates a frameshift in the manN gene, resulting in a shortening of the protein (manN 13741 4. The use of embodiment 3, comprising:

[0155] 5. Use according to embodiment 3 or 4, wherein the strain comprises at least a mutation in the glcK gene encoding glucokinase, which mutation leads to a reduction in glucokinase activity, but not to zero.

[0156] 6. The use according to embodiments 3 to 5, wherein the strain comprises at least a mutation in the ccpA gene selected from a nonsense mutation or a mutation located from nucleotide 1 to nucleotide 270 of SEQ ID NO: 65 of the coding sequence of the ccpA gene and resulting in a frameshift in the open reading frame of the ccpA gene, any of which mutations results in the expression of a truncated CcpA protein.

[0157] 7. The mutant glcK gene is a) a glucokinase having at position 275 an amino acid which is not glutamic acid, in particular an amino acid which is not an acidic amino acid, in particular an amino acid which is lysine; b) at position 275, an amino acid that is not glutamic acid, in particular an amino acid that is not an acidic amino acid, in particular an amino acid that is lysine (glcK 378 ), a glucokinase having an arginine at position 278 and / or a serine at position 279, and c) a glucokinase having at its position 144 an amino acid which is not glycine, in particular an amino acid which is not an aliphatic amino acid, in particular an amino acid which is serine 7. The use according to any one of embodiments 1 to 6, wherein the glucokinase encodes a gene selected from the group consisting of:

[0158] 8. The mutant glcK gene encodes glucokinase, the sequence of which is a) the sequence defined in SEQ ID NO: 25, in which the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine, and b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of said glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of said glucokinase) is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine; c) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is not a glycine, in particular is not an aliphatic amino acid, in particular is a serine; and d) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid of said glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of said glucokinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine. The use according to any one of embodiments 1 to 7, wherein the compound is selected from the group consisting of:

[0159] 9. The sequence of the mutant ccpA gene is a) the sequence defined in SEQ ID NO: 71, and b) a ccpA variant sequence having at least 90%, such as at least 95%, identity to SEQ ID NO: 71 The use according to any one of embodiments 1 to 8, wherein the compound is selected from the group consisting of:

[0160] 10. Use according to any one of embodiments 1 to 9, wherein the strain is capable of fermenting milk as described in test 2 described herein during 24 hours of fermentation, and the concentration of lactose remaining in the fermented milk is less than 30 g / kg.

[0161] 11. Use according to any one of embodiments 1 to 10, wherein the strain has the ability to over-consume lactose compared to a parent strain lacking said one or more mutations, e.g. the amount of lactose remaining when milk is fermented as described in test 2 is less than 20 g / kg, 10 g / kg, 8 g / kg, 6 g / kg, 4 g / kg, 2 g / kg or 1 g / kg.

[0162] 12. The use according to any one of embodiments 1 to 11, wherein the strain is galactose negative.

[0163] 13. Streptococcus thermophilus strains - strain deposited at the Leibniz-Institut DSMZ on August 15, 2017 under the name DuPont Nutrition Biosciences ApS with accession number DSM32587; - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS and with the accession number DSM34909; - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS and with the accession number DSM34910; - the strain deposited at the Leibniz-Institut DSMZ on 23 January 2024 under the name of International N&H Denmark ApS under the accession number DSM 34911; - a strain corresponding to the Streptococcus thermophilus strain deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany) on 14 January 2014 under the accession number DSM 28255, in which the sequence of the glcK gene encoding glucokinase contains the sequence defined in SEQ ID NO: 22 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto, and in which the amino acid at position 275 is lysine (glcK 378 ), and / or IIC, for example, by insertion of a nucleotide at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene. Man Mutations that result in a frameshift in the open reading frame of a protein-coding gene (manM 12997 ) and / or resulting in a stop codon at position 306 of the protein of SEQ ID NO: 80 (IIAB Man ), substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ) and / or a substitution of the nucleotide G at position 625 of manM by the nucleotide T (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167; Man Insertion of a single nucleotide A in a stretch of five nucleotides A at positions 37-41, which generates a frameshift in the manN gene, resulting in a truncation of the protein (manN 13741 and / or comprising the ccpA sequence of SEQ ID NO: 71, or a ccpA variant sequence having at least about 90%, e.g., at least 95%, identity to SEQ ID NO: 71; and 2. A strain corresponding to the Streptococcus thermophilus strain deposited on February 16, 2022 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under accession number DSM 34172, in which the sequence of the glcK gene encoding glucokinase contains the sequence set forth in SEQ ID NO: 22 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto, and in which the amino acid at position 275 is lysine (glcK 378 ), and / or IIC, for example, by insertion of a nucleotide at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene. Man Mutations that result in a frameshift in the open reading frame of a protein-coding gene (manM 12997 ) and / or resulting in a stop codon at position 306 of the protein of SEQ ID NO: 80 (IIAB Man ), substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL 13740 ) and / or a substitution of the nucleotide G at position 625 of manM by the nucleotide T (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167; Man Insertion of a single nucleotide A in a stretch of five nucleotides A at positions 37-41, which generates a frameshift in the manN gene, resulting in a truncation of the protein (manN 13741 13. The use according to any one of embodiments 1 to 12, wherein the strain is selected from the group consisting of: a strain comprising a ccpA sequence of SEQ ID NO: 71, or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71;

