EP4669118A1

EP4669118A1 - New use of lactic acid bacteria

Info

Publication number: EP4669118A1
Application number: EP24707155.8A
Authority: EP
Inventors: Armelle COCHU-BLACHÈRE; Mikael PIANFETTI
Original assignee: International N&H Denmark ApS
Current assignee: International N&H Denmark ApS
Priority date: 2023-02-22
Filing date: 2024-02-21
Publication date: 2025-12-31
Also published as: JP2026505444A; AU2024224154A1; WO2024175663A1; CN121311117A; MX2025008970A; KR20250148606A

Abstract

The present invention relates to a specific use of lactose-positive Streptococcus thermophilus strains in the production of cheese. The strains used according to the present invention may carry one or more mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, a mutation in the ccpA gene, and/or a muta- tion in at least one of the genes encoding the proteins of the mannose-glucose-specific PTS. The present invention further relates to compositions comprising the inventive strains used according to the present invention in combination with at least one strain of Lactobacillus genus and/or one strain of mesophilic lactic acid bacteria, methods for manufacturing of cheese, and the cheese obtained by the methods.

Description

NEW USE OF LACTIC ACID BACTERIA

FIELD OF THE INVENTION

The present invention relates to a specific use of lactose-positive Streptococcus thermophilus strains in the production of cheese. The strains used according to the present invention may carry one or more mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, a mutation in the ccpA gene encoding a catabolite control protein A CcpA, and/or a mutation in at least one of the genes encoding the proteins of the mannose-glucose-specific PTS.

The present invention further relates to compositions comprising the inventive strains used according to the present invention in combination with at least one strain of Lactobacillus genus and/or one strain of mesophilic lactic acid bacteria, methods for manufacturing of cheese, and the cheese obtained by the methods.

BACKGROUND TO THE INVENTION

The food industry uses bacteria in order to improve the taste and the texture of food or feed products. In the case of the dairy industry, lactic acid bacteria are commonly used to, for example, bring about the acidification of milk (by fermentation of lactose) and to texturize the product into which they are incorporated. For example, the lactic acid bacteria of the species Streptococcus thermophilus (S. thermophilus') are used extensively, alone or in combination with other bacteria, in the manufacture of fresh fermented dairy products, such as cheese or yoghurt.

One of the challenges in the production of semi-hard cheese is that the starter cultures normally used are mainly 100% mesophilic cultures, such as Lactococcus lactis (Lc. Lactis) that only consume part of lactose with the consequences of residual lactose present in cheese that promotes the growth of undesirable microorganisms. This may in turn lead to undesirable postacidification, undesirable taste, flavor, issues with texture and proteolysis development and reduce shelf life. Existing solution includes the addition of a curd washing step to the cheese process to limit the residual lactose in cheese with the consequences of increased time of cheese making process, use of a huge amount of water and energy.

The present invention has addressed these issues by providing a new specific use of lactose-positive S. thermophilus strains.

DETAILED DESCRIPTION

Starter culture used in the production of semi-hard cheese are mainly 100% mesophilic cultures, such as Lc. lactis that only consume part of lactose with the following consequences of residual lactose present in cheese promotes the growth of undesirable microorganisms that can lead to undesirable post-acidification, undesirable taste, flavor, texture and proteolysis development and reduce shelf life. Existing solution consists in the addition of a curd washing step to the cheese process to limit the residual lactose in cheese with the following consequences:

Curd washing step increases time of cheese making process

Curd washing step uses huge amount of water and energy (to heat water) which is both expensive for the cheese maker, and not good for the environment.

The present invention has addressed this problem in the art providing solutions in terms of the use of specific mutations of Streptococcus thermophilus strains modifying the sugar metabolism, and in particular modifications that will increase the lactose consumption.

Accordingly, in a broad aspect the present invention relates to the use of a lactosepositive, Streptococcus thermophilus strain in the production of a cheese, such as continental cheese, such as a semi-hard cheese or a soft-cheese.

In a first aspect the present invention relates to the use of a lactose-positive, Streptococcus thermophilus strain in the production of a cheese, such as a semi-hard cheese or a soft-cheese, which strain has the ability to consume at least about 35% lactose when grown in raw whole cow's milk inoculated at 1% v/v or about 10⁹ CFU/ml of the Streptococcus thermophilus strain, such as measured by Test 2 described herein.

In a second aspect the present invention relates to composition comprising at least one lactose-positive, Streptococcus thermophilus strain as described according to the present invention; in combination with at least one other strain of the Lactobacillus genus, such as Lactobacillus delbrueckii subspecies bulgaricus, and/or a mesophilic lactic acid bacteria, such as a species of the genus Lactococcus, such as a species of Lactococcus lactis, such as a strain of Lactococcus lactis subspecies cremoris, a strain of Lactococcus lactis subspecies hor- dinae, or Lactococcus lactis subspecies lactis, or a strain of the Bifidobacterium genus, or a strain of the Leuconostoc genus, such as Leuconostoc mesenteroides subspecies cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

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

In a further aspect the present invention relates to a method for eliminating or for 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 in semi-hard cheese making and/or soft-cheese, which method comprises the step of inoculating and fermenting a milk substrate with the Streptococcus thermophilus strain as defined herein, or a composition according to the present invention.

In a further aspect the present invention relates to a cheese, such as a semi-hard cheese and/or soft-cheese comprising at least one Streptococcus thermophilus strain as defined herein, a composition according to the present invention, or as obtained by a method according to the present invention. As used herein "cheese" refers to the term in its normal meaning of the dairy product comprising proteins and fat from milk, usually the milk of cows, buffalo, goats, camel or sheep, produced in wide ranges of flavors, textures, and forms by the coagulation of the milk protein casein, and produced by methods wherein milk is usually acidified and the casein caused to coagulate, whereafter the solid curds are separated from the liquid whey and pressed into finished cheese. Some cheeses have aromatic molds on the rind, the outer layer, or throughout. Specific examples of fresh or aged cheese may be any one selected from the group consisting of natural cheese, mozzarella cheese, fresh cheese, camembert cheese, Berg cheese, semi-hard cheeses, such as Gouda, Edam, Jarlsberg, Maasdam, Raclette, Cheddar cheese, and Tilsiter, hard cheeses such as Emmental, Parmesan and Gruyere, and Appen- zeller cheese. Semi-hard cheeses refer to cheeses with a firm, slightly springy texture, but dense in consistency, often made by compressing curds into a solid, draining them of any remaining whey in the process. A "soft-cheese" is used in its traditional meaning to refer to a cream cheese, such as Brie and Neufchatel. As used herein a "soft-cheese also includes Semi- soft cheese, such as cheeses having a high moisture content including Havarti, Munster, Port- Salut and Butterkase.

A "curd-washing-step" or "curd rinsing" is a step in the normal conventional process for the manufacture of cheeses, such as semi-hard cheese to decrease the residual lactose content and, thereby, decrease 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 (aka "cheese-before-brining") upon 6 hours of fermentation. This step of cheese making process is also referred to as "delactosing" or a delactosing step. The term "delactosing" may also refer to any step of lactose dilution by adding water during the standardization of milk or the cheese-making process, such as diafiltration, concentrated milk dilution, and curd washing.

Curd washing generally refers to a specific part of the process applied in the manufacture of cheese, such as Dutch-style cheeses including Gouda, Edam and Maasdammer, and some Swiss/Dutch-style cheeses such as Samso, Havarti and Danbo. It generally involves removal of part (30-45% of the milk volume) of the whey after cutting and its replacement by hot water (55-60-C). It is a means of dilution of the lactose content in the moisture phase of the cheese curd. The whey is partially pumped, and curd is washed with water addition (10% to 50% of total volume). This water addition will help to remove lactose from the curd. The energy and environmental cost of curd washing is extremely heavy for the cheese maker. Indeed, the volumes of water to be heated as well as the increase in the volumes of whey to be treated are extremely expensive. Avoiding the curd-washing step would be beneficial from both an economical and an environmental prospective. Some embodiment herein refers to a "variant sequence" of the strain as defined herein has a sequence identity of at least 90%, or at least 95% with reference to a specific SEQ ID NO referred to, or with the genome sequence of the parent strain from which the variant sequence is obtained including or without including the specific mutation or insertion of the strain, in particular an identity of 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% with the specific SEQ ID NO referred to, or with the genome sequence of the parent strain from which the variant is obtained. The identity is described in comparing the two sequences over their full-length (global alignment) and may be calculated using any program based on the Needleman-Wunsch algorithm.

In some embodiments, the present invention is directed to the use of a lactose-positive Streptococcus thermophilus strain carrying a mutation in one, two, three or more genes selected from the group consisting of 1) one or more mutation in the glcK gene encoding a glucokinase, 2) one or more mutation in the ccpA gene encoding the catabolite control protein A, CcpA, selected from a non-sense mutation or a mutation leading to a frameshift of the open reading frame of the ccpA gene, 3) one or more mutation in at least one of the genes encoding the proteins of the mannose-glucose-specific PTS, such as one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene, which mutation results in the expression of a truncated version of any one of the IIAB^man, IIC- ^man or IID^man proteins, respectively, 4) any combination thereof, such as one or more mutation in the glcK gene encoding a glucokinase, and a least one gene encoding a protein of the mannose-glucose-specific PTS, 5) one or more mutation in the ccpA gene encoding the catabolite control protein A, CcpA, and a least one gene encoding a protein of the mannose-glu- cose-specific PTS, 6) one or more mutation in the glcK gene encoding a glucokinase and a ccpA gene, and 7) one or more mutation in the glcK gene encoding a glucokinase, and a gene encoding a protein of the mannose-glucose-specific PTS, and a ccpA gene encoding the catabolite control protein A, CcpA, as described in the examples of WO2019122365, W02019197051, and European Patent Application 22159955.8.

In some embodiments, the lactose-positive Streptococcus thermophilus strain is carrying a mutation in one gene encoding a protein of the mannose-glucose-specific PTS. In an 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 and the manO gene. In an 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 an embodiment, the lactose-positive Streptococcus thermophilus strain is carrying a mutation in one gene selected from the group consisting of manL gene, the manM gene and the manN gene. In an embodiment, the lactose-positive Streptococcus thermophilus strain is carrying a mutation in 2 genes selected from the group consisting of manL gene, the manM gene and the manN gene. In an embodiment, the lactose-positive Streptococcus thermophilus strain is carrying a mutation in the manL gene, the manM gene and the manN gene. In some embodiments, the lactose-positive Streptococcus thermophilus strain is carrying a mutation in only one gene encoding a protein of the mannose-glucose-specific PTS being the manM gene, as described in the examples of WO2019122365, W02019197051, and European Patent Application 22159955.8.

Lactose Overconsumption Assay:

In order to evaluate the impact of specific mutations on lactose overconsumption, the following assay may be used wherein engineered strains and appropriate control strains are used to ferment milk as described in Test 2 in Example 2. Accordingly, the amounts of lactose may be measured by HPLC (High Liquid Performance Chromatography) on a Vanquish system provided by Thermo Fisher Scientific equipped with a refractometer detector. Five grams of inoculated milk before incubation (TO sample) and of inoculated milk after incubation (sample T24h) or five grams of the curd/cheese are diluted (or crushing with an IKA Ultra-Turrax at 13500 rpm for cheese samples) in 25 mL sulfuric acid solution 0.025 N. After homogenization, samples are centrifuged 10 min at 4600 rpm and the supernatant is filtered on nylon 0.2 pm filter into HPLC vials. Five microliters of sample are injected on an H+ ion exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm x 7.8 mm). The elution is performed in isocratic mode with sulfuric acid 0.025 N at 0.7 mL/min. Molecules are separated in 20 min at 40°C. Carbohydrates are detected with refractometer at 35°C.

In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention do not carry any mutation in a gene selected from a gene encoding a protein of the mannose-glucose-specific PTS and a ccpA gene encoding the catabolite control protein A, CcpA.

For the avoidance of doubt, the Streptococcus thermophilus species is to be understood as a Streptococcus salivarius subsp. thermophilus strain. By the expression "lactose-positive", is meant a Streptococcus thermophilus strain which is able to grow on lactose as a sole source of carbohydrate source, in particular on a M17 medium supplemented with 3% lactose. In one embodiment, by "lactose-positive" is meant a phenotype of a Streptococcus thermophilus strain which, when assayed by inoculating - into a M17 broth containing 3% lactose - with an overnight culture of this S. thermophilus strain tested at a rate of 1%, and incubating for 20 hours or alternatively 24 hours at 37°C, a pH of 5.5 or lower is reached at the end of incubation.

By the expression "galactose-negative" , it is meant a Streptococcus thermophilus strain which is not able to grow on galactose as a sole source of carbohydrate source, in particular on a M17 medium supplemented with 2% galactose. In a particular embodiment, the "galactose-negative" phenotype is assayed by inoculating - into a M17 broth containing 2% galactose - an overnight culture of the S. thermophilus strain to be tested at 1% and incubating for 20 hours at 37°C, and wherein a pH of 6 or above at the end of incubation is indicative of a galactose-negative phenotype.

By the expression "derivative" in reference to a parental strain (e.g., DSM28255-de- rivative), it is meant a strain obtained from an original strain (e.g., from the DSM28255- strain) by replacement of one of its genes (such as glcK, manM, ...) by another allele (in particular an allele carrying some specified mutations) of the same gene. In an embodiment, the derivative is obtained by the replacement of the gene and its promoter of the original strain by another allele of that gene and promoter. In an embodiment, the derivative is obtained by the replacement of all or part of the coding sequence of a gene of the original strain by another allele of that gene or part of it.

For embodiments requiring any of glcK sequencing, transfer of the glcK allele of a suitable strain into the genome of other S. thermophilus strains, screening and selection of Streptococcus thermophilus strains for relevant properties, identification of relevant mutations in the glcK gene, the identification of and use of further glcK mutations, such as the non-con- servative amino acid difference G144S, the identification of and use of specific mutations in the glcK gene, such as the non-conservative amino acid difference, E275K, in the GlcK sequence, reference is made to co-pending international patent application with publication number WO2019122365 and W02019197051, as well as European Patent Application 22159955.8.

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

Milk acidifying performance

The acidifying properties of S. thermophilus strains to be used according to the invention may be evaluated by recording the pH over time, during milk fermentation as described in the Milk Acidifying Assay as follows: raw whole cow's milk previously pasteurized 1 min at 74 °C was inoculated at 1% (v/v, about 10⁹ CFU/ml) with a culture of the S. thermophilus strain to be assayed (overnight culture grown in semi-skimmed milk 10% BBA heated 20 min 120°C inoculated at 1% from a frozen vial). Pasteurized milk contains around 48 g/Kg of lactose. The inoculated milk flasks were incubated statically in a water bath at 32°c for lh30, then heated for 15 min to reach and remain at 36°C for 1 hour, then slowly lowered to 28°C for up to 24h to obtain fermented milk. The pH is monitored for 24 hours using the CINAC system (Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain). The pH is measured and recorded every 5 or 25 minutes.

In some embodiments, the strains used according to the present invention comprises a mutation in the gene encoding a protein of the mannose-glucose-specific PTS that either alone or in combination with mutations of other genes may lead to the ability to overconsume lactose from the medium into the bacteria. In some embodiments, a mutated glcK gene encodes a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null. In some embodiments, said lactose-positive Streptococcus thermophilus strain carries a mutation in a gene encoding a protein of the mannose-glucose-specific PTS leading to the ability to overconsume lactose from the medium and carrying a mutation in the glcK gene encodes a glucokinase, such that the glucokinase activity of which in said strain is significantly reduced but not null in said strain. 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.

In some embodiments, the mutation in the gene encoding a protein of the mannose-glu- cose-specific PTS either alone or in combination with mutations of other genes leading to the ability to overconsume lactose from the medium. In some embodiments, a mutated glcK gene encodes a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null in said strain. In some embodiments, said lactose-positive Streptococcus thermophilus strain carrying a mutation in the glcK gene also carries a mutation in the ccpA gene.

The following parts describe respectively mutations of the glcK gene, mutations of the gene encoding a protein of the mannose-glucose-specific PTS (such as mutations of the manL, manM and manN genes) and mutations of the ccpA gene.

Though these mutations are disclosed herein separately (for sake of clarity), any embodiment of one part can be combined with any embodiment of another part or with any embodiment of the two other parts, to design a lactose-positive Streptococcus thermophilus strain as defined herein.

