WO2012130965A1 - Dérivé naturel d'une souche probiotique bien connue et efficace déficiente en production d'acide d-lactique - Google Patents

Dérivé naturel d'une souche probiotique bien connue et efficace déficiente en production d'acide d-lactique Download PDF

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WO2012130965A1
WO2012130965A1 PCT/EP2012/055672 EP2012055672W WO2012130965A1 WO 2012130965 A1 WO2012130965 A1 WO 2012130965A1 EP 2012055672 W EP2012055672 W EP 2012055672W WO 2012130965 A1 WO2012130965 A1 WO 2012130965A1
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lactobacillus johnsonii
cncm
composition
lactic acid
derivative
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PCT/EP2012/055672
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English (en)
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Raymond-David Pridmore
Francis FOATA
Michèle Delley
Ivana Jankovic
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Nestec S.A.
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Publication of WO2012130965A1 publication Critical patent/WO2012130965A1/fr

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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 THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • 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
    • 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 THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • 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
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/02—Oxidoreductases acting on the CH-OH group of donors (1.1) with a cytochrome as acceptor (1.1.2)
    • C12Y101/02004—D-lactate dehydrogenase (cytochrome) (1.1.2.4)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/151—Johnsonii
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • 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
    • C12R2001/225—Lactobacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention generally relates to the field of probiotic bacteria.
  • the present invention relates to probiotic bacteria deficient in D-lactic acid production.
  • the present invention discloses natural derivatives of the well known probiotic Lactobacillus johnsonii CNCM 1-1225 that have this property.
  • Lactobacillus johnsonii CN CM 1-1225 also known as Lactobacillus johnsonii NCC533, or as Lactobacillus acidophilus Lai, or as Lactobacillus johnsonii Ljl, a human isolate (Bernet-Camard, M. F., et al., (1997) Appl. Environ. Microbiol. 63, 2747-2753), is a probiotic that is currently commercialized very successfully under the trademark Lcl.
  • Lactobacillus johnsonii C N C M 1-1225 has several well documented health benefits, among them, for example activities for immunomodulation (Haller, D., et al., 2000, Infect. Immun. 68:752-759; Haller, D., et al., 2000, Gut 47:79-87; or Ibnou- Zekri, N ., et al., 2003, Infect. Immun. 71:428-436), or pathogen inhibition (Bernet, M. F., et al., 1994, Gut, 35:483-489), and a long history of safe use.
  • Lactobacillus johnsonii CNCM I- 1225 One aspect that has limited the application of Lactobacillus johnsonii CNCM I- 1225 in some product categories, e.g., in products intended for you ng children and i nfa nts, is the prod uction of predom i na ntly the D-lactic acid isome r from the fermentation of sugars.
  • Lactobacillus johnsonii CNCM 1-1225 for example, if grown in MRS medium, ferments lactose to D- and L-lactic acid in a 60:40% ratio.
  • the CODEX I nfant Formula Directive recommends against the consumption of D-lactic acid and D-lactic acid producing bacteria by infants of less than three years of age due to their limited D-lactic acid elimination that may result in D-lactate acidosis.
  • the CODEX has essential ly excluded D-lactic acid producing probiotics as supplements in infant formulae but has inspired the genetic engineering of strains that produce only L-lactic acid.
  • An example of such a development is the generation of a genetical ly modified orga nism (G MO), in particu lar a genetica lly modified Lactobacillus johnsonii strain, where the d-lactate dehydrogenase (D-LDH) gene (IdhD) was isolated, and an in v/ ' iro-truncated cloned copy of the IdhD gene was used to inactivate the genomic copy by gene replacement (Lapierre, L, et al., 1999, Appl. Environ. Microbiol. 65:4002-4007).
  • G MO genetical ly modified orga nism
  • IdhD d-lactate dehydrogenase gene
  • This genetically engineered strain was only produced for laboratory purposes and has never been used in food products since its genetic material has been altered using recombinant DNA technologies and the strain is consequently considered a GMO.