[0164] 14. The use according to any one of embodiments 1 to 13, wherein the cheese is a semi-hard cheese, such as Gouda cheese, or a soft cheese.

[0165] 15. At least one other strain of the genus Lactobacillus, such as Lactobacillus delbrueckii subsp. bulgaricus, and / or mesophilic lactic acid bacteria, for example a strain of Lactococcus lactis subsp. cremoris, a strain of Lactococcus lactis subsp. hordinae, or a species of Lactococcus lactis, such as a strain of Lactococcus lactis subsp. lactis, or a strain of the genus Bifidobacterium, or Leuconostoc mesenteroides. 14. A composition comprising at least one lactose-positive Streptococcus thermophilus strain according to any one of embodiments 1 to 13 in combination with a strain of the genus Leuconostoc, such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

[0166] 16. The composition of embodiment 15, wherein the lactose-positive Streptococcus thermophilus strain is as described in any one of embodiments 2 to 13.

[0167] 17. A method for producing cheese, such as semi-hard and / or soft cheese, comprising the steps of inoculating a milk substrate with the Streptococcus thermophilus strain of any one of embodiments 1 to 13, or the composition of embodiment 15 or 16, and fermenting the inoculated milk to obtain cheese.

[0168] 18. A method for eliminating or reducing the need for a delactosizing step, such as a curd washing step or any other lactose dilution, in a cheese making process, such as semi-hard cheese making and / or soft cheese making, comprising the step of inoculating and fermenting a dairy substrate with the Streptococcus thermophilus strain of any one of embodiments 1 to 13, or the composition of embodiment 15 or 16.

[0169] 19. Cheese, such as semi-hard and / or soft cheese, comprising at least one Streptococcus thermophilus strain according to any one of embodiments 1 to 13, the composition according to embodiment 15 or 16, or obtainable by the method according to embodiment 17 or 18.

[0170] 20. IIC consisting of the amino acid sequence of SEQ ID NO: 213 or a sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 213 Man A lactose-positive Streptococcus thermophilus strain containing the manM gene encoding the protein.

[0171] 21.- Strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM34909, - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM34910, and - Strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM34911 A Streptococcus thermophilus strain selected from the list consisting of: [Example]

[0172] Example 1: Selection of "lactose-overconsuming" S. thermophilus strains Lactose-positive, galactose-negative Streptococcus thermophilus strains carrying mutations in the glcK gene and / or the ccpA gene and / or a mutation in one of the genes encoding proteins of the mannose-glucose specific PTS (manL, manM, manN) have been shown to be interesting for over-consuming lactose in fermented milk products (WO 2019197051, WO 2019122365, and EP 22159955.8). To evaluate the impact of these mutations, alone or in various combinations, on the lactose consumption capacity of S. thermophilus strains in milk, the glcK gene was used as a model for the analysis of the lactose-consuming capacity of S. thermophilus strains in milk, as shown in Table 1. 378 , ccpA 855 , manL 13740 , manM 16404 , manM 12997 and / or manN 13741 Derivatives of the parent S. thermophilus strains ST21 (DSM28255), ST22 (DSM34172), and ST23 were constructed in which mutations replaced the native gene alleles.

[0173] ccpA 855 The mutation is defined by a deletion of nucleotide A in a stretch of seven nucleotide A at positions 114-120 (defined in SEQ ID NO: 71), resulting in a frameshift in the open reading frame of the ccpA gene. 855 Mutations were introduced to construct strains ST21-C and ST23-C.

[0174] glcK 378 The mutation generates a mutant glcK gene encoding glucokinase in which the glutamic acid (E) at position 275 is replaced by the amino acid lysine (K) as defined in SEQ ID NO: 22. 378 The mutations were introduced into the genomes of ST21 and ST23 to construct strains ST21-G (DSM32587) and ST23-G, and into the genomes of strains ST21-C and ST23-C to construct strains ST21-GC and ST23-GC (DSM34909). 378 Mutations and ccpA 855 (carrying both the ccpA and ccpB mutations). 855 Mutations and glcK 378 Mutations were introduced into the genomes of ST24 and ST25 to construct strains ST24-GC (DSM34910) and ST25-GC (DSM34911).