For the avoidance of doubt, in some embodiments the present invention is directed to the use of a lactose-positive Streptococcus thermophilus strain as defined herein, wherein said strain carries: 1) one or more mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity; or

2) one or more mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity and a mutation in at least one, in particular one, gene encoding a protein of the mannose-glucose-specific PTS; or

3) one or more mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity and a mutation in 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 mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity and a mutation in its ccpA gene.

5) one or more mutation 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 mutation in at least one, in particular one, gene encoding a protein of the mannose-glucose-specific PTS;

7) one or more mutation in its ccpA gene; wherein each strain carrying such mutations obtain the ability to overconsume lactose.

Mutations of the alcK aene

This part describes mutations of the glcK gene affecting the glucose porter which can be used either alone, in combination with one or more mutation of a gene encoding a protein of the mannose-glucose-specific PTS, in combination with one or more mutation of the ccpA gene as defined herein, or in combination with both one or more mutation of a gene encoding a protein of the mannose-glucose-specific PTS as defined herein and one or more mutation of the ccpA gene as defined herein, in the context of a lactose-positive Streptococcus thermophilus strain used according to the invention.

In some embodiments, a mutated glcK gene of a strain used according to the invention encodes a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null. Indeed, the inventors have put in evidence that some mutated alleles of the glcK gene codes for a glucokinase (GlcK), the glucokinase activity of which is significantly reduced but not null, when said mutated glcK gene is present in a lactose-positive Streptococcus thermophilus strain.

The expression "glcK gene encoding a glucokinase" means any DNA sequence of a Streptococcus thermophilus strain encoding the glucokinase enzyme which catalyses the conversion of glucose and ATP to glucose-6-phosphate (G6P) and ADP. Non-limitative examples of Streptococcus thermophilus glucokinase sequences are disclosed as SEQ ID Nos: 2, 4, 6, 8, 10 12, 14, 16, 18 and 20. In some embodiments within the present invention, the glucokinase activity in a Streptococcus thermophilus strain is significantly reduced but not null as a consequence of a mutation in its glcK gene. In other words, the allele of the glcK gene carried by said strain is such that the glucokinase activity in said strain is significantly reduced but not null.

The expressions "reduced glucokinase activity, but not null" or "glucokinase activity in said strain is significantly reduced but not null" refers to a strain the glucokinase activity of which is both:

- significantly reduced in said strain, in particular as compared to the glucokinase activity in a strain carrying a non-mutated glcK gene; and

- not null, i.e., that an activity is detectable by the Glucokinase Activity Assay as defined herein.

According to embodiments of the invention, the feature "glucokinase activity in said strain is significantly reduced but not null" can be determined by methods well known in the art. Thus, methods for measuring the glucokinase activity in a Streptococcus thermophilus strain are known and include enzyme assays with commercially available reactants. Reference is made herein to the paragraph 2.4 of Pool et al. (2006. Metabolic Engineering 8(5); 456-464) (incorporated herein by reference). In a particular embodiment, the glucokinase activity in a Streptococcus thermophilus strain used according to the invention is assayed by the Glucokinase Activity Assay [i.e. the Glucokinase Activity Assay is carried out using the Streptococcus thermophilus strain used according to the invention].

Glucokinase Activity Assay:

A fresh overnight culture of the Streptococcus thermophilus strain to be assayed in M17 containing 30 g/L lactose is obtained and used to inoculate at 1% (vol/vol) 10 ml of fresh M17 30 g/L lactose. Cells are harvested by centrifugation (6000 g, 10 min, 4°C) at a 600 nm optical density (OD600) of 0.8 +/- 0.2, washed in 5 ml cold GLCK buffer (5 mM MgCI2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]), and resuspended in 500 pl cold GLCK buffer. EDTA-free protease inhibitors "complete™" (Roche, supplier reference 04693132001) is added in GLCK buffer as described by the provider. Cells are disrupted by the addition of 100 mg glass beads (ISO- 212 pm, Sigma G1145) to 200 pl resuspended cells and oscillation at a frequency of 30 cy- cles/s for 6 min in a MM200 oscillating mill (Retsch, Haan, Germany). Cell debris and glass beads are removed by centrifugation (14000 g, 15 min, 4°C), and supernatant transferred into a clean 1.5 mL centrifuge tube kept on ice. Total protein content is determined by using the FLUKA Protein Quantification Kit-Rapid (ref 51254). The glucokinase activity in the cell extracts is determined spectrophotometrically by a glucose-6-phosphate dehydrogenase (G- 6PDH, EC1.1.1.49) :NADPH-coupled assay (Porter et al., 1982), essentially as described by Pool et al. (2006). Each sample (5, 10 and 20 pL) is added to assay buffer (10 mM K₂HPO₄ / KH2PO4 [pH 7.2], 5 mM MgCI2, 1 mM ATP, 20 mM glucose, 1 mM NADP, 1 U G-6PDH) in a 250 pL final volume, and the mixture was left for 5 min at 30°C. The optical density at 340 nm is measured for 5 minutes by using a Synergy HT multi-detection microplate reader (BIO- TEK). One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 pmole of D-glucose to D-Glucose 6-phosphate per minute under the assay conditions. Glucokinase activity is calculated as follows:

Glucokinase activity (U/g of total protein extract) = dOD x V / [dt x I x £ x Qprot], wherein:

- dOD is the variation of optical density (OD) at 340 nm

- V is the volume of the reaction (herein 250 pL) dt = measurement time (in minutes)

I = optical path length (herein 0.73 cm)

E = molar attenuation coefficient of NADPH; H⁺ (herein 6220 cm2 I pmol)

Qprot = quantity of protein in the cuvette (in g) Measurements are triplicated for each sample, and the glucokinase specific activity values given herein under the Glucokinase Activity Assay are the mean of three independent experiments.

In a first particular embodiment of the feature "glucokinase activity in said strain is significantly reduced but not null", the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 200 and 1500 U/g of total protein extract, as assayed by the Glucokinase Activity Assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 300 and 1200 U/g of total protein extract, as assayed by the Glucokinase Activity Assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 400 and 1000 U/g of total protein extract, as assayed by the Glucokinase Activity Assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between a minimal value selected from the group consisting of 200, 300 and 400 U/g of total protein extract and a maximal value selected from the group consisting of 1000, 1200 and 1500 U/g of total protein extract, as assayed by the Glucokinase Activity Assay. It is noteworthy that, as mentioned in the Glucokinase Activity Assay, the glucokinase activity values disclosed herein are the mean of three independent experiments (triplicates).

In a second particular embodiment of the feature "glucokinase activity in said strain is significantly reduced but not null", the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 5 and 60% the activity of the glucokinase activity of the DGCC7710 strain deposited at the DSMZ under accession number DSM28255 on January 14^th, 2014. By "glucokinase activity of the DSM28255 strain", it is meant the activity of the DSM28255 strain glucokinase (/.e., with SEQ ID NO:2) as assayed by the Glucokinase Activity Assay in the DSM28255 strain [i.e., the the Glucokinase Activity Assay is carried out using the DSM28255 strain]. The percentage value is calculated based on the glucokinase activity in the strain used according to the invention and the glucokinase activity of the DSM28255 strain, both assayed by the Glucokinase Activity Assay. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 10 and 50% the glucokinase activity of the DSM28255 strain. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strain used according to the invention is between 15 and 40% the glucokinase activity of the strain DSM28255. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain used according to the invention is between a minimal percentage selected from the group consisting of 5, 10 and 15 % the glucokinase activity of the DSM28255 strain and a maximal percentage selected from the group consisting of 40, 50 and 60 % the glucokinase activity of the DSM28255 strain. In a particular embodiment and whatever the range of percentages, the activity of the glucokinase activity is assayed by the Glucokinase Activity Assay as described herein. It is noteworthy that the percentage values disclosed herein are calculated based on glucokinase activity values which are the mean of three independent experiments (triplicates) as assayed by the Glucokinase Activity Assay.

In the first and second particular embodiments, the following strains can be used as controls in the Glucokinase Activity Assay:

- as a positive control (i.e., a Streptococcus thermophilus strain, which is representative of strains carrying a non-mutated glcK gene) : strain DGCC7710 deposited at the DSMZ under accession number DSM28255 on January 14^th, 2014.

The feature "glucokinase activity in said strain is significantly reduced but not null" can also be characterized by the maximum forward velocity of the glucokinase (herein called Vmax, and defined as the velocity of the Glucose + ATP conversion to G6P + ADP) or by the inverse of the affinity of the glucokinase (called Km) for one or two of its substrates, i.e., glucose and ATP. In some embodiments, the feature "glucokinase activity in said strain is significantly reduced but not null" for the strain used according to the invention is further characterized by the maximum forward velocity (Vmax) of its glucokinase in said strain.

Therefore, in combination with the first or second particular embodiment of the feature "glucokinase activity in said strain is significantly reduced but not null" defined herein, the maximum forward velocity (Vmax) of the glucokinase in the lactose-positive Streptococcus thermophilus strain used according to the invention is significantly reduced but not null. The feature "glucokinase Vmax in said strain is significantly reduced but not null" can be defined by one or two of these parameters:

- the Vmax is between 200 and 1500 U/g total protein extract, as assayed by The Glucokinase Vmax Assay.

- the Vmax is between 5 and 60 % the Vmax of the glucokinase of the DSM28255 strain deposited at the DSMZ under accession number DSM28255 on January 14^th, 2014, when both assayed by The Glucokinase Vmax Assay.

In a particular embodiment, a mutated glcK gene of a lactose-positive Streptococcus thermophilus strain used according to the invention encodes a glucokinase, wherein the glucokinase activity in said strain is significantly reduced but not null (as defined herein), and wherein the maximum forward velocity (Vmax) of its glucokinase in said strain is significantly reduced but not null and defined by one or two of these parameters:

The glucokinase maximum forward velocity (Vmax) in a Streptococcus thermophilus strain described herein is assayed by The Glucokinase Vmax Assay [carried out using the Streptococcus thermophilus strain used according to the invention].

The Glucokinase Vmax Assays

The maximal forward velocity (Vmax) is determined by using various concentrations of glucose (0, 5, 10, 15, 20 mM) on crude extract prepared as described in the Glucokinase Activity Assay. Measurements are triplicated for each sample, and the Vmax values given under The Glucokinase Vmax Assay are the mean of three independent experiments. The linear regression representing the inverse of the specific velocity in function of the inverse of the glucose concentration gives the inverse of the maximal forward velocity at the intersection with the Y- axis of the graphic.

In a particular embodiment of the maximum forward velocity of the glucokinase in the Streptococcus thermophilus strain used according to the invention, the Vmax is between 200 and 1500 U/g total protein extract, as assayed by The Glucokinase Vmax Assay. In a particular embodiment, the Vmax is between 300 and 1200 U/g total protein extract, as assayed by The Glucokinase Vmax Assay. In a particular embodiment, the Vmax is between 400 and 1000 U/g total protein extract. In a particular embodiment, the Vmax of the glucokinase in the Streptococcus thermophilus strain used according to the invention is between a minimal value selected from the group consisting of 200, 300 and 400 U/g of total protein extract and a maximal value selected from the group consisting of 1000, 1200 and 1500 U/g of total protein extract, as assayed by The Glucokinase Vmax Assay.

In a particular embodiment of the maximum forward velocity of the glucokinase in the Streptococcus thermophilus strain used according to the invention, the Vmax is between 5 and 60 % the Vmax of the glucokinase of the DSM28255 strain. By "Vmax of the glucokinase of the DSM28255 strain", it is meant the Vmax of the DSM28255 strain glucokinase (/.e., with SEQ ID NO:2) as assayed by The Glucokinase Vmax Assay in the DSM28255 strain [i.e., the The Glucokinase Vmax Assay is carried out using the DSM28255 strain]. The percentage value is calculated based on the Vmax of the glucokinase in the strain used according to the invention and the Vmax of the DSM28255 strain, both assayed by The Glucokinase Vmax Assay. In a particular embodiment, the glucokinase Vmax in the Streptococcus thermophilus strain used according to the invention is between 10 and 50 % the Vmax of the glucokinase of the DSM28255 strain, when both assayed by The Glucokinase Vmax Assay. In a particular embodiment, the glucokinase Vmax in the Streptococcus thermophilus strain used according to the invention is between 15 and 40% the Vmax of the glucokinase of the DSM28255 strain. In a particular embodiment, the Vmax of the glucokinase in the Streptococcus thermophilus strain used according to the invention is between a minimal percentage selected from the group consisting of 5, 10 and 15 % the Vmax of the glucokinase activity of the DSM28255 strain and a maximal percentage selected from the group consisting of 40, 50 and 60 % the Vmax of the glucokinase activity of the DSM28255 strain.

The lactose-positive Streptococcus thermophilus strain used according to the invention may carry one or more mutation in the glcK gene encoding a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null as defined herein and optionally wherein the maximum forward velocity of the glucokinase in said strain is significantly reduced but not null as defined herein.

By "mutation in the glcK gene" within the present invention, it is meant any nucleotide variation within the glcK gene, wherein said variation at the nucleotide level leads to a glucokinase activity in a strain carrying this mutated glcK gene (as the sole glcK gene) which is significantly reduced but not null as defined herein and optionally leads to a maximum forward velocity of the glucokinase in said strain which is significantly reduced but not null as defined herein. In a particular embodiment, by "mutation in the glcK gene" within the present invention, it is meant any nucleotide variation within the open reading frame of the glcK gene, wherein said variation at the nucleotide level leads to a glucokinase activity in a strain carrying this mutated glcK gene (as the sole glcK gene) which is significantly reduced but not null as defined herein and optionally leads to a maximum forward velocity of the glucokinase in said strain which is significantly reduced but not null as defined herein.

Thus, though two Streptococcus thermophilus strains may differ by the sequence of their respective glcK gene, this does not necessarily mean that one of these two glcK genes is mutated in the sense of the invention. Indeed, are not considered as mutations within the present invention:

- variations at the nucleotide level which do not lead to any change at the protein level (silent variation) and which do not impact the translation of the glcK RNA; and

- variations at the nucleotide level which do lead to a change at the protein level, but wherein this change does not impact the glucokinase activity of the resulting GlcK protein and optionally the maximum forward velocity of the resulting GlcK protein, as defined herein. Indeed, such variations can be observed at the level of the glcK gene of the Streptococcus thermophilus described herein without impacting the scope of protection.

Non-limitative examples of glcK genes which are not considered as mutated in the sense of the use according to the present invention are:

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO:2, in particular the polynucleotide as defined in SEQ ID NO: 1; this GlcK type is the one of DGCC7710 strain deposited at the DSMZ under accession number DSM28255 on January 14^th, 2014; - the polynucleotide encoding the glucokinase as defined in SEQ ID NO:4, in particular the polynucleotide as defined in SEQ ID NO:3;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO:6, in particular the polynucleotide as defined in SEQ ID NO:5;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO:8, in particular the polynucleotide as defined in SEQ ID NO:7;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO: 10, in particular the polynucleotide as defined in SEQ ID NO:9;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO: 12, in particular the polynucleotide as defined in SEQ ID NO: 11;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO: 14, in particular the polynucleotide as defined in SEQ ID NO: 13;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO: 16, in particular the polynucleotide as defined in SEQ ID NO: 15;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO: 18, in particular the polynucleotide as defined in SEQ ID NO: 17;

- the polynucleotide encoding the glucokinase as defined in SEQ ID NO:20, in particular the polynucleotide as defined in SEQ ID NO: 19.

Moreover, some nucleotide mutations within the glcK gene are not considered suitable to the invention, because they lead to a glucokinase, the activity of which is null or is under the minimal value defined herein, as assayed by the Glucokinase Activity Assay. In some embodiments, the Streptococcus thermophilus described herein does not carry a mutation selected from the group consisting of a mutation leading to the knock-out of the glcK gene and large deletions within the glcK gene.

