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WO2024175663A1 - Nouvelle utilisation de bactéries lactiques - Google Patents

Nouvelle utilisation de bactéries lactiques Download PDF

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Publication number
WO2024175663A1
WO2024175663A1 PCT/EP2024/054428 EP2024054428W WO2024175663A1 WO 2024175663 A1 WO2024175663 A1 WO 2024175663A1 EP 2024054428 W EP2024054428 W EP 2024054428W WO 2024175663 A1 WO2024175663 A1 WO 2024175663A1
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seq
gene
strain
amino acid
mutation
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Armelle COCHU-BLACHÈRE
Mikael PIANFETTI
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International N&H Denmark ApS
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Priority to AU2024224154A priority Critical patent/AU2024224154A1/en
Priority to KR1020257028218A priority patent/KR20250148606A/ko
Publication of WO2024175663A1 publication Critical patent/WO2024175663A1/fr
Priority to MX2025008970A priority patent/MX2025008970A/es
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/068—Particular types of cheese
    • A23C19/0688—Hard cheese or semi-hard cheese with or without eyes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/068—Particular types of cheese
    • A23C19/0682—Mould-ripened or bacterial surface ripened cheeses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/202—Genetic engineering of microorganisms used in dairy technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01002—Glucokinase (2.7.1.2)