[0175] The manL, manM, or manN gene is part of the mannose-glucose specific PTS IIAB Man , IIC Man and IID Man Each gene encodes a protein. 13740 The mutation is a substitution of the nucleotide G at position 916 of SEQ ID NO:79 of manL with the nucleotide T, resulting in a stop codon at position 306 of the protein SEQ ID NO:80 [the resulting protein is IIAB Man 305 was named manM. 16404The mutation is a substitution of the nucleotide G at position 625 of manM with the nucleotide T (SEQ ID NO: 157), resulting in a stop codon at position 209, as defined in SEQ ID NO: 158. [The resulting protein is IIC Man 208 was named manM. 12997 The mutation is the insertion of a single nucleotide C at any one of positions 438, 439, or 440 of SEQ ID NO: 129, which creates a frameshift in manM (SEQ ID NO: 212), resulting in a stop codon at position 180 of SEQ ID NO: 130 [the resulting protein is expressed as IIC, as defined in SEQ ID NO: 213]. Man 179 was named manN. 13741 The mutation is an insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41 of SEQ ID NO: 166 (resulting in a stretch of six nucleotide A's), generating a frameshift in manN and an IID at position 28 of SEQ ID NO: 167 as defined in SEQ ID NO: 207. Man The ST21, ST22 and ST23 derivatives contain the manL gene either in the genome of the parent strain (giving rise to strains ST21-M1, ST21-Mm1 and ST21-Mn), or in the genome of a strain already mutated in the glcK gene (giving rise to ST21-GMm1, ST22-GMm2, ST23-GMm2), or in the genome of a strain already mutated in the ccpA gene (giving rise to ST21-CM1, ST21-CMm1, ST21-CMn, ST22-CMm2, ST23-CMm2), or in the genome of a strain already mutated in both the glcK and ccpA genes (giving rise to ST21-GCMm1). 13740 , manM 12997 , manM 16404 or manN 13741 Mutations were introduced in the manL, manM, or manN genes, respectively.

[0176] All strains were grown at 37°C in M17 broth (Oxoid, supplier reference number CM0817) supplemented with 30 g / L sucrose and, where necessary, 15 g / L agar bacterial type A (Biokar, supplier reference number #A1010HA). These cultures were harvested by centrifugation and then resuspended in carbohydrate-free M17 medium and used to inoculate milk, as described in Test 1 and Test 2 defined in Example 2.

[0177] Example 2: Characterization of "lactose-overconsuming" S. thermophilus strains To evaluate the ability of the derivative strains ST21, ST22 and ST23 described in Example 1 to consume lactose, milk was fermented using the engineered and parent strains as described in Tests 1 and 2 below.

[0178] Quantification of lactose during milk fermentation [Test 1] UHT semi-skimmed milk "Le Petit Vendeen" containing 3% (w / v) milk powder (BBA, Lactalis), previously pasteurized at 90°C for 10 min, was infused with 1% (v / v, approximately 10% of the culture of the S. thermophilus strain to be assayed (cells resuspended in carbohydrate-free M17 from an overnight culture as described in Example 1). 7 The milk was inoculated with 10 ... -1Lactose was quantified by high-performance liquid chromatography (Agilent 1200 HPLC) equipped with a refractive index detector. Results were processed using Chemstation reprocessing software (Agilent).

[0179] Quantification of lactose during milk fermentation [Test 2] Raw whole milk, previously pasteurized at 74°C for 1 min, was inoculated with a culture of the S. thermophilus strain to be assayed (overnight culture grown in semi-skimmed milk 10% BBA inoculated at 1% from a frozen vial and heated at 120°C for 20 min) at 1% (v / v, approximately 10 9 The milk was inoculated with 1000 CFU / ml of lactose (CFU / ml). Pasteurized milk contains approximately 48 g / kg of lactose. The inoculated milk flasks were statically incubated in a 32°C water bath for 1 hour and 30 minutes, then heated for 15 minutes to reach 36°C, maintained for 1 hour, and then slowly cooled to 28°C over a maximum of 24 hours to obtain fermented milk. The amount of lactose was measured by HPLC (high performance liquid chromatography) on a Vanquish system provided by Thermo Fisher Scientific equipped with a refractive index detector. Five grams of the inoculated milk before incubation (TO sample) and five grams of the inoculated milk after incubation (sample T24h) were diluted in 25 mL of 0.025N sulfuric acid solution. After homogenization, the samples were centrifuged at 4600 rpm for 10 minutes, and the supernatant was filtered through a nylon 0.2 μm filter into an HPLC vial. Five microliters of the sample was injected onto an H+ ion exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm × 7.8 mm). Elution was performed in isocratic mode with 0.025 N sulfuric acid at 0.7 mL / min. Molecules were separated at 20°C at 40°C. Carbohydrates were detected by refractometry at 35°C.