In some embodiments, a lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in the open reading frame of the glcK gene leading to the substitution of an amino acid in the GlcK protein, the glucokinase activity of which in said strain carrying a mutated glcK gene is significantly reduced but not null (as defined herein) and optionally wherein the maximum forward velocity of the glucokinase in said strain is significantly reduced but not null as defined herein. In a particular embodiment, a lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in the glcK gene leading to the substitution of an amino acid in the GlcK protein, the glucokinase activity of which in said strain carrying a mutated glcK gene is significantly reduced but not null (as defined herein) and optionally wherein the maximum forward velocity of the glucokinase in said strain is significantly reduced but not null as defined herein. In a particular embodiment, the Streptococcus thermophilus strain used according to the invention carries a mutation in the glcK gene such that the GlcK protein is 322-amino acids in length and wherein the glucokinase activity in said strain is significantly reduced but not null as defined herein and optionally wherein the maximum forward velocity of the glucokinase in said strain is significantly reduced but not null as defined herein. As discussed above, some DNA modifications can be observed at the level of the glcK gene of the Streptococcus thermophilus described herein which do not impact the glucokinase activity of the strain. Based on the Glucokinase Activity Assay defined herein together with the control strains defined herein, the person skilled in the art would know how to identify 1) a glcK gene encoding a glucokinase, the glucokinase activity of which in a strain carrying this glcK gene is significantly reduced but not null (as defined herein) and optionally wherein the maximum forward velocity of the glucokinase in a strain carrying this mutated glcK gene is significantly reduced but not null (as defined herein), 2) a glcK gene bearing a modification having no impact on the glucokinase activity in a strain carrying this modification or 3) a glcK gene encoding a glucokinase, the glucokinase activity of which in a strain carrying this glcK gene is null (as defined herein).

The present inventors have identified two positions within the glucokinase, for which the amino acid nature has been shown to impact the activity of the glucokinase, such that the glucokinase activity is significantly reduced but not null as defined herein and to impact the Vmax of the glucokinase such that the Vmax is significantly reduced but not null as defined herein: position 144 and position 275 of the glucokinase (i.e., codon 144 and 275 of the glcK gene). It is noteworthy that based on the Glucokinase Activity Assay and the Glucokinase Vmax Assay defined herein together with the control strains, the person skilled in the art would know how to identify other positions and appropriate amino acids within the glucokinase, to obtain a glucokinase activity significantly reduced but not null (as defined herein) and optionally a maximum forward velocity which is significantly reduced but not null, and thus the corresponding glcK gene.

In some embodiments, the amino acid at position 275 of the glucokinase (encoded by the glcK gene of the Streptococcus thermophilus strain used according to the invention) is not a glutamic acid (i.e., is any amino acid except a glutamic acid); thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is neither GAA nor GAG. In a particular embodiment, the amino acid at position 275 of the glucokinase is not an acidic amino acid (i.e., is any amino acid except an acidic amino acid); thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon encoding a non-acidic amino acid. In a particular embodiment, the amino acid at position 275 of the glucokinase is selected from the group consisting of lysine and any of its conservative amino acids; thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon encoding an amino acid selected from the group consisting of a lysine and any of its conservative amino acids. In a particular embodiment, the amino acid at position 275 of the glucokinase is a lysine; thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is either AAA or AAG. In a particular embodiment, the nucleotides 823-825 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention are AAA or AAG.

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) a sequence as defined in SEQ ID NO:25, wherein the amino acid at position 275 is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine; and b) a GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25, wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In a particular embodiment, the GlcK variant sequence is 322-amino acids in length.

In another embodiment, the amino acid at position 144 of the glucokinase (encoded by the glcK gene of the Streptococcus thermophilus strain used according to the invention) is not a glycine (/.e., is any amino acid except a glycine); thus, in some embodiments, the codon 144 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is not GGT, GGC, GGA or GGG. In a particular embodiment, the amino acid at position 144 of the glucokinase is not an aliphatic amino acid (/.e., is any amino acid except an aliphatic amino acid). In a particular embodiment, the amino acid at position 144 of the glucokinase is selected from the group consisting of serine and any of its conservative amino acids; thus, in some embodiments, the codon 144 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon encoding an amino acid selected from the group consisting of a serine and any of its conservative amino acids. In a particular embodiment, the amino acid at position 144 of the glucokinase is a serine; thus, in some embodiments, the codon 144 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is AGT, AGO, TOT, TOO, TCA or TOG. In a particular embodiment, the nucleotides 430-432 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention are AGT, AGO, TOT, TOO, TCA or TCG.

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) a sequence as defined in SEQ ID NO:46, wherein the amino acid at position 144 is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine; and b) a GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46, wherein the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In a particular embodiment, the GlcK variant sequence is 322-amino acids in length. For the definition of the GIcK variant having at least 90% similarity or identity with SEQ ID NO:25, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]; the position 275 as defined in SEQ ID NO:25 is not considered for the calculation of the similarity or of the identity. In a particular embodiment, the GIcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO:25, wherein the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In some embodiments, the GIcK variant sequence has at least 95% similarity or identity with SEQ ID NO:25, wherein the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In some embodiments, the GIcK variant sequence has at least 97% similarity or identity with SEQ ID NO:25, wherein the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine.

In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:25 by from 1 to 30 amino acid substitutions wherein the amino acid at position 275 of said GIcK variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine (the position 275 is not considered for the calculation of the number of substitution(s)). In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:25 by from 1 to 20 amino acid substitutions, wherein the amino acid at position 275 of said GIcK variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:25 by from 1 to 15 amino acid substitutions wherein the amino acid at position 275 of said GIcK variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:25 by from 1 to 10 amino acid substitutions wherein the amino acid at position 275 of said GIcK variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine. In a particular embodiment, the GIcK 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, wherein the amino acid at position 275 of said GIcK variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine.

For the definition of the GIcK variant having at least 90% similarity or identity with SEQ ID NO:46, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]; the position 144 as defined in SEQ ID NO:46 is not considered for the calculation of the similarity or of the identity. In a particular embodiment, the GIcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO:46, wherein the amino acid corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In some embodiments, the GIcK variant sequence has at least 95% similarity or identity with SEQ ID NO:46, wherein the amino acid corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In some embodiments, the GIcK variant sequence has at least 97% similarity or identity with SEQ ID NO:46, wherein the amino acid corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine.

In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:46 by from 1 to 30 amino acid substitutions wherein the amino acid at position 144 of said GIcK variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine (the position 144 is not considered for the calculation of the number of substitution(s)). In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:46 by from 1 to 20 amino acid substitutions, wherein the amino acid at position 144 of said GIcK variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:46 by from 1 to 15 amino acid substitutions wherein the amino acid at position 144 of said GIcK variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In a particular embodiment, the GIcK variant sequence differs from SEQ ID NO:46 by from 1 to 10 amino acid substitutions wherein the amino acid at position 144 of said GIcK variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine. In a particular embodiment, the GIcK 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, wherein the amino acid at position 144 of said GIcK variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine.

In some embodiments, the sequence of the GIcK protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, wherein the amino acid at position 275 of said variant is any amino acid except a glutamic acid, in particular is any amino acid except an acidic amino acid, in particular is a lysine; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GIcK protein, the sequence of which is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, wherein the amino acid at position 275 of the glucokinase is not a glutamic acid, in particular is not an acidic amino acid, in particular is a lysine respectively.

In a particular embodiment, either as the SEQ ID NO:25 or any GIcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25 as defined herein (in particular SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is not a glutamic acid; thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is neither GAA nor GAG; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25 as defined herein (in particular SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is not a glutamic acid.

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

In a particular embodiment, either as the SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25 as defined herein (in particular SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is selected from the group consisting of lysine and any of its conservative amino acids; thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon encoding an amino acid selected from the group consisting of lysine and any of its conservative amino acids; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25 as defined herein (in particular SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is a lysine and any of its conservative amino acids.

In a particular embodiment, either as the SEQ ID NO:25 or any GIcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25 as defined herein (in particular SEQ ID NO:26, 27, 28, 29, 30, 31, 32, 33 or 34), the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of the glucokinase) is a lysine; thus, in some embodiments, the codon 275 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon encoding a lysine respectively, in particular is AAA or AAG, respectively; thus, 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 SEQ ID NOs: 22, 35, 36,

37, 38, 39, 40, 41, 42 and 43; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which 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 the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO:21.

In another 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 SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, wherein the amino acid at position 144 of said variant is any amino acid except a glycine, in particular is any amino acid except an aliphatic amino acid, in particular is a serine; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, wherein the amino acid at position 144 of the glucokinase is not a glycine, in particular is not an aliphatic amino acid, in particular is a serine.

In a particular embodiment, either as the SEQ ID NO:46 or any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46 as defined herein (in particular SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is not a glycine; thus, in some embodiments, the codon 144 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is not GGT, GGC, GGA or GGG; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO:46 and any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46 as defined herein (in particular SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is not a glycine. In a particular embodiment, either as the SEQ ID NO:46 or any GIcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46 as defined herein (in particular SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is not an aliphatic amino acid; thus, in some embodiments, the codon 144 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is a codon which does not encode an aliphatic amino acid; thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain used according to the invention encodes a GlcK protein, the sequence of which is selected from the group consisting of SEQ ID NO:46 and any GlcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46 as defined herein (in particular SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 (or the amino acid at position 144 of the glucokinase) is not an aliphatic amino acid.

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

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

When defining the sequence of the GlcK protein of a lactose-positive-Streptococcus thermophilus strain used according to the invention, it is according to the teaching of this application that the glucokinase activity in the strain expressing this GlcK protein is significantly reduced but not null as defined herein and optionally that the Vmax of the glucokinase in this strain is significantly reduced but not null as defined herein.

Mutations of a oene encoding a Drotein of the mannose-alucose-soecific PTS, in oar- ticular mutations of the manL, manM and manN genes.

This part describes mutations of a gene encoding a protein of the mannose-glucose- specific PTS, in particular mutations of the manL, manM and manN genes, which can be used either in combination with a mutation of a glcK gene as defined herein, or in combination with a mutation of a ccpA gene as defined herein, or in combination with both a mutation of a glcK gene and a mutation of a ccpA gene as defined herein, in the context of a lactose-positive Streptococcus thermophilus strain used according to the invention.

Any mutation in a gene encoding a protein of the mannose-glucose-specific PTS is appropriate, may be combined with a mutated glcK gene as defined herein, or combined with a mutation of a ccpA gene as defined herein, or combined with both a mutated glcK gene and a mutated ccpA gene as defined herein in a lactose-positive Streptococcus thermophilus strain.

The inventors have shown that mutations in a gene encoding a protein of the mannose- glucose-specific PTS, in particular in a mutated manL gene, a mutated manM gene, or a mutated manN gene, which leads to the ability to overconsume lactose from the medium in a lactose-positive Streptococcus thermophilus strain, are particularly advantageous within the invention.

In some embodiments, the mutation of the gene encoding a protein of the mannose-glu- cose-specific PTS, in particular of the manL gene, manM gene or manN gene, is a mutation leading to the knock-out (i.e., the complete disruption) of the gene.

In some embodiments, the mutation of the gene encoding a protein of the mannose-glu- cose-specific PTS, in particular of the manL gene, manM gene or manN gene, is a mutation of the promoter of the gene, in particular a mutation of the promoter of the gene reducing or inhibiting the transcription of the gene.

In some embodiments, the mutation of the gene encoding a protein of the mannose-glu- cose-specific PTS, in particular of the manL gene, manM gene or manN gene, is a mutation introduced into the coding sequence of the gene, in particular a mutation leading to the ability to overconsume lactose..

In some embodiments, the mutation of the gene encoding a protein of the mannose-glu- cose-specific PTS, in particular of the manL gene, manM gene or manN gene is a mutation in the coding sequence of the gene, leading to a truncated protein, in particular to a truncated IIAB^Man protein, a truncated IIC^Man protein or a truncated IID^Man protein, in particular to a truncated protein (such as a truncated IIAB^Man protein, a truncated IIC^Man protein or a truncated IID^Man protein) leading to the ability to overconsume lactose. Whatever the position of the truncation, the mutation introduced into the gene is either a nucleotide substitution leading to a STOP codon or a deletion, insertion or deletion/insertion leading to 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 leading to a STOP codon. In some embodiments, the mutation introduced into the gene is a deletion, insertion or deletion/insertion leading to a frameshift of the open reading frame and a premature STOP codon.

Though two Streptococcus thermophilus strains may differ by the sequence of their respective manL, manM or manN gene, this does not necessarily mean that one of these genes is mutated in the sense of the invention. Indeed, not considered as mutations of the manL, manM or manN gene suitable within the present invention are:

- variations at the nucleotide level which do not lead to any change at the protein level (silent variation) and which do not impact the translation of the manL, manM or manN RNA; and

- variations at the nucleotide level which do lead to a change at the protein level, but wherein this change does not lead to a lactose overconsumption, when combined with a mutated glcK gene as defined herein, or combined with a mutation of a ccpA gene as defined herein, or when combined with both a mutated glcK gene and a mutated ccpA gene in a lactose-positive phenotype of a Streptococcus thermophilus strain.

Non-limitative examples of manL, manM and manN genes (respectively encoding the IIAB^Man protein, the IIC^Man protein and the IID^Man protein) which are not considered as mutated to be suitable according to the present invention are:

- the polynucleotide encoding the IIAB^Man protein as defined in SEQ ID NO:78, in particular the polynucleotide as defined in SEQ ID NO:77; this IIAB^Man type is the one of the DSM28255 strain;

- the polynucleotide encoding the IIAB^Man protein as defined in SEQ ID NO:80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110, in particular the polynucleotide as defined in SEQ ID NO:79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109. The sequence of the IIAB^Man proteins as defined in SEQ ID NO:80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110 is from 98.4 to 99.6% identical to SEQ ID NO:78;

- the polynucleotide encoding the IIC^Man protein as defined in SEQ ID NO: 130, in particular the polynucleotide as defined in SEQ ID NO: 129; this IIC^Man type is the one of DSM28255;

- the polynucleotide encoding the IIC^Man protein as defined in SEQ ID NO: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156, in particular the polynucleotide as defined in SEQ ID NO: 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 and 155. The sequence of the IIC^Man proteins as defined in SEQ ID NO: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156 is from 98.5 to 99.6% identical to SEQ ID NO: 130;

- the polynucleotide encoding the IID^Man protein as defined in SEQ ID NO: 167, in particular the polynucleotide as defined in SEQ ID NO: 166; this IID^Man type is the one of DSM28255 strain;

- the polynucleotide encoding the IID^Man protein as defined in SEQ ID NO: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205 , in particular the polynucleotide as defined in SEQ ID NO: 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204. The sequence of the IID^Man proteins as defined in SEQ ID NO: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205 is from 97.3 to 99.6% identical to SEQ ID NO: 167.

The present inventors have identified at least one mutation in the manL gene, which 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 as compared to the original strain, when assayed by test 2 of the example 2.

In some embodiments, the mutation in the manL gene leads to the truncation of the IIAB^Man protein at position 305. In some embodiments, the mutation in the manL gene is the substitution of the nucleotide G in the nucleotide T at position 916 (leading to a stop codon at position 306). A Streptococcus thermophilus IIAB^Man protein truncated at position 305 is referred herein as IIAB^Man ₃o₅.

In some embodiments, the sequence of said IIAB^Man protein truncated in position 305 is selected from the group consisting of: a) a sequence as defined in SEQ ID NO: 112; and b) a IIAB^Man variant sequence having at least about 90%, such as at least 95% similarity or identity with SEQ ID NO: 112, in particular being 305 amino acids in length.

For the definition of the IIAB^Man variant having at least about 90%, such as at least 95% similarity or identity with SEQ ID NO: 112, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]. In a particular embodiment, the IIAB^Man variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO: 112.

In a particular embodiment, the IIAB^Man variant sequence differs from SEQ ID NO: 112 by from 1 to 30 amino acid substitutions. In a particular embodiment, the IIAB^Man variant sequence differs from SEQ ID NO: 112 by from 1 to 20 amino acid substitutions. In a particular embodiment, the IIAB^Man variant sequence differs from SEQ ID NO: 112 by from 1 to 15 amino acid substitutions. In a particular embodiment, the IIAB^Man variant sequence differs from SEQ ID NO: 112 by from 1 to 10 amino acid substitutions. In a particular embodiment, the IIAB^Man 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 sequence of the IIAB^Man protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NOs: 112 to 128.