  • natu ra l probiotic strain in particular a natu ral derivative of the Lactobacillus johnsonii strain CNCM 1-1225, which is deficient in D-lactic acid production and which is nevertheless viable.
  • the present inventors were surprised to see that they cou ld achieve the objective of the present invention by the subject matter of the independent claims.
  • the dependant claims further develop the idea of the present invention.
  • the inventors have investigated the possibility to isolate a natural (non-GMO), viable and genetically stable variant of Lactobacillus johnsonii Lai that produces only L-lactic acid.
  • a DNA seq uence analysis identified predomi na ntly point m utations in the lactate dehydrogenase gene that alter the amino acid sequence of the enzyme and hence its catalytic properties.
  • one embodiment of the present invention is a natural derivative of the Lactobacillus johnsonii strai n CN CM 1-1225, wherein the derivative of the Lactobacillus johnsonii strain CNCM 1-1225 is deficient in D-lactic acid production.
  • “Deficient in D-lactic acid production” means for the purpose of the present invention that a strain prod uces less than 5%, preferably less than 2%, even more preferred less than 1.1%, and ideally 0% of D-lactic acid compared to the total lactic acid production.
  • the D- and L-lactic acid concentrations can be measured in the cell- free culture supernatant.
  • the total quantity of lactic acid produced by the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 is initially related to cell growth. When cells enter into the stationary phase and stop dividing, they continue to metabolise sugar and produce more lactic acid. The ratio of D- and L- lactic acid produced was, however, found to be constant.
  • a "natural" derivative of the Lactobacillus johnsonii strain CNCM 1-1225 means a strain which is not considered a GMO.
  • Such a natural derivative may for example be obtained by screening colonies with are subject to changes in the genome sequence that occur naturally, e.g., due to mis-repair of damaged DNA or errors in DNA replication. This natural occurrence of errors may be enhanced by subjecting the colonies to stress conditions, for example by the application of ethyl methane sulfonate, EMS.
  • GMO 'genetically modified organism
  • nucleic acid techniques involving the formation of new combinations of genetic material by the insertion of nucleic acid molecules produced by whatever means outside an organism, into any virus, bacterial plasmid or other vector system and their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation;
  • a strain is considered a "derivative" of the Lactobacillus johnsonii strain CNCM 1-1225, if it has a nucleic acid identity of at least 99.95%, for example of at least 99.99%, preferably of at least 99.995%.
  • a strain is considered a derivative of the Lactobacillus johnsonii strain CNCM 1-1225 if it has no more than 500, for example no more than 100, preferably no more than 50 nucleic acid changes compared to the nucleic acid sequence of Lactobacillus johnsonii CNCM 1-1225.
  • Lactobacillus johnsonii strain CNCM 1-1225 of the present invention exhibited mutations in the d-ldh gene responsible for the D-lactic acid deficient phenotype.
  • the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 may have an altered the D-lactate dehydrogenase enzyme sequence.
  • the D-lactate dehydrogenase enzyme sequence of the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 may comprise a serine to leucine amino acid change at amino acid position 235, and/or an arginine to cystine amino acid change at amino acid position 236, and/or a glycine to aspartic acid amino acid change at amino acid position 155.
  • the changes in the D-lactate dehydrogenase enzyme protein sequence are based on changes in the nucleic acid sequence of the D-lactate dehydrogenase gene.
  • any natural changes in the nucleic acid sequence of the D-lactate dehydrogenase gene that inactivate the resulting enzyme may achieve the subject matter of the present invention. Also at least one deletion of one or more subsequent nucleotides within the wild-type sequence, or part or the whole of the D- lactate dehydrogenase gene may achieve the subject matter of the present invention.
  • the present inventors have analyzed the nucleic acid sequence of the D-lactate dehydrogenase gene in the natural derivative of Lactobacillus johnsonii CNCM 1-1225 of the present invention.