[0180] The results are shown in Table 1. The five parent strains (ST21, ST22, ST23, ST24 and ST25) consumed limited amounts of lactose (approximately 30%). 378 , ccpA 855 or one of the manLMN mutations (manL13740 , manM 12997 , manM 16404 or manN 13741 All single mutants carrying either glcK or glcK were able to consume more lactose (ranging from 35% to 45%). 378 Mutations and ccpA 855 Introducing both mutations into the genomes of ST21 (producing ST21-GC), ST23 (producing ST23-GC), ST24 (producing ST24-GC), and ST25 (producing ST25-GC) resulted in strains that consumed even more lactose (ranging from 51 to 59%). 378 Mutations in the manM gene (manM 12997 or manM 16404 ) yielded strains ST21-GMm1, ST22-GMm2, and ST23-GMm2, which were capable of hydrolyzing up to 84% of the lactose present in milk. ST21 (yielding ST21-CMm1), ST22 (yielding ST22-CMm2), and ST23 (yielding ST23-CMm2) contained ccpA in their genomes. 855 Mutations in the manM gene (manM 12997 or manM 16404 ) resulted in similar levels of lactose overconsumption. 378 , ccpA 855 and manM 16404 When all mutations were correlated within the genome, ST21-GCM hydrolyzed up to 89% of the lactose present in milk, resulting in the highest level of lactose overconsumption.

[0181] In addition, our results indicate that the manL introduced into the genome of ST21 13740 , manM 16404 or manN 13741 Mutations alone (generating ST21-Ml, ST21-Mm1, ST21-Mn) or ccpA 855Even with the associated mutations (producing ST21-CMl, ST21-CMm1, and ST21-CMn), similar levels of lactose overconsumption were measured, shown to be approximately 40% and 85%, respectively.

[0182] [Table 1]

[0183] Strains ST21, ST22 and their derivatives were evaluated under the conditions defined in Test 1, and strains ST23, ST24 and ST25 and their derivatives were evaluated under the conditions defined in Test 2.

[0184] Example 3: Use of "lactose-overconsuming" S. thermophilus strains in / for the production of Gouda cheese The classical Gouda production process requires the use of a mesophilic starter culture and includes several steps as shown in Table 2.

[0185] [Table 2]

[0186] The two "lactose-overconsuming" strains (ST21-G and ST21-GC) constructed in Example 1 and further analyzed in Example 2 were tested under experimental Gouda production conditions as described in Example 3 (Table 2). Fermentations were carried out using a reference mesophilic culture (designated Meso) or the parent ST21 strain or its "lactose-overconsuming" derivatives (ST21-G or ST21-GC).

[0187] To further evaluate the benefits of the "lactose-overconsuming" strain, in one experimental setup the full classical Gouda process was applied, and in another the curd washing step was omitted. The efficiency of the process in combination with the starter culture in reducing the lactose concentration in the product was evaluated by lactose quantification (as described in test 3). This lactose quantification was performed i) in the whey at the time of forming (2 hours after inoculation with the starter culture; see Table 3) and ii) in the curd (6 hours after inoculation with the starter culture; see Table 4).

[0188] Quantification of Lactose During Cheesemaking [Test 3] Raw whole milk, previously pasteurized at 74°C for 1 minute, was infused with a S. thermophilus strain and a mesophilic culture (designated Meso) at 1.10°C. 10 CFU / 100L ~ 1.10 11 The inoculation dose was determined at the respective CFU / 100L dose. Pasteurized milk contains approximately 48 g / kg lactose. A Gouda-type process was applied, as shown in Table 2. Five grams of whey at the time of molding and curd before brining were collected for sugar measurement. The amount of lactose was measured by HPLC (high-performance liquid chromatography) on a Vanquish system provided by Thermo Fisher Scientific, equipped with a refractive index detector. Five grams of curd / cheese was diluted in 25 mL of 0.025N sulfuric acid solution (or, for cheese samples, ground using an IKA Ultra-Turrax at 13,500 rpm). After homogenization, the sample was centrifuged at 4,600 rpm for 10 minutes, and the supernatant was filtered through a nylon 0.2 μm filter into an HPLC vial. Five microliters of sample was injected onto an H+ ion-exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm x 7.8 mm). Elution was performed in isocratic mode with 0.025N sulfuric acid at 0.7 mL / min. The molecules were separated in 20 minutes at 40° C. The carbohydrates were detected by refractometry at 35° C.