In some embodiments, the manL gene carried by the Streptococcus thermophilus strain used according to the invention encodes a IIAB^Man protein, the sequence of which is selected from the group consisting of SEQ ID NO: 112 and any IIAB^Man variant sequence having at least about 90%, such as at least 95% similarity or identity with SEQ ID NO: 112 as defined herein (in particular SEQ ID NO: 113 to 128). In some embodiments, the manL gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO: 111.

The inventors have identified at least two mutations in the manM gene, which when each is 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] enables the strain to overconsume lactose as compared to the original strain, when assayed by test 2 of the example 2.

In some embodiments, the mutation in the manM gene leads to the truncation of the IIC^Man protein at position 208. In some embodiments, the mutation in the manM gene is the substitution of the nucleotide G in the nucleotide T at position 625 (leading to a stop codon at position 209). A Streptococcus thermophilus IIC^Man protein truncated at position 208 is referred herein as nc^Man208.

In some embodiments, the sequence of said IIC^Man protein truncated in position 208 is selected from the group consisting of: a) a sequence as defined in SEQ ID NO: 158; and b) a IIC^Man variant sequence having at least 90% similarity or identity with SEQ ID

NO: 158, in particular being 208 amino acids in length.

For the definition of the IIC^Man variant having at least 90% similarity or identity with SEQ ID NO: 158, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]. In a particular embodiment, the IIC^Man variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO: 158.

In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO: 158 by from 1 to 30 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO: 158 by from 1 to 20 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO: 158 by from 1 to 15 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO: 158 by from 1 to 10 amino acid substitutions. In a particular embodiment, the IIC^Man 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 sequence of the IIC^Man protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NO: 158 to 165.

In some embodiments, the manM gene carried by the Streptococcus thermophilus strain used according to the invention encodes a IIC^Man protein, the sequence of which is selected from the group consisting of SEQ ID NO: 158 and any IIC^Man variant sequence having at least about 90%, such as at least 95% similarity or identity with SEQ ID NO: 158 as defined herein (in particular SEQ ID NO: 159 to 165). In some embodiments, the manM gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO: 157.

In some embodiments, the mutation in the manM gene leads to the truncation of the IIC^Man protein at position 179 or 180 of SEQ ID NO: 130.

In some embodiments, the mutation in the manM gene is an insertion of one nucleotide C at any one of positions 438, 439, or 440 of SEQ ID NO: 129 creating a frameshift in manM (SEQ ID NO:212), leading to a stop codon at position 180 of SEQ ID NO: 130 (manM₁₂997')- A resulting Streptococcus thermophilus IIC^Man protein truncated at position 179 is referred herein as nc^Man179 (SEQ ID NO:213).

In some embodiments, the sequence of said IIC^Man protein truncated in position 179 is selected from the group consisting of: a) a sequence as defined in SEQ ID NO:213; and b) a IIC^Man variant sequence having at least 90% similarity or identity with SEQ ID NO:213, in particular being 179 amino acids in length.

For the definition of the IIC^Man variant having at least 90% similarity or identity with SEQ ID NO:213, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]. In a particular embodiment, the IIC^Man variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO:213.

In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO:213 by from 1 to 30 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO:213 by from 1 to 20 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO:213 by from 1 to 15 amino acid substitutions. In a particular embodiment, the IIC^Man variant sequence differs from SEQ ID NO:213 by from 1 to 10 amino acid substitutions. In a particular embodiment, the IIC^Man 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 sequence of the IIC^Man protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NO:213.

In some embodiments, the manM gene carried by the Streptococcus thermophilus strain used according to the invention encodes a IIC^Man protein, the sequence of which is selected from the group consisting of SEQ ID NO:213 and any IIC^Man variant sequence having at least about 90%, such as at least 95% similarity or identity with SEQ ID NO:213 as defined herein.

The inventors have identified at least one mutation in the manN gene, which 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 as compared to the original strain, when assayed by test 2 of the example 2.

In some embodiments, the mutation in the manN gene leads to the truncation of the IID^Man protein at position 28 of SEQ ID NO: 167. In some embodiments, the mutation in the manN gene is an insertion of a nucleotide A in the stretch of 5 nucleotides A at positions 37- 41 (leading to a stretch of 6 nucleotides A, a frameshift of the open reading frame and a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167). This Streptococcus thermophilus IID^Man protein truncated at position 28 of SEQ ID NO: 167 is referred herein as IID^Man ₂₈.

In some embodiments, the sequence of said IID^Man protein truncated in position 28 is selected from the group consisting of: a) a sequence as defined in SEQ ID NO:207; and b) an IID^Man variant sequence having at least 90% similarity or identity with SEQ ID NO:207, in particular being 28 amino acids in length.

For the definition of the IID^Man variant having at least 90% similarity or identity with SEQ ID NO:207, the similarity or identity is calculated herein over the whole length of the 2 sequences after optimal alignment [i.e., number of similar or identical amino acid residues in the aligned parts(s) of the sequences]. In a particular embodiment, the IIC^Man variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO:207.

In a particular embodiment, the IID^Man variant sequence differs from SEQ ID NO:207 by from 1 to 10 amino acid substitutions. In a particular embodiment, the IID^Man 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 sequence of the IID^Man protein of a lactose-positive Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NO:207 to 211.

In some embodiments, the manN gene carried by the Streptococcus thermophilus strain used according to the invention encodes an IID^Man protein, the sequence of which is selected from the group consisting of SEQ ID NO:207 and any IID^Man variant sequence having at least 90% similarity or identity with SEQ ID NO:207 as defined herein (in particular SEQ ID NO: 208 to 211). In some embodiments, the manN gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO:206. Whatever the mutation of the glcK gene as defined herein and the mutation of the ccpA gene as defined herein (present alone or combined in a lactose-positive Streptococcus thermophilus strain used according to the invention), at least one gene encoding a protein of the mannose-glucose-specific PTS is mutated as defined herein. Whatever the embodiment, the invention encompasses a lactose-positive Streptococcus thermophilus strain carrying a mutation 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, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manL. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manM. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manN. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manL and a mutation in manM. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manL and a mutation in manN. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manM and a mutation in manN. In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manL, a mutation in manM and a mutation in manN.

Any method can be used to identify a mutation in a gene encoding a protein of the mannose-glucose-specific PTS, in particular in the manL gene, manM gene or manN gene suitable within the lactose-positive Streptococcus thermophilus strain used according to the invention.

As an example, to identify a suitable mutation in the manL gene, manM gene or manN gene, the person skilled in the art can proceed by the following method : a) Provide a parent strain, such as any one of the DSM32587, DSM34909, DSM34910, and the DSM34911 strains (mutated in their glcK gene); b) carry out mutagenesis on the manL, manM or manN gene of the strain in a), for example by random or directed mutagenesis, to obtain a manL, manM or manN gene, the sequence of which is different from the sequence of the manL, manM or manN gene of DSM32587 or another original strain, to obtain a man-mutated strain, such as a man- mutated DSM32587 strain; c) determining if a phenotype enabling the strain to overconsume lactose as compared to the original strain is accomplished; and d) optionally determining if a PHSTOP phenotype is accomplished.

Alternatively, the person skilled in the art can proceed by the following method: a) Provide a parent strain, such as the DSM32587 strain or a DSM28255 strain in which its ccpA gene has been replaced by the mutated ccpA gene as defined in SEQ ID NO:71 (ccpAaiAi 14-120) , called herein DGCC771O-CCPAAIAH4-I2O strain (corresponding to strain ST21-C mentioned herein); b) carry out mutagenesis on the manL, manM or manN gene of the strain in a), for example by random or directed mutagenesis, to obtain a manL, manM or manN gene, the sequence of which is different from the sequence of the manL, manM or manN gene of the DGCC771O-ccpA\iAii4-i2o strain, to obtain a man-mutated DGCC771O-ccpA\iAii4-i2o strain; c) determining if a phenotype enabling the strain to overconsume lactose as compared to the original strain is accomplished; and d) optionally determining if a PHSTOP phenotype is accomplished.

Once identified, the mutated manL, manM or manN gene according to the invention can be introduced in lieu of the manL, manM or manN of a lactose-positive Streptococcus thermophilus strain, to obtain a lactose-positive Streptococcus thermophilus strain used according to the invention.

Mutations of the ccoA aene

This part describes mutations of the ccpA gene which can be used either alone, in combination with one or more mutation of a gene encoding a protein of the mannose-glu- cose-specific PTS as defined herein, in combination with one or more mutation of the glcK gene as defined herein, or in combination with both one or more mutation of a gene encoding a protein of the mannose-glucose-specific PTS as defined herein and one or more mutation of the glcK gene as defined herein, in the context of a lactose-positive Streptococcus thermophilus strain used according to the invention.

Any mutation in the ccpA gene is appropriate, as long as it either alone, in combination with one or more mutation of a gene encoding a protein of the mannose-glucose-specific PTS as defined herein, in combination with one or more mutation of the glcK gene as defined herein, or in combination with both one or more mutation of a gene encoding a protein of the mannose-glucose-specific PTS as defined herein and one or more mutation of the glcK gene as defined herein in a lactose-positive Streptococcus thermophilus strain, promote an over- consumption of lactose, when grown in milk.

In some embodiments, the ccpA gene mutation is not a mutation leading to the knock-out (i.e., the complete disruption) of the gene.

In some embodiments, the ccpA gene mutation is a mutation in the coding sequence of the ccpA gene, in particular 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 non-sense mutation (i.e. leading to a STOP codon) located between the nucleotide 1 and the nucleotide 270 of the coding sequence of the ccpA gene; and b) a mutation, located in the first quarter of the coding sequence of the ccpA gene (i.e., between nucleotide 1 and the nucleotide 250), leading to a frameshift of the open reading frame of the ccpA gene. In some embodiments, the mutation leading to a frameshift of the open reading frame of the ccpA gene is located between nucleotide 50 and the nucleotide 200 of the coding sequence of the ccpA gene. In some embodiments, the mutation leading to a frameshift of the open reading frame of the ccpA gene is located between nucleotide 100 and the nucleotide 150 of the coding sequence of the ccpA gene. Whatever the location of the mutation leading to a frameshift, the mutation is selected from the group consisting of a deletion, an insertion or a deletion/insertion (which all are not a multiple of 3).

Though two Streptococcus thermophilus strains may differ by the sequence of their respective ccpA gene, this does not necessarily mean that one of these two ccpA genes is mutated in the sense of the invention. Indeed, not considered as mutations of the ccpA gene suitable for the present invention are variations at the nucleotide level which do lead to a change at the protein level.

Non-limitative examples of ccpA genes which are not considered as mutated to be suitable according to the sense of the invention are:

- the polynucleotide as defined in SEQ ID NO:65 (ccpA type STI); this ccpA type is the one of the DSM28255 strain;

- the polynucleotide as defined in SEQ ID NO:66 (ccpA type ST2), which has 99.8% identity with SEQ ID NO:65;

- the polynucleotide as defined in SEQ ID NO:67 (ccpA type ST3), which has 99.8% identity with SEQ ID NO:65; this ccpA type is the one of the DSM33651 strain;

- the polynucleotide as defined in SEQ ID NO:68 (ccpA type ST4), which has 99.7% identity with SEQ ID NO:65;

- the polynucleotide as defined in SEQ ID NO:69 (ccpA type ST5), which has 99.8% identity with SEQ ID NO:65; and

- the polynucleotide as defined in SEQ ID NO:70 (ccpA type ST6), which has 99.7% identity with SEQ ID NO:65.

The invention may in some embodiments be directed to the use of a lactose-positive Streptococcus thermophilus strain carrying a mutation in the ccpA gene selected from the group consisting of a non-sense mutation located between the nucleotide 1 and the nucleotide 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, leading to a frameshift of the open reading frame of the ccpA gene.

In some embodiments, the mutation of the ccpA gene is a deletion of a nucleotide A in the stretch of 7 nucleotides A at positions 114-120 (leading to a frameshift of the open reading frame of the ccpA gene). Such Streptococcus thermophilus mutated ccpA gene is referred herein as CCPAAIAH4-I2O-

In some embodiments, the sequence of said ccpA gene with a STOP codon at codon 66 is selected from the group consisting of: a) a sequence as defined in SEQ ID NO:71; and b) a ccpA variant sequence having at least 90% identity with SEQ ID NO:71. The ccpA variant as defined herein carries a mutation as defined above, i.e., is selected from the group consisting of a non-sense mutation located between the nucleotide 1 and the nucleotide 270 of the coding sequence of the ccpA gene and a mutation, located in the first quarter of the coding sequence of the ccpA, leading to a frameshift of the open reading frame of the ccpA gene.

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

The person skilled in the art is given, in this part of the application, guidance on how to identify mutations of the ccpA gene other than the one specifically disclosed.

Thus, the person skilled in the art can proceed by the following method: a) Provide a parent strain, such as the DSM28255 strain in which its manL gene has been replaced by the mutated manL gene as defined in SEQ ID NO: 111 (manL gene encoding the IIAB^Man305 protein), called herein DGCC7710-IIAB^Man ₃₀5 strain; b) carry out mutagenesis on the ccpA gene of the strain in a), for example by random or directed mutagenesis, to obtain a ccpA the sequence of which is different from the sequence of the ccpA gene of the DGCC771O-IIAB^Man ₃o5 strain, to obtain a ccpA-mutated strain, such as a ccpA-mutated DGCC771O-IIAB^Man ₃o₅ strain; c) determining if a phenotype enabling the strain to overconsume lactose as compared to the original strain is accomplished; and d) optionally determining if a PH_STOP phenotype is accomplished.

Alternatively, the person skilled in the art can proceed by the following method: a) Provide a parent strain, such as the DSM28255 strain in which its manM gene has been replaced by the mutated manM gene as defined in SEQ ID NO: 157 (manM gene encoding the IIC^Man 208 protein), called herein DGCC7710-IIC^Man208 strain; b) carry out mutagenesis on the ccpA gene of the strain in a), for example by random or directed mutagenesis, to obtain a ccpA the sequence of which is different from the sequence of the ccpA gene of the DGCC771O-IIC^Man ₂o8 strain, to obtain a ccp/l-mutated strain, such as a ccp/l-mutated DGCC771O-IIC^Man ₂o8 strain; c) determining if a phenotype enabling the strain to overconsume lactose as compared to the original strain is accomplished; and d) optionally determining if a PHSTOP phenotype is accomplished.

The person skilled in the art can also proceed by the following method: a) Provide a parent strain, such as the DSM28255 strain in which its manN gene has been replaced by the mutated manN gene as defined in SEQ ID NO:206 (manN gene encoding the IID^Man ₂₈ protein), called herein DGCC7710-IID^Man ₂₈ strain; b) carry out mutagenesis on the ccpA gene of the strain in a), for example by random or directed mutagenesis, to obtain a ccpA the sequence of which is different from the sequence of the ccpA gene of the DGCC7710-IID^Man ₂₈ strain, to obtain a ccp/l-mutated strain, such as a ccp/l-mutated DGCC7710-IID^Man ₂₈ strain; c) determining if a phenotype enabling the strain to overconsume lactose as compared to the original strain is accomplished; and d) optionally determining if a PHSTOP phenotype is accomplished.

Once identified, a mutated ccpA gene - as identified herein - can be introduced in lieu of the ccpA gene of a lactose-positive Streptococcus thermophilus strain, to obtain a lactosepositive Streptococcus thermophilus strain used according to the invention.

Further characterization of the strains of the invention

The present inventors have shown that strains having one or more mutation in the glcK gene encoding a glucokinase, 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) enable, when they are used to ferment milk:

1) an increase of lactose consumption in the fermented milk; optionally

2) obtaining a fermented milk not undergoing post-acidification, even when stored at fermentation temperature for strains having one or more mutation in the glcK gene combined with a further mutation in a gene encoding a protein of the mannose-glucose-specific PTS, optionally further having a mutation in the ccpA gene.

In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention is characterized by the fact that the strain leads to a low lactose fermented milk, when used to ferment milk.

In some embodiments, the lactose-positive Streptococcus thermophilus strain used according to the invention is characterized by the fact that the strain leads to a fermented milk, not undergoing post-acidification when stored at fermentation temperature.