  • the present i nve ntion a lso relates to a n at u ra l d e rivative of t he Lactobacillus johnsonii strain CNCM 1-1225, wherein the nucleic acid sequence of the D-lactate dehydrogenase gene in the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 comprises a C to T transition at nucleic acid position 744, and/or a C to T transition at nucleic acid position 746, and/or a G to A transition at nucleic acid position 504, and/or a single base-pair deletion at position 363, and/or a partial or complete deletion of the D-lactate dehydrogenase gene.
  • D-lactate dehydrogenase While such changes that result in the inactivation of D-lactate dehydrogenase may occur spontaneously at low frequencies in nature, they are random, and can be repaired back to the parent sequence (wild type) at the same frequency.
  • CNCM 1-1225 that achieve the object of the present invention were isolated, purified and characterized in detail.
  • the present invention hence, relates to natural derivative of the Lactobacillus johnsonii strain CN CM 1-1225 that may be selected from the grou p consisting of Lactobacillus johnsonii C N C M 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii C N C M 1-4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii CNCM 1-4440, Lactobacillus johnsonii CNCM 1-4442, and Lactobacillus johnsonii CNCM 1-4443.
  • Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii C N C M 1-4440, Lactobacillus johnsonii CNCM 1-4442, and Lactobacillus johnsonii CNCM 1-4443 were deposited on February 8 th , 2011, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Do Frankfurt Roux, F-75724 Paris Cedex 15, France, under the Budapest Treaty.
  • CNCM Collection Nationale de Cultures de Microorganismes
  • Lactobacillus johnsonii CNCM 1-1225 was deposited on 30 June 1992, with the
  • the natu ral derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention may be cultured according to any suitable method and may be prepared for addition to the compositions of the present invention by freeze-drying or spray-drying for example.
  • the probiotic strain Lactobacillus johnsonii CNCM 1-1225 provides numerous well documented health benefits some of which are detailed above.
  • Probiotic means microbial cell preparations or components of microbial cells with a beneficial effect on the health or well-being of the host. (Salminen S, et al. "Probiotics: how should they be defined” Trends Food Sci. Technol. 1999:10 107-10).
  • the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention may be regarded essentially as bioequivalent in view of the provided health benefits.
  • the present invention relates to a composition
  • a composition comprising the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell- free culture supernatant in accordance with the present invention for use in the preparation of a composition for use in a method for treatment of the h u ma n or animal body by therapy.
  • the present invention also relates to the use of a composition comprising the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell- free culture supernatant in accordance with the present invention in the preparation of a pharmaceutical composition or a medicament.
  • Lactobacillus johnsonii C N C M 1-1225 has been extensively studied for its probiotic-associated activities, including immunomodulation (Haller, D., et al., 2000, Infect. Immun. 68, 752-759; Haller, D., et al., 2000, Gut 47, 79-87; Ibnou-Zekri, N., et al., 2003, Infect. Immun. 71, 428-436), pathogen inhibition (Bernet, M. F., et al., 1994, G u t 35, 483-489), and epithelial cell attachment (Neeser, J. R., et al., 2000, Glycobiology 10, 1193-1199; Granato, D., et al., 1999, Appl. Environ. Microbiol. 65, 1071-1077).
  • immunomodulation Haller, D., et al., 2000, Infect. Immun. 68, 752-759
  • Haller
  • the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell-free culture supernatant thereof in accordance with the present invention provide the same health benefits.
  • the present invention relates to a composition
  • a composition comprising the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell- free cu ltu re su pernata nt in accordance with the present i nvention for use i n the treatment or prevention of disorders linked to a weakened immune system.
  • the present invention also relates to the use of the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell-free culture supernatant in accordance with the present invention for the preparation of a composition to treat or prevent disorders linked to a weakened immune system.