[0189] Table 3 shows the amount of lactose present in whey at the time of extrusion. The amount of lactose measured for the sample obtained with curd washing (approximately 33 g / Kg) was significantly different from that obtained without curd washing (50 g / Kg). This difference is primarily a result of the dilution factor (approximately 25%) caused by curd washing. In contrast, there was little, if any, difference in the amount of lactose measured in whey obtained from fermentations with various starter cultures at this early stage of the process.

[0190] [Table 3]

[0191] Similarly, Table 4 shows the amount of lactose present in the curd 6 hours after bacterial inoculation and before brining. When fermentation was performed with the classical mesophilic starter culture MESO, the lactose content in the curd was approximately six-fold lower in the product obtained with the process that included a curd washing step (1.1 g / kg) compared to the product obtained with a process that did not involve curd washing (6.3 g / kg). These data clearly demonstrate the delactosizing efficiency of the curd washing step to reduce the amount of residual lactose in the "pre-brining cheese." For curds obtained from fermentation with the parent S. thermophilus ST21, the lactose content in the curd was higher than that measured in fermentations with classical mesophilic starter cultures, regardless of the process used: 1.9 g / kg with the curd washing step and 4.7 g / kg without the curd washing step compared to 1.1 g / kg. Replacing the parent ST21 with one of the selected "lactose-overconsuming" strains (ST21-G or ST21-GC) had a dramatic effect on the lactose content in the curd. For both ST21-G and ST21-GC, the lactose content in the curd obtained from the process including the curd washing step was very low (0.3 g / Kg). Furthermore, even if the curd washing step was omitted, the lactose content in the curd was low, at 1.3 and 0.5 g / Kg, in fermentations with ST21-G or ST21-GC, respectively. This is similar to or lower than that measured in the curd obtained from the classical Gouda cheese process with a classical mesophilic starter culture in the process including the curd washing step.

[0192] These data suggest that glcK 378 It has been clearly demonstrated that the use of S. thermophilus strains with mutations introduced into their genome allows the curd washing step of the classical Gouda cheese process to be omitted. 855 Mutations and glcK 378 Even higher lactose reduction can be achieved by introducing combinations of mutations into the strain genome.

[0193] [Table 4]

[0194] Example 4: Use of "lactose-overconsuming" S. thermophilus strains associated with warm cultures in Gouda cheese production To further validate the experimental data, the parent S. thermophilus strain ST21 or its "lactose-overconsuming" derivative ST21-GC, combined with a classical mesophilic starter culture designated Meso, was evaluated in Gouda cheesemaking. Cheese was produced with and without a 25% curd washing step (1 hour 30 minutes after inoculation with bacteria), and lactose was quantified in the curd before the brining step as described in Test 3 of Example 3 (Table 5). The residual amount of lactose in the curd obtained with mesophilic culture alone was 6.9 g / kg when the curd washing step was omitted; curd washing reduced this amount to 3.7 g / kg. Addition of ST21 to the mesophilic culture only slightly reduced these values ​​to 5.8 g / kg and 3.5 g / kg, respectively, indicating that the addition of the parent S. thermophilus strain did not prevent the curd washing step from being omitted. In contrast, the addition of the "lactose-overconsuming" ST21-GC to mesophilic cultures had a dramatic effect on the lactose remaining in the curd, as lactose was below detectable levels in the curd even when the curd washing step was omitted. These results support the existence of the ccpA gene in the S. thermophilus genome. 855 glcK combined with mutations 378 The mutation clearly demonstrated the benefit of removing lactose, thus allowing the omission of the curd washing step in the Gouda cheese making process.

[0195] [Table 5]

[0196] In another assay, a similar Gouda cheese production was performed using a mesophilic culture (designated Meso) supplemented with a S. thermophilus strain of another genetic background (ST23) instead of ST21 or its "lactose-overconsuming" derivatives, namely ST23-G and ST23-GC (described in Example 2). Lactose quantification was performed on samples from a process in which the curd washing step was omitted. This is shown in Table 6. As previously observed, the level of residual lactose in the curd obtained from fermentation with mesophilic culture alone was high (6.7 g / Kg). Supplementing the fermentation with the parent strain ST23 made it possible to reduce the residual lactose level to 1.7 g / Kg. Supplementing the mesophilic culture with either ST23-G or ST23-GC provided the best performance in reducing the level of lactose in the curd (which was almost undetectable). These results support the role of glcK in S. thermophilus. 378 Mutation alone or ccpA 855 The effect of the mutation combinations on lactose consumption in the Gouda cheesemaking process was confirmed to be independent of the parent strain and could be obtained in multiple S. thermophilus genetic backgrounds, including the various parent strains (ST21, ST22, and ST23) described in Example 2, and each "lactose-overconsuming" derivative behaved similarly in the tests.