The expression "not undergoing post-acidification" means a milk product which, when inoculated with a strain used according to the invention and fermented as described under the section of "Milk acidifying performance", has its pH decreased to a specific pH value (i.e. the PHSTOP value) at which value the speed of acidification definitively becomes less than 0.1 mUpH/min, wherein said PHSTOP value is between 4.4 and 5.3, and optionally the slope between pH6 and pH 5.5 is at least -0.008 UpH/min.

The term "PHSTOP phenotype" as used herein refers to a strain of Streptococcus thermophilus, which when used in fermenting milk as described under the section of "Milk acidifying performance" will produce a milk product "not undergoing post-acidification" as defined herein.

Thus, the absence of post-acidification is characterized by the fact that the pH of the fermented milk stops between 4.4 and 5.3. The pH is considered to be stopped (PHSTOP), when the speed of acidification (ApH/Atime) definitively becomes less than 0.1 mUpH/min (less than 0.0001 UpH/min). By "definitively becomes", it is meant that the speed of acidification stays less than 0.1 mUpH/min for the remaining time of the fermentation described in the Milk Acidifying Assay (i.e. up to 24h at fermentation temperature), once the PHSTOP is obtained.

In some embodiments, the PHSTOP obtained using a strain used according to the invention is between 4.7 and 5.2. In some embodiments, the PHSTOP obtained using a strain used according to the invention is between 4.8 and 5.1. In some embodiments, the PHSTOP obtained using a strain used according to the invention is between a minimal value selected from the group consisting of 4.4, 4.5, 4.6, 4.7 and 4.8 and a maximal value selected from the group consisting of 5.1, 5.2 and 5.3.

In some embodiments, the fermented milk not undergoing post-acidification is also characterized by the slope between pH6 and pH5.5. The slope represents the inverse of the velocity (speed 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. method and use with lactose-i strains of the invention.

The invention is also directed to the use of a bacterial composition comprising or consisting of at least one, in particular one, lactose-positive Streptococcus thermophilus strain used according to the invention. In a particular embodiment, the bacterial composition is a pure culture, i.e., comprises or consists of a single bacterium strain. In another embodiment, the bacterial composition is a mixed culture, i.e., comprises or consists of a lactose-positive Streptococcus thermophilus strain(s) of the invention and at least one other bacterium strain. By "at least" (in reference to a strain or bacterium), it is meant 1 or more, and in particular 1, 2, 3, 4 or 5 strains.

Thus, in some embodiments, a bacterial composition of the invention comprises or consists of a lactose-positive Streptococcus thermophilus strain(s) of the invention and at least one lactic acid bacterium of the species selected from the group consisting of a Lactococcus species, a Streptococcus species, a Lactobacillus species including Lactobacillus acidophilus, an Enterococcus species, a Pediococcus species, a Leuconostoc species, a Bifidobacterium species and an 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 includes Bifidobacterium animalis, in particular Bifidobacterium animalis subsp lactis. Other lactic acid bacteria species include Leuconostoc sp., Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus.

In some embodiments, the bacterial composition comprises or consists of a lactose-positive Streptococcus thermophilus strain(s) of the invention, and at least one Streptococcus thermophilus strain, different from the S. thermophilus strain(s) of the invention and/or at least one strain of the Lactobacillus species, and/or any combination thereof. In a particular embodiment, the bacterial composition comprises or consists of the Streptococcus thermophilus strain(s) of the invention, one or several strain(s) of the species Lactobacillus delbrueckii subsp. bulgaricus and/or one or several strain(s) of the species Lactobacillus helveticus and/or any combination thereof, and optionally at least one Streptococcus thermophilus strain, different from the S. thermophilus strain(s) of the invention. In a particular embodiment, the bacterial composition comprises or consists of the Streptococcus thermophilus strain(s) of the invention, at least one strain of species Streptococcus thermophilus, different from the S. thermophilus strain(s) of the invention, and a strain of the species Lactobacillus delbrueckii subsp. bulgaricus. In another particular embodiment, the bacterial composition comprises or consists of the Streptococcus thermophilus strain(s) of the invention, and a strain of the species Lactobacillus delbrueckii subsp. bulgaricus.

In some embodiments, the bacterial composition comprises or consists of the Streptococcus thermophilus strain(s) of the invention, a Lactococcus lactis subsp. lactis and/or a Lactococcus lactis subsp. cremoris.

In a particular embodiment of any bacterial composition defined herein, either as a pure or mixed culture, the bacterial composition further comprises at least one probiotic strain such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paraca- sei, or Lactobacillus casei.

In a particular embodiment, the bacterial composition, either as a pure or mixed culture as defined above is under frozen, dried, freeze-dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder. In a particular embodiment, the bacterial composition of the invention is in a frozen format or in the form of pellets or frozen pellets, in particular contained into one or more box or sachet. In another embodiment, the bacterial composition as defined herein is under a powder form, such as a dried or freeze-dried powder, in particular contained into one or more box or sachet.

In a particular embodiment, the bacterial composition of the invention, either as a pure culture or mixed culture as defined above, and whatever the format (frozen, dried, freeze- dried, liquid or solid format, in the form of pellets or frozen pellets, or in a powder or dried powder) comprises a lactose-positive Streptococcus thermophilus strain(s) of the invention in a concentration comprised in the range of 10⁵ to 10¹² cfu (colony forming units) per gram of the bacterial composition. In a particular embodiment, the concentration of a lactose-positive Streptococcus thermophilus strain(s) within the bacterial composition of the invention is in the range of 10⁷ to 10¹² cfu per gram of the bacterial composition, and in particular at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹ CFU/g of the bacterial composition. In a particular embodiment, when in the form of frozen or dried concentrate, the concentration of a lactose-positive Streptococcus thermophilus strain(s) - as pure culture or as a mixed culture - within the bacterial composition is in the range of 10⁸ to 10¹² cfu/g of frozen concentrate or dried concentrate, and more preferably at least 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹ or at least 10¹² cfu/g of frozen concentrate or dried concentrate.

The invention also concerns a method for manufacturing a fermented product being a cheese, comprising a) inoculating a substrate with a lactose-positive Streptococcus thermophilus strain(s) of the invention and b) fermenting said inoculated substrate, to obtain a fermented cheese product. In a particular embodiment, a lactose-positive Streptococcus thermophilus strain(s) described herein is inoculated as a bacterial composition as defined herein, such as a pure culture or a mixed culture. In some embodiments, the substrate into which the S. thermophilus strain(s) or bacterial composition described herein is added to is milk substrate. By "milk substrate", it is meant milk of animal and/or plant origin. In a particular embodiment, the milk substrate is of animal origin, such as cow, goat, sheep, buffalo, zebra, horse, donkey, or camel, and the like. The milk may be in the native state, a reconstituted milk, a skimmed milk, or a milk supplemented with compounds necessary for the growth of the bacteria or for the subsequent processing of fermented milk. Therefore, in a particular embodiment, the invention also provides a method for manufacturing a cheese product, comprising a) inoculating a milk substrate with a lactose-positive Streptococcus thermophilus strain(s) or bacterial composition described herein and b) fermenting said inoculated milk substrate, to obtain a fermented cheese product.

Various features and embodiments of the present invention will now be described by way of non-limiting examples.

Numbered embodiments of the invention:

1. Use of a lactose-positive, Streptococcus thermophilus strain in the production of a cheese, such as a semi-hard cheese or a soft-cheese, which strain has the ability to consume at least about 35% lactose when grown in raw whole cow's milk inoculated at 1% v/v or about 10⁹ CFU/ml of the Streptococcus thermophilus strain, such as measured by Test 2 described herein. 2. The use according to embodiment 1, which use is for eliminating or for reducing the need for a delactosing step, such as a curd-washing-step or any other lactose dilution in the production of a cheese, such as in semi-hard cheese making.

3. The use according to embodiments 1 or 2, which strain comprises one or more mutations selected from: a) a mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, but not null; and/or b) a mutation in the ccpA gene selected from a non-sense mutation or a mutation leading to a frameshift of the open reading frame of the ccpA gene, each mutation located between the nucleotide 1 and the nucleotide 270 of SEQ ID:65 of the coding sequence of the ccpA gene, any of these mutations resulting in the expression of a truncated CcpA protein; and/or c) a mutation in at least one of the genes encoding the proteins of the mannose-glucose-spe- cific PTS, such as one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene which mutation results in the expression of a truncated version of any one of the IIAB^Man, IIC^Man or IID^Man proteins, respectively.

4. The use according to embodiment 3, which strain comprises a mutation in the gene encoding a protein of the mannose-glucose-specific PTS, such as one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene, which mutation results in the expression of a truncated version of any one of IIAB^Man, IIC^Man or IID^Man, respectively, such as a mutation by substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene (manL₁₃74o) leading to a stop codon at position 306 of SEQ ID NO:80 of the protein [IIABMan³⁰⁵); a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439, or 440 of SEQ ID NO: 129 in the manM gene (manMiggg?') leading to a truncation of the IIC^Man protein at position 179 of SEQ ID NO: 130; and/or by insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37-41 of SEQ ID NO: 166 creating a frameshift in manN gene (manNi₃₇4i') leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167.

5. The use according to embodiments 3 or 4, which strain comprises at least a mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, but not null.

6. The use according to embodiments 3-5, which strain comprises at least a mutation in the ccpA gene selected from a non-sense mutation or a mutation leading to a frameshift of the open reading frame of the ccpA gene located between the nucleotide 1 and the nucleotide 270 of SEQ ID:65 of the coding sequence of the ccpA gene, any of these mutations resulting in the expression of a truncated CcpA protein. 7. The use according to any one of embodiments 1-6, wherein said mutated glcK gene encodes a glucokinase selected from the group consisting of: a) a glucokinase having an amino acid at position 275 which is not a glutamic acid, in particular which is not an acidic amino acid, in particular which is a lysine, b) a glucokinase having an amino acid at position 275 which is not a glutamic acid, in particular which is not an acidic amino acid, in particular which is a lysine (gld^s), and having an arginine at position 278 and/or a serine at position 279; and c) a glucokinase having an amino acid at its position 144 which is not a glycine, in particular which is not an aliphatic amino acid, in particular which is a serine.

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

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

10. The use according to any one of embodiments 1-9, wherein, when said strain is able to ferment milk as described in Test 2 described herein during 24 hours of fermentation, wherein the concentration of lactose remaining in the fermented milk is less than 30 g/kg.

11. The use according to any one of embodiments 1-10, wherein said strain has the ability to overconsume lactose as compared to a parental strain without said one or more mutations, such as wherein the remaining amount of lactose when fermenting milk as described in Test 2 is lower than 20 g/kg, 10 g/kg, 8 g/kg, 6 g/kg, 4 g/kg, 2 g/kg or 1 g/kg. 12. The use according to any one of embodiments 1-11, which strain is galactose-negative.

13. The use according to any one of embodiments 1-12, which Streptococcus thermophilus strain is selected from the group consisting of:

- a strain deposited in the name of DuPont Nutrition Biosciences ApS at Leibniz-Institut DSMZ on August 15th, 2017 under accession number DSM32587;

- a strain deposited in the name of International N&H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34909;

- a strain deposited in the name of International N&H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34910;

- a strain deposited in the name of International N&H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34911;- a strain corresponding to the Streptococcus thermophilus strain deposited under accession number DSM28255 on January 14^th, 2014 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany]; into which the sequence of the glcK gene encoding a glucokinase, contains the sequence as defined by SEQ ID NO:22 or a variant sequence being at least about 90%, such as at least about 95% identical herewith, wherein the amino acid at position 275 is a lysine (g/c ^s), and/or comprising a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439 or 440 of SEQ ID NO: 129 in the manM gene (manM^gg?),' and/or comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO: 79 of the manL gene (manL^o), leading to a stop codon at position 306 of the protein of SEQ ID:80 (IIAB^Man/; and/or comprising a substitution of nucleotide G at position 625 of manM by a nucleotide T (manM₁₆₄04), leading to a stop codon at position 209; and/or comprising the insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37- 41 creating a frameshift in manN gene (ma nN 13741) leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167; and/or 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 with SEQ ID NO:71; and a strain corresponding to the Streptococcus thermophilus strain deposited under accession number DSM34172 on February 16, 2022, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany]; into which the sequence of the glcK gene encoding a glucokinase, contains the sequence as defined by SEQ ID NO:22 or a variant sequence being at least about 90%, such as at least about 95% identical herewith, wherein the amino acid at position 275 is a lysine (glcK₃₇8), and/or comprising a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439 or 440 of SEQ ID: 129 in the manM gene (manMi₂997) and/or comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene (manL^o) leading to a stop codon at position 306 of the protein of SEQ ID:80 (IIAB^Man); and/or comprising a substitution of nucleotide G at position 625 of manM by a nucleotide T (manM_1S404), leading to a stop codon at position 209 ; and/or comprising the insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37-41 creating a frameshift in manN gene (resulting in manN^i mutated allele) leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167; and/or 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 with SEQ ID NO:71.

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

15. A composition comprising at least one lactose-positive, Streptococcus thermophilus strain as defined in any one of embodiments 1-13; in combination with at least one other strain of the Lactobacillus genus, such as Lactobacillus delbrueckii subspecies bulgaricus, and/or a mesophilic lactic acid bacteria, such as a species of the genus lactococcus, such as a species of Lactococcus lactis, such as a strain of Lactococcus lactis subspecies cremoris, a strain of Lactococcus lactis subspecies hordinae, or Lactococcus lactis subspecies lactis, or a strain of the Bifidobacterium genus, or a strain of the Leuconostoc genus, such as Leuconostoc mesen- teroides subspecies cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

16. The compositions according to embodiment 15, which lactose-positive, Streptococcus thermophilus strain is as defined in any one of embodiments 2-13.

17. A method for manufacturing a cheese, such as a semi-hard cheese and/or a soft-cheese, comprising the step of inoculating a milk substrate with the Streptococcus thermophilus strain as defined in any one of embodiments 1-13, or a composition according to embodiments 15 or 16, and fermenting said inoculated milk, to obtain the cheese.

18. A method for eliminating or for reducing the need for a delactosing step, such as a curdwashing-step or any other lactose dilution in a cheese making process, such as in semi-hard cheese making and/or soft-cheese, which method comprises the step of inoculating and fermenting a milk substrate with the Streptococcus thermophilus strain as defined in any one of embodiments 1-13, or a composition according to embodiments 15 or 16.

19. A cheese, such as a semi-hard cheese and/or a soft-cheese comprising at least one Streptococcus thermophilus strain as defined in any one of embodiments 1-13, a composition according to embodiments 15 or 16, or as obtained by a method according to embodiments 17 or 18. 20. A lactose-positive, Streptococcus thermophilus strain comprising a manM gene encoding an IIC^Man protein consisting of an amino acid sequence of SEQ ID NO:213, or a sequence having at least about 90%, such as at least 95% identity with SEQ ID NO:213.

21. A Streptococcus thermophilus strain selected from the list consisting of

- a strain deposited in the name of International N&H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34910; and

- a strain deposited in the name of International N&H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34911.

EXAMPLES

Example 1: Selection of "lactose overconsuming" S. thermophilus strains

Lactose-positive, galactose-negative, Streptococcus thermophilus strains carrying a mutation in glcK gene and/or in ccpA gene and/or in one of the genes encoding a protein of the mannose-glucose-specific PTS (manL, manM, ma nN) were shown to be of interest to overconsume lactose in dairy fermented products (W02019197051, WO2019122365 and European Patent Application 22159955.8). In order to assess the impact of these mutations alone or in various combinations on the ability of S. thermophilus strains to consume lactose in milk, derivatives of parental S. thermophilus strains ST21 (DSM28255), ST22 (DSM34172) and ST23 were constructed into which the glcK₃₇g, ccpAgss, manL₁₃₇4o, manM_1S404, manM₁₂997 and/or manN₁₃74i mutations replaced the native gene alleles, as presented in Table 1.

The ccpAgss mutation is defined by the deletion of a nucleotide A in the stretch of 7 nucleotides A at positions 114-120 (as defined in SEQ ID NO:71), leading to a frameshift of the open reading frame of the ccpA gene. The ccpAgss mutation was introduced into the genome of ST21 and ST23 to create the ST21-C and ST23-C strains.