  • Typical exam ples of disorders lin ked to a weakened imm une system may be selected from the grou p consisting of flu, rhinitis, com mon cold, and combinations thereof.
  • the com position com prising the natu ra l derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell-free culture supernatant in accordance with the present invention may also be for use in the treatment or prevention of disorders linked to the cell attachment and cell invasion by enterovirulent bacteria or viruses.
  • the present invention also extends to the use of the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell-free culture supernatant in accordance with the present invention for the preparation of a composition to treat or prevent disorders linked to the cell attachment and cell invasion by enterovirulent bacteria or viruses.
  • the enterovirulent bacterial or viral species may for example be selected from the group consisting of Salmonella; Campylobacter; Listeria; Escherichia coli strains, such as ETEC, EH EC, EP EC, or EI EC strains, for example; Yersinia; Shigella; Toxin producing bacteria, such as Staphylococcus aureus, Clostridium botulinum, or Bacillus cereus; Vibrio vulnifucus/parahaemolyticus; rotavirus; norovirus; verotoxigenic E. coli; Enterobacter sakazakii; toxigenic C.
  • perfringens type A and B
  • food-borne parasites such as Echinococcus, Toxoplasma, or Giardia
  • Helicobacter pylori Helicobacter pylori
  • Clostridium difficile Clostridium tetani; or combinations thereof.
  • the disorder linked to the cell attachment and cell invasion by enterovirulent bacterial or viral species may be selected from the group consisting of lower respiratory tract infections, gastro-intestinal tract infections, otitis media, and combinations thereof.
  • composition of the present invention may be any kind of composition as long as it is suitable for administration to humans or animals.
  • the com position of the prese nt i nve ntio n may i n pa rticu la r be to be administered orally, enterally, parenterally or topically.
  • the compositions may be provided in any galenical form normally available for the selected mode of administration.
  • composition of the present invention may be administered to any age group.
  • composition of the present invention is to be administered during the cold season, e.g., from autumn to spring.
  • composition of the present invention may also be consumed at any time. It may be preferred to consume the composition of the present invention in the morning, e.g., to boost the immune system for the day.
  • the composition may, e.g., be selected from the group consisting of food compositions, petfood compositions, drinks, dairy products, nutritional formulas, infant formulas, food additives, nutraceuticals, pharmaceutical compositions, food ingredients and/or cosmetic compositions.
  • the composition may be selected from the group consisting of acidified milk products, such as yoghurts or yoghurt drinks; or milk based powders.
  • the composition may be provided in the form of a shelf stable powder.
  • the composition may be provided with a water activity smaller than 0.2, for example in the range of 0.19-0.05, preferably smaller than 0.15.
  • Water activity or a w is a measurement of the energy status of the water in a system. It is defined as the vapor pressure of water deriving from the powder/product divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.
  • the com positions of the present invention may be cleansing, protective, treatment or care creams, skincare lotions, gels or foams, such as clea nsing or disinfecting lotions, bath compositions or deodorant compositions.
  • rd s m o re pa rticu l a rly th e co m positio n s fo r exte rn a l to pica l administration they may be aqueous, aqueous-alcoholic or oily solutions, solutions or d ispersions of the lotion or seru m type, em u lsions of l iq u id or semi-liquid consistency, of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice-versa (W/O), or suspensions or emulsions of soft, semi-solid or solid consistency, of the cream type, aqueous or anhydrous gels, microemulsions, microcapsules, microparticles, or vesicular dispersions of ionic and/or non-ionic type.
  • a topical com position according to the invention may advantageously be formulated in any galenical form that is suitable for haircare, especially in the form of a hair lotion, a shampoo, especially an antidandruff shampoo, a hair conditioner, a detangler, a hair cream or gel, a styling lacquer, a hairsetting lotion, a treating lotion, a dye composition (especial ly for oxidation dyeing) optional ly in the form of a colouring shampoo, a hair-restructuring lotion, a permanent-waving composition, a lotion or gel for combating hair loss, an antiparasitic sha mpoo or a medicated shampoo, especially an anti-seborrhoea shampoo, a scalp care prod uct, which is especially anti-irritant, anti-ageing or restructuring, or which activates the blood circulation.