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 10 9 CFU/ml of the Streptococcus thermophilus strain, such as measured by Test 2 described herein.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 glc
  • the lactose-positive Streptococcus thermophilus strain is carrying a mutation in one gene encoding a protein of the mannose-glucose-specific PTS.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HPLC High Liquid Performance Chromatography
  • 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.
  • 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.
  • the Streptococcus thermophilus species is to be understood as a Streptococcus salivarius subsp. thermophilus strain.
  • 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.
  • 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.
  • 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.
  • 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.
  • a parental strain e.g., DSM28255-de- rivative
  • a strain obtained from an original strain e.g., from the DSM28255- strain
  • another allele in particular an allele carrying some specified mutations
  • 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.
  • 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.
  • 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.
  • 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 10 9 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.
  • 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.
  • a mutated glcK gene encodes a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null.
  • 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.
  • 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.
  • a mutated glcK gene encodes a glucokinase, the glucokinase activity of which in said strain is significantly reduced but not null in said strain.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • G6P glucose-6-phosphate
  • 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.
  • the glucokinase activity in a Streptococcus thermophilus strain is significantly reduced but not null as a consequence of a mutation in its glcK gene.
  • 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 feature "glucokinase activity in said strain is significantly reduced but not null" can be determined by methods well known in the art.
  • 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).
  • 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].
  • 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 "completeTM” (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).
  • G- 6PDH glucose-6-phosphate dehydrogenase
  • :NADPH-coupled assay (Porter et al., 1982), essentially as described by Pool et al. (2006).
  • Assay buffer (10 mM K 2 HPO 4 / 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).
  • BIO- TEK Synergy HT multi-detection microplate reader
  • 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:
  • 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.
  • 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.
  • 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.
  • 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).
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • strains can be used as controls in the Glucokinase Activity Assay:
  • 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.
  • Vmax the maximum forward velocity of the glucokinase
  • Km affinity of the glucokinase
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • Vmax glucokinase maximum forward velocity 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 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.
  • 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.
  • 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.
  • the Vmax is between 5 and 60 % the Vmax of the glucokinase of the DSM28255 strain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Non-limitative examples of glcK genes which are not considered as mutated in the sense of the use according to the present invention are:
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleotides 823-825 of the glcK gene carried by the Streptococcus thermophilus strain used according to the invention are AAA or AAG.
  • 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.
  • the GlcK variant sequence is 322-amino acids in length.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the GlcK variant sequence is 322-amino acids in length.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 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), 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 amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 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
  • the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 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
  • the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 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,
  • 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,
  • the glcK gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO:21.
  • 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.
  • the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 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:
  • the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 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), 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
  • the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 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:
  • the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a truncated protein in particular to a truncated IIAB Man protein, a truncated IIC Man protein or a truncated IID Man protein
  • 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.
  • the mutation introduced into the gene is a nucleotide substitution leading to a STOP codon.
  • 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.
  • 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:
  • 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:
  • 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.
  • 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;
  • 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;
  • 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.
  • the mutation in the manL gene leads to the truncation of the IIAB Man protein at position 305.
  • 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 3 o 5 .
  • 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.
  • 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].
  • 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.
  • 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.
  • 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).
  • 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.
  • the mutation in the manM gene leads to the truncation of the IIC Man protein at position 208.
  • 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 .
  • 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; and b) a IIC Man variant sequence having at least 90% similarity or identity with 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.
  • 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 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.
  • 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.
  • 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).
  • the manM gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO: 157.
  • 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.
  • 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 (manM 12 997')- A resulting Streptococcus thermophilus IIC Man protein truncated at position 179 is referred herein as nc Man179 (SEQ ID NO:213).
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the mutation in the manN gene leads to the truncation of the IID Man protein at position 28 of SEQ ID NO: 167.
  • 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 .
  • 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.
  • 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 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.
  • 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.
  • 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).
  • the manN gene carried by the Streptococcus thermophilus strain used according to the invention is as defined in SEQ ID NO:206.
  • 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.
  • the lactose-positive Streptococcus thermophilus strain used according to the invention carries a mutation in manL.
  • 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.
  • 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.
  • a suitable mutation in the manL gene, manM gene or manN gene 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 phen
  • 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);
  • 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.
  • 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.
  • the ccpA gene mutation is not a mutation leading to the knock-out (i.e., the complete disruption) of the gene.
  • 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.
  • the mutation is a mutation selected from the group consisting of: a) a non-sense mutation (i.e.
  • 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.
  • 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.
  • 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).
  • 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:
  • ccpA type STI the polynucleotide as defined in SEQ ID NO:65
  • this ccpA type is the one of the DSM28255 strain
  • ccpA type ST3 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 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.
  • 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-
  • 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.
  • 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].
  • the ccpA variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO:71.
  • the ccpA variant sequence differs from SEQ ID NO:71 by from 1 to 30 nucleotide substitutions.
  • 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.
  • 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.
  • 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 Man 305 protein), called herein DGCC7710-IIAB Man 30 5 strain
  • 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 Man 208 strain;
  • 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 phen
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • a specific pH value i.e. the PHSTOP value
  • PSTOP phenotype 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.
  • 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).
  • PHSTOP stopped
  • 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.
  • 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).
  • the slope is at least -0.009 UpH/min.
  • 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.
  • the bacterial composition is a pure culture, i.e., comprises or consists of a single bacterium strain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the bacterial composition further comprises at least one probiotic strain such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paraca- sei, or Lactobacillus casei.
  • probiotic strain such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paraca- sei, or Lactobacillus casei.
  • 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.
  • 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.
  • 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.
  • 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 10 5 to 10 12 cfu (colony forming units) per gram of the bacterial composition.
  • the concentration of a lactose-positive Streptococcus thermophilus strain(s) within the bacterial composition of the invention is in the range of 10 7 to 10 12 cfu per gram of the bacterial composition, and in particular at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 or at least 10 11 CFU/g of the bacterial composition.
  • 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 10 8 to 10 12 cfu/g of frozen concentrate or dried concentrate, and more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 or at least 10 12 cfu/g 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.
  • 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.
  • the substrate into which the S. thermophilus strain(s) or bacterial composition described herein is added to is milk substrate.
  • milk substrate it is meant milk of animal and/or plant origin.
  • 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.
  • 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 10 9 CFU/ml of the Streptococcus thermophilus strain, such as measured by Test 2 described herein.
  • 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.
  • 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
  • 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 (manL 13 74o) leading to a stop codon at position 306 of SEQ ID NO:80 of the protein [IIABMan 305 ); 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
  • strain comprises at least a mutation in the glcK gene encoding a glucokinase, which mutation induces a reduction in glucokinase activity, but not null.
  • 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.
  • 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
  • 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.
  • 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.
  • strain is galactose-negative.
  • 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 Leuconosto
  • compositions according to embodiment 15, which lactose-positive, Streptococcus thermophilus strain is as defined in any one of embodiments 2-13.
  • a method for manufacturing a 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.
  • 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.
  • 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.
  • 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.
  • a Streptococcus thermophilus strain selected from the list consisting of
  • 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).
  • ManL, manM, ma nN mannose-glucose-specific PTS
  • thermophilus strains ST21 (DSM28255), ST22 (DSM34172) and ST23 were constructed into which the glcK 37 g, ccpAgss, manL 137 4o, manM 1S4 04, manM 12 997 and/or manN 13 74i 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 glcK 37 g 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 glcK 37 g 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 glcK 37 g mutation and the ccpAgss mutation).
  • the ccpAgss mutation and the glcK 37 g 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 manL 137 4o 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- Man 3os] ⁇
  • the manM 1S 404 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 Man 2os], as defined in SEQ ID NO: 158.
  • the manMi 29 97 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 Man i79 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 manLi 37 4o, manMi 29 97, manMi6404 or manNi 3 74i 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-GMm 2 , ST23-GMm 2 ) or into the genome of strains already mutated in the ccpA gene (resulting in ST21-CMI, ST21-CMmi, ST21-CMn, ST22-CMm 2 , ST23-CMm 2 ) or into the genome of a strain already mutated in both the glcK and the ccpA genes (resulting in ST21-GCMmi).
  • 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 10 7 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.
  • sample 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, Aillesburg) 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).
  • Raw whole cow's milk previously pasteurized 1 min at 74 °C was inoculated at 1% (v/v, about 10 9 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.
  • lactose were measured by HPLC (High Liquid Performance Chromatography) on a Vanquish system provided by Thermo Fisher Scientific equipped with a refractometer detector.
  • HPLC High Liquid Performance Chromatography
  • 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 refrac
  • 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 glcK 37 8 the ccpAsss or one of the manLMN mutations (manLi 37 4o, manMi 2 997, manM 1S4 04 or manN i i) were able to consume more lactose (ranging between 35% and 45%).
  • Table 1 Analysis of the impact of the glcK 378 , ccpAgss, manM 16 404, manL 137 4o, manN 1374 i, and manMi299 7 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.
  • Example 2 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).
  • 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 1.10 10 CFU/100 L and 1.10 11 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.
  • 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.
  • 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).
  • Table 4 displayed the quantity of lactose present in the curds six hours after the bacterial inoculation and before brining.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • a classical mesophilic starter culture named Meso
  • 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.
  • MESO mesophilic starter culture
  • 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)
  • 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.
  • 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).
  • DSM33651 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].
  • DSM34172 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].