The glcK₃₇g mutation generates a mutated glcK gene that encodes a glucokinase with the glutamic acid (E) at position 275 being replaced by the amino acid lysine (K) as defined in SEQ ID NO:22). The glcK₃₇g mutation was introduced into the genome of ST21 and ST23 to create the ST21-G (DSM32587) and ST23-G strains and of ST21-C and ST23-C strains to create the ST21-GC and ST23-GC (DSM34909) strains, (all carrying both the glcK₃₇g mutation and the ccpAgss mutation). The ccpAgss mutation and the glcK₃₇g mutation were introduced into the genome of ST24 and ST25 to create the ST24-GC (DSM34910) and ST25-GC (DSM34911) strains.

The manL, manM, or manN genes encode the IIAB^Man, IIC^Man and IID^Man proteins, respectively, which are part of the mannose-glucose-specific PTS. The manL₁₃₇4o mutation is a substitution of nucleotide G at position 916 of SEQ ID NO:79 of manL by a nucleotide T, leading to a stop codon at position 306 of SEQ ID NO:80 of the protein [the resulting protein was named IIAB- ^Man3os] ■ The manM_1S404 mutation is a substitution of nucleotide G at position 625 of manM by a nucleotide T (SEQ ID NO: 157), leading to a stop codon at position 209 [the resulting protein was named IIC^Man2os], as defined in SEQ ID NO: 158. The manMi₂₉97 mutation is an insertion of one nucleotide C at any one of positions 438, 439, or 440 of SEQ ID NO: 129 creating a frameshift in manM (SEQ ID NO:212), leading to a stop codon at position 180 of SEQ ID NO: 130 [the resulting protein was named IIC^Mani79 as defined in SEQ ID NO:213]. The manNi374i mutation is an insertion of one nucleotide A in the stretch of 5 nucleotides A at positions 37-41 (resulting to a stretch of 6 nucleotides A) of SEQ ID NO: 166, creating a frameshift in manN leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167 as defined in SEQ ID NO:207). ST21, ST22 and ST23 derivatives were mutated in the manL, manM, or manN genes by respectively introducing the manLi₃₇4o, manMi₂₉97, manMi6404 or manNi₃74i mutations either into the genome of parental strains (resulting in strains ST21-MI, ST21-Mmi and ST21-Mn), or into the genome of strains already mutated in the glcK gene (resulting in ST21-GMml, ST22-GMm₂, ST23-GMm₂) or into the genome of strains already mutated in the ccpA gene (resulting in ST21-CMI, ST21-CMmi, ST21-CMn, ST22-CMm₂, ST23-CMm₂) or into the genome of a strain already mutated in both the glcK and the ccpA genes (resulting in ST21-GCMmi).

All strains were grown at 37°C in M17 broth (OxoTd, supplier reference CM0817) supplemented with 30 g/L of sucrose and if necessary, addition of 15 g/L Agar bacteriologic Type A (Biokar, supplier reference #A1010HA). These cultures were harvested by centrifugation then resuspended in a M17-carbohydrate-free medium that was used to seed milk as described in Test 1 and Test 2 defined in the Example 2.

Example 2: Characterization of "lactose overconsuming" S. thermophilus strains

In order to evaluate the ability of the derivative strains of ST21, ST22 and ST23 described in the Example 1 to consume lactose, the engineered strains and parental strains were used to ferment milk as described in Tests 1 and 2 below.

Quantification of lactose during milk fermentation [Test 11

UHT semi-skimmed milk"Le Petit Vendeen containing 3% (w/v) milk powder (BBA, Lactalis), previously pasteurized 10 min at 90 °C, was inoculated at 1% (v/v, about 10⁷ CFU/ml) with a culture of the S. thermophilus strain to be assayed (M17-carbohydrate-free resuspended cells from overnight culture as described in Example 1). Milk contains about 58 g/Kg of lactose. The inoculated milk flasks were statically incubated in a water bath at 43°C during 24h, to obtain fermented milk. Five grams of inoculated milk before incubation (TO sample) and of inoculated milk after incubation (sample T24h) were diluted in 25 g 0.025 N sulfuric acid solution, before being centrifuged at 4600 rpm for 10 minutes at 4 °C. The supernatant was filtered through a 0.2 pm Nylon filter (Phenomenex, Germany, Aschaffenburg) directly into a 2 ml HPLC vial. Lactose was quantified by high performance liquid chromatography (Agilent 1200 HPLC) equipped with a refractive index detector using an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories Inc.) at 35°C, with 12.5 mM sulfuric acid solution as the elution fluid and a flow rate of 0.6 ml min^-1. The exploitation of results was made with Chemstation reprocessing software (Agilent).

Quantification of lactose during milk fermentation (Test 21

Raw whole cow's milk previously pasteurized 1 min at 74 °C was inoculated at 1% (v/v, about 10⁹ CFU/ml) with a culture of the S. thermophilus strain to be assayed (overnight culture grown in semi-skimmed milk 10% BBA heated 20 min 120°C inoculated at 1% from a frozen vial). Pasteurized milk contains around 48 g/Kg of lactose. The inoculated milk flasks were incubated statically in a water bath at 32°c for lh30, then heated for 15 min to reach and remain at 36°C for 1 hour, then slowly lowered to 28°C for up to 24h to obtain fermented milk. The amounts of lactose were measured by HPLC (High Liquid Performance Chromatography) on a Vanquish system provided by Thermo Fisher Scientific equipped with a refractometer detector. Five grams of inoculated milk before incubation (TO sample) and of inoculated milk after incubation (sample T24h) were diluted in 25 mL sulfuric acid solution 0.025 N. After homogenization, samples were centrifuged 10 min at 4600 rpm and the supernatant was filtered on nylon 0.2 pm filter into HPLC vials. Five microliters of sample were injected on an H+ ion exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm x 7.8 mm). The elution was performed in isocratic mode with sulfuric acid 0.025 N at 0.7 mL/min. Molecules were separated in 20 min at 40°C. Carbohydrates were detected with refractometer at 35°C.

The results are displayed in Table 1. The five parental strains (ST21, ST22, ST23, ST24 and ST25) consumed a limited amount of lactose (about 30%). All the single mutants carrying either the glcK₃₇8 the ccpAsss or one of the manLMN mutations (manLi₃₇4o, manMi₂997, manM_1S404 or manN i i) were able to consume more lactose (ranging between 35% and 45%). Introducing both the glcK₃₇8 and the ccpAsss mutations into the genome of ST21 (generating ST21- GC),ST23 (generating ST23-GC), ST24 (generating ST24-GC) and ST25 (generating ST25-GC) resulted in strains that were consuming even more lactose (ranging between 51 and 59%). The combination of the glcK₃₇8 mutation with a mutation in the manM gene (manMi₂₉97 or manMi6404) into the genome of ST21, ST22 and ST23 was resulting in strains ST21-GMmi, ST22-GMm₂, ST23-GMm₂ capable of hydrolyzing up to 84% the lactose present in milk. Similar levels of lactose overconsumption were obtained when the ccpAsss mutation was combined with a mutation in the manM gene (manMi₂₉97 or manMis ) into the genome of ST21 (generating ST21-CMmi), ST22 (generating ST22-CMm₂) and ST23 (generating ST23-CMm₂). Finally, the highest level of lactose overconsumption was obtained when all the glcK₃₇8, ccpAsss and manMi6404 mutations were associated into the genome, since ST21-GCM hydrolyzed up to 89% the lactose present in milk.

In addition, our results showed that whatever the manLi₃₇4o, manMi_S404 or manNi₃₇4i mutations introduced into the genome of ST21, either alone (generating ST21-MI, ST21-Mmi, ST21-Mn) or associated with the ccpAgss mutation (generating ST21-CMI, ST21-CMmi, ST21-CMn), similar levels of lactose overconsumption were measured : about 40% and 85%, respectively.

Table 1 : Analysis of the impact of the glcK₃₇₈, ccpAgss, manM₁₆404, manL₁₃₇4o, manN₁₃₇₄i, and manMi299₇ mutations alone or combined on the ability of S. thermophilus to consume lactose when grow in milk. Strains ST21, ST22 and their derivative strains were evaluated under conditions defined in the Test 1, and strains ST23, ST24 and ST25 and their derivative strains were evaluated under conditions defined in the Test 2.

Example 3: Use of "lactose overconsuming" S. thermophilus strains in/for Gouda cheese making

A classic Gouda making process requires the use of a mesophilic starter culture and includes the multiple steps as shown in Table 2.

Table 2: Conventional Gouda making process

Two "lactose overconsuming" strains (ST21-G and ST21-GC) constructed in Example 1 and further analyzed in Example 2 were tested in experimental Gouda manufacturing conditions as described in Example 3 (Table 2). Fermentations were performed using a reference mesophilic culture (named Meso) or the parental ST21 strain or its "lactose overconsuming" derivatives (ST21-G or ST21-GC).

To further evaluate the benefit of the "lactose overconsuming" strains, in one experimental setting the complete classic Gouda process was applied and in another experimental setting the curd-washing step was omitted. Efficiency of the process combined to the starter culture on the reduction of lactose concentration in the product was evaluated through lactose quantification (as described in Test 3). This quantification of lactose was performed i) in the whey at molding (two hours upon inoculation with the starter culture; see Table 3), and ii) in the curd (six hours upon inoculation with the starter culture; see Table 4)

Quantification of lactose during cheese making TTest 31

Raw whole cow's milk previously pasteurized 1 min at 74 °C was inoculated with a dosage of the S. thermophilus strain and mesophilic culture (named Meso) comprised between 1.10¹⁰ CFU/100 L and 1.10¹¹ CFU/100 L for each. Pasteurized milk contains around 48 g/Kg of lactose. A Gouda-type process as indicated in Table 2 was applied. Five grams of whey at moulding and curd before brining were sampling for sugar measurement. The amounts of lactose were measured by HPLC (High Liquid Performance Chromatography) on a Vanquish system provided by Thermo Fisher Scientific equipped with a refractometer detector. Five grams of the curd/ cheese were diluted (or crushing with an IKA Ultra-Turrax at 13500 rpm for cheese samples) in 25 mL sulfuric acid solution 0.025 N. After homogenization, samples were centrifuged 10 min at 4600 rpm and the supernatant was filtered on nylon 0.2 pm filter into HPLC vials. Five microliters of sample were injected on an H+ ion exchange column (Rezex ROA-Organic Acid H+, 8%, 150 mm x 7.8 mm). The elution was performed in isocratic mode with sulfuric acid 0.025 N at 0.7 mL/min. Molecules were separated in 20 min at 40°C. Carbohydrates were detected with refractometer at 35°C.

Table 3 displayed the quantity of lactose present in the whey at molding. The quantities of lactose measured were significantly different for samples obtained with curd-washing (about 33 g/Kg) and without curd washing (50 g/Kg); this difference being mostly a consequence of a dilution factor (about 25%) resulting from the curd-washing. On the contrary, there was little (if any) differences on the quantity of lactose measured in the whey obtained from the fermentation with various starter cultures at this early stage of the process.

Table 3: Lactose quantification in whey at moulding during experimental Gouda process (as defined in Table 2) with or without a curd-washing step upon an inoculation with the reference mesophilic culture (Meso) or with the parental S. thermophilus strain ST21 or its "lactose overconsuming" derivatives (ST21-G or ST21-GC).

Similarly, Table 4 displayed the quantity of lactose present in the curds six hours after the bacterial inoculation and before brining. When fermentation was performed with the classical mesophilic starter culture MESO, the lactose content in the curd was about 6 times lower in the product obtained in a process including a curd-washing step (1.1 g/Kg) compared to the product obtained in a process without curd-washing (6.3 g/kg). These data clearly demonstrating the delactosing efficiency of the curd-washing step to reduce the amount of residual lactose in the "cheese-before-brining". For curds obtained from fermentation with the parental S. thermophilus ST21, the lactose content in the curd was higher than that measured for fermentation with the classical mesophilic starter culture whatever the process used, 1.9 g/Kg with a curd-washing step and 4.7 g/Kg without a curd-washing step, compared to 1.1 g/Kg. Replacing the parental ST21 by one of the selected "lactose overconsuming" strains (ST21-G or ST21-GC) had a dramatic effect on the lactose content in the curd. With both ST21-G and ST21-GC, the lactose content in the curd obtained from a process including a curd-washing step was very low (0.3 g/Kg). Moreover, even if the curd-washing step was omitted, the lactose content in the curd was as low as 1.3 and 0.5 g/Kg upon fermentation with ST21-G or ST21-GC, respectively; that is similar or lower to what was measured for a curd obtained from a classical Gouda cheese process with a classical mesophilic starter culture in a process including a curd-washing step.

These data clearly indicated that the curd-washing step of the classical Gouda cheese process can be omitted thanks to the use of S. thermophilus strain into which genome the glcK₃₇s mutation has been introduced. Moreover, even higher lactose reduction can be achieved thanks to the introduction of the combination of the ccpA₈₅₅ and glcK₃₇₈ mutations into the strain genome.

Table 4: Lactose quantification in curds produced using an experimental Gouda process (as defined in Table 2) with or without a curd-washing step upon an inoculation either with a reference mesophilic culture (named Meso) or with the parental strain ST21 or its "lactose overconsuming" derivatives (ST21-G or ST21-GC).

Example 4: Use of "lactose overconsuming" S. thermophilus strains associated with mesophilic culture during gouda cheese making

To further validate the experimental data, the parental S. thermophilus strains ST21 or its "lactose overconsuming" derivative ST21-GC combined to a classical mesophilic starter culture named Meso were evaluated in Gouda cheese making. Cheese making was conducted with or without a curd-washing step at 25% (lh30 upon bacterial inoculation) and lactose was quantified in the curds before the step of brining as described in Test 3 of Example 3 (Table 5). Remaining amount of lactose in the curds obtained with the mesophilic culture alone was 6.9 g/kg when the curd-washing step was omitted, and this amount was decreased to 3.7 g/kg thanks to curd-washing. Addition of ST21 to the mesophilic culture only slightly reduced these values to 5.8 g/kg and 3.5 g/kg, respectively; showing that the addition of the parental S. thermophilus strain did not allow the omission of the curd-washing step. On the contrary, addition of the "lactose overconsuming" ST21-GC to the mesophilic culture had a dramatic effect on the remaining lactose in the curd since lactose was below the level of detection in the curd even when the curd-washing step was omitted. These results clearly indicated the benefit of glcK₃₇s mutation combined to the ccpAsss mutation in S. thermophilus genome to eliminate lactose, thus allowing the omission of the curd-washing step in Gouda cheese production processes.

Table 5: Lactose quantification (as described in Test 3 in Example 3) in curds produced from Gouda cheese making (as defined in Table 2) with or without a curd-washing step upon an inoculation with a classical mesophilic starter culture (named Meso), supplemented or not with the parental strains ST21 and its "lactose overconsuming" derivative ST21-GC.

In another assay, similar Gouda cheese productions were performed using a mesophilic culture (named 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 the process during which the curd-washing step was omitted and are displayed in Table 6. As previously observed, the level of remaining lactose in the curd obtained from the fermentation with a mesophilic culture alone was high (6.7 g/Kg). Supplementation of the fermentation with the parental strain ST23 allowed to reduce the level of residual lactose to 1.7 g/Kg. Best performances in reducing the level of lactose in curd, that was barely detectable, were obtained thanks to the supplementation of a mesophilic culture with either ST23-G or ST23-GC. These results confirmed that the effect of the glcK₃₇₈ mutation alone or combined with the ccpAsss mutation in S. thermophilus on the consumption of lactose in the process of Gouda cheese production is independent of the parental strain and could be obtained with multiple S. thermophilus genetic backgrounds among which are the various parental strains listed in example 2 (ST21, ST22 and ST23) which respective "lactose overconsuming" derivatives displayed similar behavior in Test.

Gouda cheese productions were also performed using a mesophilic culture (named Meso) supplemented with the "lactose overconsuming" derivatives of two other genetic backgrounds of S. thermophilus, ST24-GC and ST25-GC. Data shown in Table 6 confirmed the previous results obtained with ST23-GC. Table 6: Lactose quantification (as described in Test 3 in Example 3) in curds produced from Gouda cheese making (as defined in Table 2) without a curd-washing step upon an inoculation with a classical mesophilic starter culture named MESO supplemented or not with the parental strain ST23 or its "lactose overconsuming" derivatives ST23-G or ST23-GC, or with the "lactose overconsuming" strains ST24-GC or ST25-GC.