  • a dye composition especial ly for oxidation dyeing
  • the proportion of the fatty phase may range from 5% to 80% by weight, and preferably from 10% to 50% by weight, relative to the total weight of the composition.
  • the oils, the emulsifiers and the coemulsifiers used in the composition in emulsion form are chosen from those conventionally used in the cosmetics and/or dermatological field.
  • the emulsifier and the coemulsifier may be present, in the composition, in a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight, relative to the total weight of the composition.
  • the fatty phase may represent more than 90% of the total weight of the composition.
  • the galenic forms for topical administration may also contain adjuvants that are customary in the cosmetics, pharmaceutical and/or dermatological field, such as hyd rophilic or lipophilic gel ling agents, hyd rophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, screens, odour absorbers and dyestuffs.
  • adjuvants that are customary in the cosmetics, pharmaceutical and/or dermatological field, such as hyd rophilic or lipophilic gel ling agents, hyd rophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, screens, odour absorbers and dyestuffs.
  • the amounts of these various adjuvants are those conventionally used in the field under consideration, and are, for example, from 0.01% to 20% of the total weight of the composition. Depending on their nature, these adjuvants may be introduced into the fatty phase and/or into the
  • mineral oils such as, for example, hydrogenated polyisobutene and liquid petroleum jelly
  • plant oils such as, for example, a liquid fraction of shea butter, sunflower oil and apricot kernel oil
  • animal oils such as, for example, perhydrosqualene
  • synthetic oils in particular Purcellin oil, isopropyl myristate and ethylhexyl palmitate
  • unsaturated fatty acids and fluoro oils such as, for example, perfluoropolyethers.
  • Use may also be made of fatty alcohols, fatty acids such as, for example, stearic acid and such as, for example, waxes, in particular paraffin wax, carnauba wax and beeswax.
  • fatty acids such as, for example, stearic acid and such as, for example, waxes, in particular paraffin wax, carnauba wax and beeswax.
  • silicone compounds such as silicone oils and, for example, cyclomethicone and dimethicone, and silicone waxes, resins and gums.
  • emulsifiers that may be used in the invention, mention may, for example, be made of glyceryl stearate, polysorbate 60, the mixture of cetylstearyl alcohol/oxyethylenated cetylstearyl alcohol comprising 33 mol of ethylene oxide, sold under the name Sinnowax AO * by the company Henkel, the mixture of PEG-6/PEG- 32/glycol stearate sold under the name Tefose * 63 by the company Gattefosse, PPG-3 myristyl ether, silicone emulsifiers such as cetyl dimethicone copolyol and sorbitan monostearate or tristearate, PEG-40 stearate, or oxyethylenated sorbitan monostearate (20 EO).
  • glyceryl stearate polysorbate 60
  • cetylstearyl alcohol/oxyethylenated cetylstearyl alcohol comprising 33 mol of ethylene oxide
  • composition of the invention may also advantageously contain a spring and/or mineral water, in particular chosen from Vittel water, waters from the Vichy basin, and la Roche Posay water.
  • hydrophilic gelling agents such as carbomer, acrylic copolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides, and in particular the mixture of polyacrylamide, C13-14 isoparaffin and Laureth-7 so l d u n d e r t h e n a m e Se p ige l 305 * by the company SEPPIC, polysaccharides, for instance derivatives such as hydroxyalkylcellu loses, and in particular hydroxypropylcellulose and hydroxyethylcellulose, natural gums such as guar gum, locust bean gum, carob and xanthan gum, and clays.