[0197] Gouda cheese production was also performed using mesophilic cultures (designated Meso) supplemented with "lactose-overconsuming" derivatives of two other genetic backgrounds of S. thermophilus, ST24-GC and ST25-GC. The data, shown in Table 6, confirmed the previous results obtained with ST23-GC.

[0198] [Table 6]

[0199] array SEQ ID NO: 2 [ka] SEQ ID NO: 22 [ka] SEQ ID NO: 25 (bold underlined X corresponds to aa at position 275 and can be any naturally occurring amino acid but is not Glu) [ka] SEQ ID NO: 45 [ka] SEQ ID NO: 46 (bold underlined X corresponds to aa at position 144 and can be any naturally occurring amino acid but is not Glu) [ka] SEQ ID NO: 65 [ka] SEQ ID NO: 71 [ka] SEQ ID NO:79 [ka] SEQ ID NO: 80 [ka] SEQ ID NO: 111 [ka] SEQ ID NO: 112 [ka] SEQ ID NO: 129 [ka] SEQ ID NO: 130 [ka] SEQ ID NO: 157 [ka] SEQ ID NO: 158 [ka] SEQ ID NO: 166 [ka] SEQ ID NO: 167 [ka] SEQ ID NO: 206 [ka] SEQ ID NO: 207 MAEKIQLSQADRKKGLVALTILARCMEL SEQ ID NO: 208 MAEKIQLSQADRKKGLVALTILARFMEL SEQ ID NO: 209 MSEKIQLSQADRKKGLVALTILARFMEL SEQ ID NO: 210 MAEKIQLSQADRRKGLVALTILARFMEL SEQ ID NO: 211 MSEKIQLSQADHKKGLVALTILARFMEL SEQ ID NO: 212 [ka] SEQ ID NO: 213 [ka]

[0200] KK DGCC numbers are internal references to the DuPont Danisco collection. DSM numbers are numbers assigned by the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Inhoffenstr. 7B, D-38124 Braunschweig) following deposits made pursuant to the Budapest Treaty.

[0201] With respect to Streptococcus thermophilus strain DGCC7710 (also referred to herein as ST21), deposited in accordance with the Budapest Treaty at the Leibniz-Institut DSMZ-Deutsche Sammlung von Microorganismen und Zellkulturen, GmbH on January 14, 2014, under the number DSM 28255, the applicant confirms that the depositor, Danisco Deutschland GmbH, Busch-Johannsen-Strasse 1, D-25899 Niebuell, Germany, has authorized the applicant (DuPont Nutrition Biosciences ApS) to refer to the deposited biological material in the present application. The terms "strain DGCC7710" and "DGCC7710 derivative" are used interchangeably with the terms "strain DSM 28255" and "DSM 28255 derivative."

[0202] We designed a derivative of strain DGCC7710 in which the glcK gene encodes glucokinase and the glutamic acid (E) at position 275 was replaced with the amino acid lysine (K). This derivative (DGCC12534) was deposited at the DSMZ on August 15, 2017, under accession number DSM32587 (herein referred to as ST21-G, E275K, mutated in its glcK gene). - A Streptococcus thermophilus strain deposited on September 29, 2020 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany] under accession number DSM33651 (herein referred to as ST3 or DSM33651).

[0203] The following strains have been deposited in accordance with the Budapest Treaty under the name of DuPont Nutrition Biosciences ApS: - A Streptococcus thermophilus strain deposited on February 16, 2022 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany] under accession number DSM34172 (referred to herein as ST22 or DSM34172). - Streptococcus thermophilus strain DGCC13706, deposited on January 23, 2024 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany] under accession number DSM34909 (referred to herein as ST23-GC or DSM34909). - Streptococcus thermophilus strain DGCC14231, deposited on January 23, 2024 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany] under accession number DSM34910 (referred to herein as ST24-GC or DSM34910). - Streptococcus thermophilus strain DGCC14283, deposited on January 23, 2024 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany] under accession number DSM 34911 (referred to herein as ST25-GC or DSM 34911).

[0204] Applicant requests that until the date a patent is granted, only samples of the deposited microorganisms described herein may be made available to practitioners.