SEQUENCES

SEQ ID NO:2

MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLE-

LYGLTAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVG

AGANNRNVVFITLGTGVGGGVIADGN-

LIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTS

KDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRS-

RVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

SEQ ID NO:22

MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLE-

LYGLTAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVG

AGANNRNVVFITLGTGVGGGVIADGN-

LIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTS

KDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGKFLRS-

RVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK

SEQ ID NO:25 (X underlined in bold corresponds to the aa at position 275 and can be any naturally occurring amino acid, but not Glu)

MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGL

TAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAAL

GERWVGAGANNRNVVFITLGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCG

NKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTSKDIIVAATEGDKFADSI

VDKVSKYLGLATANISNILNPDSVVIGGGVSAAGXFLRSRVEGYFTRYAFPQVRRTTKVK

LAELGNDAGIIGAASLAYSIDK

SEQ ID NO:45

MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLE-

LYGLTAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVG

AGAN N RN VVFITLGTGVSGGVI ADGN -

LIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTS

KDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRS-

RVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK SEQ ID NO:46 (X underlined in bold corresponds to the aa at position 144 and can be any naturally occurring amino acid, but not Glu)

MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGL

TAEDFIGIGMGSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAAL

GERWVGAGANNRNVVFITLGTGVXGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCG

NKGCLETVASATGIVRVAHHLAEKYEGNSSIKAAVDNGEFVTSKDIIVAATEGDKFADSI

VDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFLRSRVEGYFTRYAFPQVRRTTKVK LAELGNDAGIIGAASLAYSIDK

SEQ ID NO:65

ATGAATACTGATGAAACAATCACAATTTATGATGTAGCGCGTGAAGCTGGAGTATCGATGG-

CAACTGTTTCTCGTGTTGTAAATGGTAACAAAAACGTAAAAGAAAACACCCGAAAAAAAGTGCTCGAA

GTCATTGATCGTTTGGATTACCGTCCAAATGCGGTTGCGCGTGGCTTGG-

CAAGTAAAAAAACAACTACTGTAGGAGTTGTCATTCCAAATATTGTAAATAGCTATTTTGCTACTCTAGC

TAAAGGTATTGATGACATTGCAACCATGTATAAGTATAATATTGTTCTTGCTTCCAG-

TGATGATAATGAGGATCATGAAGTTACAGTCATTCATTCTCTAATTTCTAAACAAGTTGATGGTATTATT

TTTATGGGACACCATCTGACAGAAAAAATCCGTGCAGAATTCTCTCGTACCCGTACAC-

CGATTGTTCTAGCAGGAACAGTTGATCTCGAACACCAATTACCAAGTGTTAACATCGACTATAAAGCTG

CCGTTGAGGATTGTGTAACGCAACTTGCTAAAAATAATGAAAAGGTTGCCTTTGTATCAG-

GACCACTAATTGATGATATTAATGGCAAACTACGTTTGGCCGGGTATAAGTCTGGACTTGAAAAGAATA

ATTTGAGCTACAACGAAGGACTTGTCTTTGAAGCTAAATATAGCTATAAAGACGGCTTTGAG-

TTAGCACAACGTGTCTTGAACTCTGGTGCCACTGCTGCCTATGTTGGGGAAGATGAATTGGCTGCAGG

TCTCTTGAATGGCCTCTTTGCTGCAGGCAAATCAGTTCCAGAAGATTTCGAAATCATCACAA-

GCAATGATTCACCGGTTACAAGCTACACACGTCCAAACCTTTCTAGTATAAACCATCCTCTCTATGATTT

AGGGGCAGTTAGCATGCGTATGTTGACTAAAATTATGCATAAGGAAGAACTTGAAGATAAA-

GACGTTATTCTTAATCATGGTCTAACTTTACGCCAGTCAACAAAATAA

SEQ ID NO:71

ATGAATACTGATGAAACAATCACAATTTATGATGTAGCGCGTGAAGCTGGAGTATCGATGG-

CAACTGTTTCTCGTGTTGTAAATGGTAACAAAAACGTAAAAGAAAACACCCGAAAAAAGTGCTCGAAG

TCATTGATCGTTTGGATTACCGTCCAAATGCGGTTGCGCGTGGCTTGG-

CAAGTAAAAAAACAACTACTGTAGGAGTTGTCATTCCAAATATTGTAAATAGCTATTTTGCTACTCTAGC

TAAAGGTATTGATGACATTGCAACCATGTATAAGTATAATATTGTTCTTGCTTCCAG-

TGATGATAATGAGGATCATGAAGTTACAGTCATTCATTCTCTAATTTCTAAACAAGTTGATGGTATTATT

TTTATGGGACACCATCTGACAGAAAAAATCCGTGCAGAATTCTCTCGTACCCGTACAC-

CGATTGTTCTAGCAGGAACAGTTGATCTCGAACACCAATTACCAAGTGTTAACATCGACTATAAAGCTG

CCGTTGAGGATTGTGTAACGCAACTTGCTAAAAATAATGAAAAGGTTGCCTTTGTATCAG-

GACCACTAATTGATGATATTAATGGCAAACTACGTTTGGCCGGGTATAAGTCTGGACTTGAAAAGAATA

ATTTGAGCTACAACGAAGGACTTGTCTTTGAAGCTAAATATAGCTATAAAGACGGCTTTGAG-

TTAGCACAACGTGTCTTGAACTCTGGTGCCACTGCTGCCTATGTTGGGGAAGATGAATTGGCTGCAGG

TCTCTTGAATGGCCTCTTTGCTGCAGGCAAATCAGTTCCAGAAGATTTCGAAATCATCACAA-

GCAATGATTCACCGGTTACAAGCTACACACGTCCAAACCTTTCTAGTATAAACCATCCTCTCTATGATTT

AGGGGCAGTTAGCATGCGTATGTTGACTAAAATTATGCATAAGGAAGAACTTGAAGATAAA-

GACGTTATTCTTAATCATGGTCTAACTTTACGCCAGTCAACAAAATAA

SEQ ID NO:79

ATGGGTATCGGTATTATTATTGCCAGCCATGGTAAGTTCGCTGAAGGAATCCACCAATCA

GGCTCTATGATTTTTGGGGACCAAGAGAAAGTTCAAGTTGTGACTTTCATGCCAAGTGAA

GGTCCTGATGATTTGTACGCTCACTTCAACAACGCCATTGCACAATTCGATGTTGATGAT

GAAATTCTTGTTTTGGCTGACCTTTGGAGTGGTTCACCATTTAACCAAGCTAGTCGAATC

GCTAGGGAAAACCCAGATCGCAAGATTGCTATCATCACAGGACTTAACTTGCCAATGCTA

ATCCAAGCATACACTGAACGTATGATGGATGCTAACGCTACTGTAGAGCAAGTTGCTGCT

AATATCATCAAGGAAGCTAAGGCTGGTATCAAGGCACTTCCAGAGGCGCTAAATCCAGCT GAGGAAACAACTGCAGCTCCTGTAGAAGCTGCAGCACCTCAAGGAGCTATCCCTGAAGGA

ACAGTCATCGGAGATGGTAAACTCAAGATTAACTTGGCACGTTTGGACACACGTCTCTTG

CATGGTCAAGTAGTAACTAACTGGGTACCTTATTCTAAAGCAGACCGTATTATTGTTGCT

TCGGATGACGTTGCCAAAGATGAGCTTCGTAAGGAATTGATCAAACAGGCTGCACCAAAC

GGTATTAAAGTAAACGTTGTTCCGATTCAAAAATTAATTGATGCTTCTAAAGACCCACGT

TTTGGAAATACACATGCGCTTGTCTTGTTCGAAACTGTTCAAGACGCACTTCGTGCTATC GAAGGTGGCGTGCCAATAAAAGAACTTAACGTTGGTTCTATGGCTCACTCAACTGGTAAA ACAATGGTTAACAACGTTTTGTCTATGGATAAAGATGATGTTGCTTGTTTTGAAAAATTA

CGTGACCTTGGCGTTGAATTTGACGTCCGTAAGGTTCCAAACGATTCTAAGAAAGACTTG

TTTGAGCTTATCAAGAAAGCTAACGTTCAATAA

SEQ ID NO:80

MGIGIIIASHGKFAEGIHQSGSMIFGDQEKVQVVTFMPSEGPDDLYAHFNNAIAQFDVDD

EILVLADLWSGSPFNQASRIARENPDRKIAIITGLNLPMLIQAYTERMMDANATVEQVAA

NIIKEAKAGIKALPEALNPAEETTAAPVEAAAPQGAIPEGTVIGDGKLKINLARLDTRLL

HGQVVTNWVPYSKADRIIVASDDVAKDELRKELIKQAAPNGIKVNVVPIQKLIDASKDPR

FGNTHALVLFETVQDALRAIEGGVPIKELNVGSMAHSTGKTMVNNVLSMDKDDVACFEKL

RDLGVEFDVRKVPNDSKKDLFELIKKANVQ*

SEQ ID NO: 111

ATGGGTATCGGTATTATTATTGCCAGCCATGGTAGGTTCGCTGAAGGAATCCAC-

CAATCAGGCTCTATGATTTTTGGGGACCAAGAGAAAGTTCAAGTTGTGACTTTCATGCCAAGTGAAGG

TCCTGATGATTTGTACGCTCACTTCAACAACGCCATTGCACAATTCGATGTT-

GATGATGAAATTCTTGTTTTGGCTGACCTTTGGAGTGGTTCACCATTTAACCAAGCTAGTCGAATCGCT

AGGGAAAATCCAGATCGCAAGATTGCTATCATCACAGGACTTAACTT-

GCCAATGCTAATCCAAGCATACACTGAACGTATGATGGATGCTAACGCTACTGTAGAGCAAGTTGCTG

CTAATATCATCAAGGAAGCTAAGGGTGGTATCAAGGCACTTCCAGAA-

GAGCTAAATCCAGCTGAGGAAACAACTGCAGCTCCTGTAGAAGCTGCAGCACCTCAAGGAGCTATCCC

TGAAGGAACAGTCATCGGAGATGGTAAACTCAAGATTAACTTGGCACGTTTGGACACAC-

GTCTCTTGCATGGTCAAGTAGTAACTAACTGGGTACCTTATTCTAAAGCAGACCGTATTATTGTTGCTT

CGGATGACGTTGCCAAAGATGAGCTTCGTAAGGAATTGATCAAACAGGCTGCACCAAACGG- TATTAAAGTAAACGTTGTTCCGATTCAAAAATTAATTGATGCTTCTAAAGACCCACGTTTTGGAAATACA CATGCGCTTGTCTTGTTCGAAACTGTTCAAGACGCACTTCGTGC-