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Abstract

La présente invention concerne une utilisation spécifique de souches de Streptococcus thermophilus positives au lactose dans la production de fromage. Les souches utilisées selon la présente invention peuvent porter une ou plusieurs mutations dans le gène glcK codant pour une glucokinase, ladite mutation induisant une réduction de l'activité de la glucokinase, une mutation dans le gène ccpA, et/ou une mutation dans au moins l'un des gènes codant pour les protéines du PTS spécifique du mannose-glucose. La présente invention concerne en outre des compositions comprenant les souches de l'invention utilisées selon la présente invention en combinaison avec au moins une souche du genre Lactobacillus et/ou une souche de bactéries lactiques mésophiles, des procédés de production de fromage et le fromage obtenu par les procédés.
PCT/EP2024/054428 2023-02-22 2024-02-21 Nouvelle utilisation de bactéries lactiques Ceased WO2024175663A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017103051A1 (fr) * 2015-12-18 2017-06-22 Chr. Hansen A/S Bactérie lactique pour préparer des produits alimentaires fermentés ayant un goût sucré naturel augmenté et une texture élevée
WO2019122365A1 (fr) 2017-12-22 2019-06-27 Dupont Nutrition Biosciences Aps Nouvelles bactéries lactiques à propriétés édulcorantes et utilisations de celles-ci
WO2019197051A1 (fr) 2018-12-21 2019-10-17 Dupont Nutrition Biosciences Aps Nouvelles bactéries d'acide lactique
WO2021239969A1 (fr) * 2020-05-28 2021-12-02 Chr. Hansen A/S Contournement du lavage de caillé dans la fabrication de fromage continental
US20220000135A1 (en) * 2018-10-30 2022-01-06 Dupont Nutrition Biosciences Aps Methods and cultures to manufacture pizza cheese

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017103051A1 (fr) * 2015-12-18 2017-06-22 Chr. Hansen A/S Bactérie lactique pour préparer des produits alimentaires fermentés ayant un goût sucré naturel augmenté et une texture élevée
WO2019122365A1 (fr) 2017-12-22 2019-06-27 Dupont Nutrition Biosciences Aps Nouvelles bactéries lactiques à propriétés édulcorantes et utilisations de celles-ci
US20220000135A1 (en) * 2018-10-30 2022-01-06 Dupont Nutrition Biosciences Aps Methods and cultures to manufacture pizza cheese
WO2019197051A1 (fr) 2018-12-21 2019-10-17 Dupont Nutrition Biosciences Aps Nouvelles bactéries d'acide lactique
WO2021239969A1 (fr) * 2020-05-28 2021-12-02 Chr. Hansen A/S Contournement du lavage de caillé dans la fabrication de fromage continental

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Title
POOL ET AL., METABOLIC ENGINEERING, vol. 8, no. 5, 2006, pages 456 - 464

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