[0205] In respect of the indication that a European patent is sought, samples of the deposited microorganisms shall, until the publication of the reference to the grant of a European patent or the date on which the application is refused or withdrawn or deemed to be withdrawn, only be made available by the furnishing of such samples to experts designated by the requester requesting samples and recognized by i) the applicant and / or ii) the European Patent Office, whichever is applicable.

Claims

1. 1. Use of a lactose-positive Streptococcus thermophilus strain in the manufacture of cheese, such as semi-hard or soft cheese, wherein the strain is present at a concentration of 1% v / v or about 10% v / v as measured by Test 2 described herein. ９ 10. The method of claim 1, wherein said strain of Streptococcus thermophilus has the ability to consume at least about 35% lactose when grown in inoculated raw whole milk at CFU / ml.

2. 2. The use according to claim 1, wherein the use is for eliminating or reducing the need for a delactosing step, such as a curd washing step or any other lactose dilution, in the manufacture of cheese, such as semi-hard cheese production.

3. The strain is a) a mutation in the glcK gene encoding glucokinase that leads to a decrease in glucokinase activity, but not to zero; and / or b) mutations in the ccpA gene selected from nonsense mutations or mutations that result in a frameshift in the open reading frame of the ccpA gene, each mutation being located in the coding sequence of the ccpA gene from nucleotide 1 to nucleotide 270 of SEQ ID NO: 65, and any of these mutations resulting in the expression of a truncated CcpA protein; and / or c) a mutation in at least one gene encoding a protein of the mannose-glucose specific PTS, for example, one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene, each of which is IIAB Ｍａｎ , II C Ｍａｎ or IID Ｍａｎ Mutations that result in the expression of a truncated form of any one of the proteins The use according to claim 1 or 2, comprising one or more mutations selected from:

4. The strain may have a mutation in one or more genes encoding proteins of the mannose-glucose specific PTS, such as the manL gene, the manM gene, and the manN gene, each of which may be a mutation in one or more genes selected from the group consisting of IIAB, ... Ｍａｎ , II C Ｍａｎ or IID Ｍａｎ and a mutation resulting in the expression of a truncated form of any one of the following: ３０５ ), a mutation resulting from a substitution of nucleotide G with T at position 916 of SEQ ID NO: 79 of the manL gene (manL １３７４０ ) e.g., IIC at position 179 of SEQ ID NO: 130; Ｍａｎ IIC by the insertion of nucleotides at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene, resulting in a truncation of the protein. Ｍａｎ Mutations that result in a frameshift in the open reading frame of a protein-coding gene (manM １２９９７ ) and / or the IID at position 28 of SEQ ID NO: 167 Ｍａｎ A mutation by insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41 of SEQ ID NO: 166, which generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ 4. The use according to claim 3, comprising:

5. 5. The use according to claim 3 or 4, wherein the strain comprises at least a mutation in the glcK gene encoding glucokinase, said mutation causing a reduction in glucokinase activity but not bringing it to zero.

6. 6. The use according to any one of claims 3 to 5, wherein the strain comprises at least a mutation in the ccpA gene selected from a nonsense mutation or a mutation located from nucleotide 1 to nucleotide 270 of SEQ ID NO: 65 of the coding sequence of the ccpA gene and resulting in a frameshift in the open reading frame of the ccpA gene, any of which mutations results in the expression of a truncated CcpA protein.

7. The mutant glcK gene a) a glucokinase having an amino acid at position 275 which is not glutamic acid, in particular an amino acid which is not an acidic amino acid, in particular an amino acid which is lysine; b) at position 275, an amino acid that is not glutamic acid, in particular an amino acid that is not an acidic amino acid, in particular an amino acid that is lysine (glcK ３７８ ), a glucokinase having an arginine at position 278 and / or a serine at position 279, and c) a glucokinase having at its position 144 an amino acid which is not glycine, in particular an amino acid which is not an aliphatic amino acid, in particular an amino acid which is serine The use according to any one of claims 1 to 6, wherein the glucokinase encodes a compound selected from the group consisting of:

8. The mutant glcK gene encodes glucokinase and its sequence is a) the sequence defined in SEQ ID NO: 25, in which the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine; and b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of said glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of said glucokinase) is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine; c) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is not a glycine, in particular is not an aliphatic amino acid, in particular is a serine; and d) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid of said glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of said glucokinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine. The use according to any one of claims 1 to 7, selected from the group consisting of:

9. The sequence of the mutant ccpA gene is a) the sequence defined in SEQ ID NO: 71, and b) a ccpA variant sequence having at least 90%, such as at least 95%, identity to SEQ ID NO: 71 The use according to any one of claims 1 to 8, selected from the group consisting of:

10. The use according to any one of claims 1 to 9, wherein the strain is galactose negative.

11. The Streptococcus thermophilus strain is - the strain deposited at the Leibniz-Institut DSMZ on August 15, 2017 under the name DuPont Nutrition Biosciences ApS under the accession number DSM 32587; - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name DuPont Nutrition Biosciences ApS under the accession number DSM 34909; - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name DuPont Nutrition Biosciences ApS under the accession number DSM 34910; - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name DuPont Nutrition Biosciences ApS under the accession number DSM 34911; Streptococcus thermophilus, deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany) on January 14, 2014 under the accession number DSM 28255. a strain corresponding to a strain of S. thermophilus, wherein the sequence of the glcK gene encoding glucokinase contains the sequence defined in SEQ ID NO: 22, or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, wherein the amino acid at position 275 is lysine (glcK378), and / or comprises a mutation resulting in a frameshift of the open reading frame of the gene encoding the IICMan protein, e.g., by an insertion of a nucleotide at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene (manM12997); and / or comprises a substitution of the nucleotide G by a T at position 916 of SEQ ID NO: 79 of the manL gene (manL13740), resulting in a stop codon at position 306 of the protein of SEQ ID NO: 80 (IIABMan); and / or comprises a substitution of the nucleotide G by a T at position 625 of manM, resulting in a stop codon at position 209 of the protein of SEQ ID NO: 80 (IIABMan); １６４０４ and / or the IID at position 28 of SEQ ID NO: 167; Ｍａｎ Insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41 (manN) which generates a frameshift in the manN gene, resulting in a shortening of the protein. １３７４１ and / or a strain comprising the ccpA sequence of SEQ ID NO: 71, or a ccpA variant sequence having at least about 90%, e.g., at least 95%, identity to SEQ ID NO: 71; and Streptococcus thermophilus, deposited on February 16, 2022 at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany) under accession number DSM 34172. a strain corresponding to a strain of S. thermophilus, wherein the sequence of the glcK gene encoding glucokinase contains the sequence defined in SEQ ID NO: 22, or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, wherein the amino acid at position 275 is lysine (glcK378), and / or comprises a mutation resulting in a frameshift of the open reading frame of the gene encoding the IICMan protein, e.g., by an insertion of a nucleotide at any one of positions 438, 439 or 440 of SEQ ID NO: 129 of the manM gene (manM12997); and / or comprises a substitution of the nucleotide G by a T at position 916 of SEQ ID NO: 79 of the manL gene (manL13740), resulting in a stop codon at position 306 of the protein of SEQ ID NO: 80 (IIABMan); and / or comprises a substitution of the nucleotide G by a T at position 625 of manM, resulting in a stop codon at position 209 of the protein of SEQ ID NO: 80 (IIABMan); １６４０４ and / or the IID at position 28 of SEQ ID NO: 167; Ｍａｎ Insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41 (manN) which generates a frameshift in the manN gene, resulting in a shortening of the protein. １３７４１ and / or comprising the ccpA sequence of SEQ ID NO: 71, or a ccpA variant sequence having at least about 90%, e.g., at least 95%, identity to SEQ ID NO:

71. The use according to any one of claims 1 to 10, selected from the group consisting of:

12. at least one other strain of the genus Lactobacillus, such as Lactobacillus delbrueckii subsp. bulgaricus, and / or a mesophilic lactic acid bacterium, for example a strain of Lactococcus lactis subsp. cremoris, a strain of Lactococcus lactis subsp. hordinae, or a strain of Lactococcus lactis subsp. lactis.

12. A composition comprising at least one lactose-positive Streptococcus thermophilus strain according to any one of claims 1 to 11 in combination with a species of the genus Lactococcus such as a species of Lactococcus lactis, or a strain of the genus Bifidobacterium, or a strain of the genus Leuconostoc, such as Leuconostoc mesenteroides subsp. cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

13. 13. A method for eliminating or reducing the need for a delactosing step such as a curd washing step or any other lactose dilution in a cheese making process such as semi-hard cheese making and / or soft cheese making, comprising the step of inoculating and fermenting a dairy substrate with a Streptococcus thermophilus strain according to any one of claims 1 to 11, or a composition according to claim 12.

14. IIC consisting of the amino acid sequence of SEQ ID NO:213 or a sequence having at least about 90%, e.g., at least 95%, identity to SEQ ID NO:

213. Ｍａｎ A lactose-positive Streptococcus thermophilus strain containing the manM gene encoding a protein.

15. - the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM 34909, the strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM 34910, and - strain deposited at the Leibniz-Institut DSMZ on January 23, 2024 under the name of International N&H Denmark ApS under the accession number DSM 34911 A Streptococcus thermophilus strain selected from the list consisting of:

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