TATCGAAGGTGGCGTGCCAATAAAAGAACTTAACGTTGGTTCTATGGCTCACTCAACTGGTAAAACAA

TGGTTAACAACGTTTTGTCTATGGATAAAGATGATGTTGCTTGTTTTGAAAAATTACGTGAC-

CTTGGCGTTTAATTTGACGTCCGTAAGGTTCCAAACGATTCTAAGAAAGATTTGTTTGAGCTTATCAAG

AAAGCTAACGTTCAATAA

SEQ ID NO: 112

MGIGIIIASHGRFAEGIHQSGSMIFGDQEKVQVVTFMPSEGPDDLYAHFNNAIA-

QFDVDDEILVLADLWSGSPFNQASRIARENPDRKIAIITGLNLPMLIQAYTERMMDANATVEQVAANIIKE

AKGGIKALPEELNPAEETTAAPVEAAAPQGAIPEGTVIGDGKLKINLARLD-

TRLLHGQVVTNWVPYSKADRIIVASDDVAKDELRKELIKQAAPNGIKVNVVPIQKLIDASKDPRFGNTHAL

VLFETVQDALRAIEGGVPIKELNVGSMAHSTGKTMVNNVLSMDKDDVACFEKLRDLGV

SEQ ID NO: 129

ATGTCAGATATGTCAATTATTTCTGCGATTTTGGTCGTAGCTGTTGCCTTCCTTGCTGGT

CTTGAAAGTATCCTTGACCAATTCCAATTCCACCAACCACTTGTTGCATGTACCCTCATC

GGTGCTGCCACAGGTAACCTCACTGCAGGTATCATGCTTGGTGGTTCTCTTCAAATGATT

ACCCTTGCTTGGGCAAACATCGGTGCTGCCGTAGCTCCTGACGTTGCCCTTGCATCTGTT

GCCGCTGCCATCATTTTGGTTAAAGGTGGTAAATTTACAGCTGAAGGTATCGGTGTTGCG

ATTGCAATAGCTATCCTGCTTGCAGTTGCAGGTCTCTTCCTAACTATGCCTGTTCGTACA

GCATCTATTGCCTTTGTTCATGCTGCAGATAAAGCTGCAGAACACGGAAACATCGCTGGT

GTTGAACGTGCATACTACCTCGCTCTCCTTCTTCAAGGTTTGCGTATTGCTGTGCCAGCA

GCCCTTCTTCTTGCCATCCCGGCCCAATCTGTTCAACATGCCCTTGGCTTGATGCCTGAC

TGGCTCACCCATGGTTTGGTTGTCGGTGGTGGTATGGTCGTAGCCGTTGGTTACGCCATG

ATTATCAATATGATGGCTACTCGTGAAGTTTGGCCATTCTTCGCCATTGGTTTTGCTTTG

GCAGCAATTAGCCAATTGACACTTATCGCTCTTAGTACCATTGGTGTTGCCATCGCCTTC

ATCTACCTCAACCTTTCTAAACAAGGTGGCGGAAATGGTGGCGGAAATGGTGGCGGAACT

TCATCTGGTTCAGGCGACCCAATCGGCGATATCTTGGAAGACTACTAG

SEQ ID NO: 130

MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIM-

LGGSLQMITLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRTASIAF

VHAADKAAEHGNIAGVERAYYLALLLQGLRIAVPAALLLAIPAQSVQHALGLMP-

DWLTHGLVVGGGMVVAVGYAMIINMMATREVWPFFAIGFALAAISQLTLIALSTIGVAIAFIYLNLSKQGG GNGGGNGGGTSSGSGDPIGDILEDY SEQ ID NO: 157

ATGTCAGATATGTCAATTATTTCTGCGATTTTGGTCGTAGCTGTTGCCTTCCTT-

GCTGGTCTTGAAAGTATCCTTGACCAATTCCAATTCCACCAACCACTTGTTGCATGTACCCTCATCGGT

GCTGCCACAGGTAACCTCACTGCAGGTATCATGCTTGGTGGTTCTCTTCAAATGAT-

TACCCTTGCTTGGGCAAACATCGGTGCTGCCGTAGCTCCTGACGTTGCCCTTGCATCTGTTGCCGCTG

CCATCATTTTGGTTAAAGGTGGTAAATTTACAGCTGAAGGTATCGGTGTTGCGATTGCAA-

TAGCTATCCTGCTTGCAGTTGCAGGTCTCTTCCTAACTATGCCTGTTCGTACAGCATCTATTGCC I I I G I

TCATGCTGCAGATAAAGCTGCAGAACACGGAAACATCGCTGGTGTTGAACGTGCATACTAC-

CTCGCTCTCCTTCTTCAAGGTTTGCGTATTGCTGTGCCAGCAGCCCTTCTTCTTGCCATCCCGGCCCAA

TCTGTTCAACATGCCCTTGGCTTGATGCCTGACTGGCTCACCCATGGTTTGGTT-

GTCGGTGGTGGTATGGTCGTAGCCGTTGGTTACGCCATGATTATCAATATGATGGCTACTCGTTAAGT

TTGGCCATTCTTCGCCATTGGTTTTGCTTTGGCAGCAATTAGCCAATTGACAC-

TTATCGCTCTTAGTACCATTGGTGTTGCCATCGCCTTCATCTACCTCAACCTTTCTAAACAAGGTGGCG

GAAATGGTGGCGGAAATGGTGGCGGAACTTCATCTGGTTCAGGCGACCCAATCGGCGA-

TATCTTGGAAGACTACTAG

SEQ ID NO: 158

MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIM-

LGGSLQMITLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRTASIAF

VHAADKAAEHGNIAGVERAYYLALLLQGLRIAVPAALLLAIPAQSVQHALGLMP-

DWLTHGLVVGGGMVVAVGYAMIINMMATR

SEQ ID NO: 166

ATGGCTGAAAAAATTCAATTATCTCAAGCGGATCGTAAAAAGGTTTGGTGGCGCTCACAA

TTCTTGCAAGGTGCATGGAACTATGAACGTATGCAAAACTTGGGTTGGGCTTACTCACTC

ATTCCTGCTATCAAAAAACTTTATACTAACAAAGAGGACCAAGCCGCAGCTCTTAAACGT

CACTTGGAATTCTTCAACACTCACCCTTACGTAGCTGCTCCTATCATAGGGGTTACCTTA

GCTCTTGAAGAAGAAAAAGCTAATGGTACTGAAATCGAAGATGCGGCTATCCAAGGGGTT

AAAATCGGTATGATGGGTCCACTTGCCGGTATCGGTGACCCTGTCTTCTGGTTCACAATT

CGTCCAATTCTTGGTGCCCTTGGTGCATCATTGGCACAAGCTGGTAACATTGCTGGTCCA

CTTATCTTCTTCATTGGTTGGAACCTTATCCGCATGGCCTTCTTGTGGTACACTCAAGAA

CTTGGTTACAAAGCAGGTTCAGAAATCACTAAAGACATATCTGGTGGTATCTTGAAAGAT

ATTACTAAAGGGGCATCAATACTTGGTATGTTCATCTTGGCCGTCCTCGTTGAACGTTGG

GTATCTGTCGTCTTCACTGTAAAGCTTCCAGGTAAAGTTTTGCCTAAAGGTGCTTATATT

GAATGGCCAAAAGGATATGTTACTGGTGACCAACTAAAAACTATCCTTGGTCAAGTCAAC

GATAAGCTTAGCTTTGATAAGATTCAAGTCGATACCCTACAAAAACAATTGGATTCATTA

ATTCCAGGTTTGACGGGACTTCTCCTTACTTTTGCATGTATGTGGTTGCTTAAGAAGAAA

GTTTCACCAATCACAATCATCATCGGACTCTTTGTAGTTGGTATTGTTGCAAGCTTCTTC GGAATCATGTAA

SEQ ID NO: 167

MAEKIQLSQADRKKVWWRSQFLQGAWNYERMQNLGWAYSLIPAIKKLYTNKEDQAAALKR

HLEFFNTHPYVAAPIIGVTLALEEEKANGTEIEDAAIQGVKIGMMGPLAGIGDPVFWFTI

RPILGALGASLAQAGNIAGPLIFFIGWNLIRMAFLWYTQELGYKAGSEITKDISGGILKD

ITKGASILGMFILAVLVERWVSVVFTVKLPGKVLPKGAYIEWPKGYVTGDQLKTILGQVN

DKLSFDKIQVDTLQKQLDSLIPGLTGLLLTFACMWLLKKKVSPITIIIGLFVVGIVASFF

GIM*

SEQ ID NO:206

ATGGCTGAAAAAATTCAATTATCTCAAGCGGATCGTAAAAAAGGTTT-

GGTGGCGCTCACAATTCTTGCAAGGTGCATGGAACTATGAACGTATGCAAAACTTGGGTTGGGCTTAC

TCACTCATTCCTGCTATCAAAAAACTTTATACTAACAAAGAGGACCAAGCCG-

CAGCTCTTAAACGTCACTTGGAATTCTTCAACACTCACCCTTACGTAGCTGCTCCTATCATAGGGGTTA

CCTTAGCTCTTGAAGAAGAAAAAGCTAATGGTACTGAAATCGAAGATGCGGC-

TATCCAAGGGGTTAAAATCGGTATGATGGGTCCACTTGCCGGTATCGGTGACCCTGTCTTCTGGTTCA

CAATTCGTCCAATTCTTGGTGCCCTTGGTGCATCATTGGCACAAGCTGGTAACATT-

GCTGGTCCACTTATCTTCTTCATTGGTTGGAACCTTATCCGCATGGCCTTCTTGTGGTACACTCAAGAA

CTTGGTTACAAAGCAGGTTCAGAAATCACTAAAGACATATCTGGTGGTATCTTGAAAGA-

TATTACTAAAGGGGCATCAATACTTGGTATGTTCATCTTGGCCGTCCTCGTTGAACGTTGGGTATCTGT CGTCTTCACTGTAAAGCTTCCAGGTAAAGTTTTGCCTAAAGGTGCTTA-

TATTGAATGGCCAAAAGGATATGTTACTGGTGACCAACTAAAAACTATCCTTGGTCAAGTCAACGATAA

GCTTAGCTTTGATAAGATTCAAGTCGATACCCTACAAAAACAATTGGAT-

TCATTAATTCCAGGTTTGACGGGACTTCTCCTTACTTTTGCATGTATGTGGTTGCTTAAGAAGAAAGTTT

CACCAATCACAATCATCATCGGACTCTTTGTAGTTGGTATTGTTGCAAGCTTCTTCGGAATCATGTAA

SEQ ID NO:207

MAEKIQLSQADRKKGLVALTILARCMEL

SEQ ID NO:208

MAEKIQLSQADRKKGLVALTILARFMEL

SEQ ID NO:209

MSEKIQLSQADRKKGLVALTILARFMEL

SEQ ID NO:210

MAEKIQLSQADRRKGLVALTILARFMEL

SEQ ID N0:211

MSEKIQLSQADHKKGLVALTILARFMEL

SEQ ID NO:212

ATGTCAGATATGTCAATTATTTCTGCGATTTTGGTCGTAGCTGTTGCCTTCCTTGCTGGT

CTTGAAAGTATCCTTGACCAATTCCAATTCCACCAACCACTTGTTGCATGTACCCTCATC

GGTGCTGCCACAGGTAACCTCACTGCAGGTATCATGCTTGGTGGTTCTCTTCAAATGATT

ACCCTTGCTTGGGCAAACATCGGTGCTGCCGTAGCTCCTGACGTTGCCCTTGCATCTGTT

GCCGCTGCCATCATTTTGGTTAAAGGTGGTAAATTTACAGCTGAAGGTATCGGTGTTGCG

ATTGCAATAGCTATCCTGCTTGCAGTTGCAGGTCTCTTCCTAACTATGCCTGTTCGTACA

GCATCTATTGCCTTTGTTCATGCTGCAGATAAAGCTGCAGAACACGGAAACATCGCTGGT

GTTGAACGTGCATACTACCCTCGCTCTCCTTCTTCAAGGTTTGCGTATTGCTGTGCCAGC

AGCCCTTCTTCTTGCCATCCCGGCCCAATCTGTTCAACATGCCCTTGGCTTGATGCCTGA

CTGGCTCACCCATGGTTTGGTTGTCGGTGGTGGTATGGTCGTAGCCGTTGGTTACGCCAT

GATTATCAATATGATGGCTACTCGTGAAGTTTGGCCATTCTTCGCCATTGGTTTTGCTTT

GGCAGCAATTAGCCAATTGACACTTATCGCTCTTAGTACCATTGGTGTTGCCATCGCCTT

CATCTACCTCAACCTTTCTAAACAAGGTGGCGGAAATGGTGGCGGAAATGGTGGCGGAAC

TTCATCTGGTTCAGGCGACCCAATCGGCGATATCTTGGAAGACTACTAG

SEQ ID NO:213

MSDMSIISAILVVAVAFLAGLESILDQFQFHQPLVACTLIGAATGNLTAGIMLGGSLQMI

TLAWANIGAAVAPDVALASVAAAIILVKGGKFTAEGIGVAIAIAILLAVAGLFLTMPVRT

ASIAFVHAADKAAEHGNIAGVERAYYPRSPSSRFAYCCASSPSSCHPGPICSTCPWLDA*

STRAINS

DGCC numbers are internal references to DuPont Danisco collection; DSM numbers are the numbers assigned by the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganis- men und Zellkulturen, GmbH (Inhoffenstr. 7B, D-38124 Braunschweig), following deposit under the Budapest Treaty.

As far as the Streptococcus thermophilus strain DGCC7710 deposited under the Budapest Treaty at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, on January 14^th, 2014 under number DSM28255 (herein also referred to as ST21) is concerned, we hereby confirm that the depositor, Danisco Deutschland GmbH (of Busch-Johannsen-Strasse 1, D-25899 Niebull, Germany) has authorised the Applicant (DuPont Nutrition Biosciences ApS) to refer to the deposited biological material in this application. The expressions "DGCC7710 strain" and "DGCC7710 derivative" are used interchangeably with the expressions "DSM28255 strain" and "DSM28255 derivative".

A derivative of the DGCC7710 strain was designed, into which the glcK gene encodes a glucokinase with the glutamic acid (E) at position 275 was replaced by the amino acid lysine (K). This derivative (DGCC12534) was deposited at the DSMZ on August 15th, 2017 under accession number DSM32587 (herein referred to as ST21-G, E275K mutated in its glcK gene).

- Streptococcus thermophilus strain deposited under accession number DSM33651 (herein referred to as ST3 or DSM33651) on September 29, 2020, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany].

The following strains have been deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS:

- Streptococcus thermophilus strain deposited under accession number DSM34172 (herein referred to as ST22 or DSM34172) on February 16, 2022, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany].

- Streptococcus thermophilus strain DGCC13706 deposited under accession number DSM

34909 (herein referred to as ST23-GC or DSM34909) on January 23, 2024, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D- 38124 Braunschweig - Germany].

- Streptococcus thermophilus strain DGCC14231 deposited under accession number DSM

34910 (herein referred to as ST24-GC or DSM34910) on January 23, 2024, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D- 38124 Braunschweig - Germany].

- Streptococcus thermophilus strain DGCC14283 deposited under accession number DSM

34911 (herein referred to as ST25-GC or DSM34911) on January 23, 2024, at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D- 38124 Braunschweig - Germany].

The applicant requests that a sample of the deposited micro-organisms stated herein may only be made available to an expert, until the date on which the patent is granted.

In respect to those designations in which a European Patent is sought, a sample of these deposited microorganisms will be made available until the publication of the mention of the grant of the European patent or until the date on which application has been refused or with- drawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample, and approved either i) by the Applicant and/or ii) by the European Patent Office, whichever applies.

Claims

1. Use of a lactose-positive, Streptococcus thermophilus strain in the production of a cheese, such as a semi-hard cheese or a soft-cheese, which strain has the ability to consume at least about 35% lactose when grown in raw whole cow's milk inoculated at 1% v/v or about 10⁹ CFU/ml of the Streptococcus thermophilus strain, such as measured by Test 2 described herein.

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

3. The use according to claims 1 or 2, which strain comprises one or more mutations selected from: a) a mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, but not null; and/or b) a mutation in the ccpA gene selected from a non-sense mutation or a mutation leading to a frameshift of the open reading frame of the ccpA gene, each mutation located between the nucleotide 1 and the nucleotide 270 of SEQ ID:65 of the coding sequence of the ccpA gene, any of these mutations resulting in the expression of a truncated CcpA protein; and/or c) a mutation in at least one of the genes encoding the proteins of the mannose-glucose-spe- cific PTS, such as one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene which mutation results in the expression of a truncated version of any one of the IIAB^Man, IIC^Man or IID^Man proteins, respectively.

4. The use according to claim 3, which strain comprises a mutation in the gene encoding a protein of the mannose-glucose-specific PTS, such as one or more genes selected from the group consisting of the manL gene, the manM gene and the manN gene, which mutation results in the expression of a truncated version of any one of IIAB^Man, IIC^Man or IID^Man, respectively, such as a mutation by substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene (manL₁₃₇4o) leading to a stop codon at position 306 of SEQ ID NO:80 of the protein [IIABMan³⁰⁵); a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439, or 440 of SEQ ID NO: 129 in the manM gene (manMi₂997') leading to a truncation of the IIC^Man protein at position 179 of SEQ ID NO: 130; and/or by insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37-41 of SEQ ID NO: 166 creating a frameshift in manN gene (manN₁₃74i) leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167.

5. The use according to claims 3 or 4, which strain comprises at least a mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, but not null.

6. The use according to claims 3-5, which strain comprises at least a mutation in the ccpA gene selected from a non-sense mutation or a mutation leading to a frameshift of the open reading frame of the ccpA gene located between the nucleotide 1 and the nucleotide 270 of SEQ ID:65 of the coding sequence of the ccpA gene, any of these mutations resulting in the expression of a truncated CcpA protein.

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

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

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

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

11. The use according to any one of claims 1-10, which Streptococcus thermophilus strain is selected from the group consisting of:

- a strain deposited in the name of DuPont Nutrition Biosciences ApS at Leibniz-Institut DSMZ on January 23, 2024 under accession number DSM 34909;

- a strain deposited in the name of DuPont Nutrition Biosciences ApS at Leibniz-Institut DSMZ on January 23, 2024 under accession number DSM 34910;

- a strain deposited in the name of DuPont Nutrition Biosciences ApS at Leibniz-Institut DSMZ on January 23, 2024 under accession number DSM 34911;

- a strain corresponding to the Streptococcus thermophilus strain deposited under accession number DSM28255 on January 14^th, 2014 at the DSMZ [Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany]; into which the sequence of the glcK gene encoding a glucokinase, contains the sequence as defined by SEQ ID NO:22 or a variant sequence being at least about 90%, such as at least about 95% identical herewith, wherein the amino acid at position 275 is a lysine glcK₃78 , and/or comprising a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439 or 440 of SEQ ID: 129 in the manM gene (manM 12997') and/or comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene (manL^o), leading to a stop codon at position 306 of the protein of SEQ ID:80 (IIAB^Man/; and/or comprising a substitution of nucleotide G at position 625 of manM by a nucleotide T (manMis ), leading to a stop codon at position 209; and/or comprising the insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37-41 creating a frameshift in manN gene (manN i) leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167; and/or 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 with SEQ ID NO:71; and a strain corresponding to the Streptococcus thermophilus strain deposited under accession number DSM34172 on February 16, 2022, at the DSMZ [Deutsche Sammlung von Mikroor- ganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig - Germany]; into which the sequence of the glcK gene encoding a glucokinase, contains the sequence as defined by SEQ ID NO:22 or a variant sequence being at least about 90%, such as at least about 95% identical herewith, wherein the amino acid at position 275 is a lysine glcK₃7s , and/or comprising a mutation leading to a frameshift of the open reading frame of the gene coding for the IIC^Man protein, such as by insertion of a nucleotide in any one of positions 438, 439 or 440 of SEQ ID: 129 in the manM gene (manM 12997) and/or comprising a substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene (manL io) leading to a stop codon at position 306 of the protein of SEQ ID:80 (IIAB^Man ; and/or comprising a substitution of nucleotide G at position 625 of manM by a nucleotide T (manM_1S404), leading to a stop codon at position 209 ; and/or comprising the insertion of one nucleotide A at the stretch of 5 nucleotides A at positions 37-41 creating a frameshift in manN gene (resulting in manN^i mutated allele) leading to a truncation of the IID^Man protein at position 28 of SEQ ID NO: 167; and/or 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 with SEQ ID NO:71.

12. A composition comprising at least one lactose-positive, Streptococcus thermophilus strain as defined in any one of claims 1-11; in combination with at least one other strain of the Lactobacillus genus, such as Lactobacillus delbrueckii subspecies bulgaricus, and/or a mesophilic lactic acid bacteria, such as a species of the genus lactococcus, such as a species of Lactococcus lactis, such as a strain of Lactococcus lactis subspecies cremoris, a strain of Lactococcus lactis subspecies hordinae, or Lactococcus lactis subspecies lactis, or a strain of the Bifidobacterium genus, or a strain of the Leuconostoc genus, such as Leuconostoc mesenteroides subspecies cremoris, Leuconostoc paramesenteroides, and Leuconostoc lactis.

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

14. A lactose-positive, Streptococcus thermophilus strain comprising a manM gene encoding an IIC^Man protein consisting of an amino acid sequence of SEQ ID NO:213, or a sequence having at least about 90%, such as at least 95% identity with SEQ ID NO:213.

15. A Streptococcus thermophilus strain selected from the list consisting of

- a strain deposited in the name of International N&.H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34909;

- a strain deposited in the name of International N&.H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34910; and

- a strain deposited in the name of International N&.H Denmark ApS at Leibniz-Institut DSMZ on January 23, 2024, under accession number DSM 34911.