  • carboxylic polymers such as carbomer, acrylic copolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides, and in particular the mixture of polyacrylamide, C13-14 isoparaffin and Laureth-7 so l d u n d e r
  • lipophilic gelling agents mention may be made of modified clays such as bentones, metal salts of fatty acids, such as aluminium stearates and hydrophobic silica, or else ethylcellulose and polyethylene.
  • compositions according to the invention may also be solid preparations constituting cleansing soaps or bars.
  • They may also be used for the scalp in the form of solutions, creams, gels, emulsions or mousses, or alternatively in the form of aerosol compositions also containing a propellant under pressure.
  • an ingestible support or carrier In the case of oral use in accordance with the invention for oral administration, the use of an ingestible support or carrier is preferred.
  • the ingestible support or ca rrier may be of diverse natu re depending on the type of com position u nder consideration.
  • composition according to the invention to be administered orally may be formulated for example in the form of coated tablets, gel capsules, gels, emulsions, tablets, capsules, hydrogels, food bars, compact or loose powders, liquid suspensions or solutions, confectionery products, fermented milks, fermented cheeses, chewing gum, toothpaste or spray solutions or food carriers.
  • Tablets or lozenges, oral supplements in dry form and oral supplements in liq uid form are suita ble for use as dietetic or pharmaceutica l su pports or food carriers.
  • the composition may be, for example, a food supplement, which may be formulated via the usual processes for in particular producing sugar-coated tablets, gel capsules, gels, emulsions, tablets, capsules and hydrogels allowing controlled release.
  • a microorganism according to the invention may be incorporated into al l forms of food su pplements or en riched foods, for exa mple food bars or compacted or non-compacted powders.
  • the powders may be diluted in water, soda, milk products or soya bean derivatives, or may be incorporated into food bars.
  • a microorganism of the invention may moreover be formulated with the usual excipients and components for such oral compositions or food supplements, i.e. in particular fatty and/or aqueous com ponents, h u mecta nts, thickeners, preservatives, textu ring agents, flavou r enhancers and/or coating agents, antioxidants, preservatives and dyes that are customary in the food sector.
  • com positions are administered in an amount sufficient to at least pa rtial ly cu re or a rrest the sym ptoms of a disease a nd its complications.
  • An a mou nt adequate to accomplish this is defined as "a therapeutically effective dose”. Amounts effective for this purpose will depend on a number of factors known to those of skill in the art such as the severity of the disease and the weight and general state of the patient.
  • compositions according to the invention are administered to a patient susceptible to or otherwise at risk of a particular disease in an amou nt that is sufficient to at least partial ly reduce the risk of developing a disease.
  • a prophylactically effective dose Such an amount is defined to be "a prophylactically effective dose”.
  • the precise amounts depend on a number of patient specific factors such as the patient's state of health and weight.
  • compositions of the present invention comprise at least one natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell-free culture supernatant of the present invention in a therapeutically or prophylactically effective dose.
  • the composition may comprise the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention in an amount of 10 6 -10 12 cfu, for example 10 8 to 10 10 cfu per daily dose.
  • L. johnsonii CNCM 1-1225 may be used in the treatment or prevention of disorders related to the immune system including infections, see WO 2010/133475, fully incorporated herein by reference. It was found that L. johnsonii CNCM 1-1225 strongly induces the constitutive hBDl expression, and that heat-treated L. johnsonii CNCM 1-1225 up- regulates hBDl more strongly than its live counterpart.
  • the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention may also be present in a non- replicating form.
  • CNCM 1-1225 include derivatives, which have been heat treated. This includes natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 that are inactivated, dead, non-viable and/or present as fragments such as DNA, metabolites, cytoplasmic compounds, and/or cell wall materials.
  • Non-replicating means that no viable cells and/or colony forming units can be detected by classical plating methods. Such classical plating methods are summarized in the microbiology book: James Monroe Jay, Martin J. Loessner, David A. Golden.2005. Modern food microbiology.7th edition, Springer Science, New York, N.Y.790 p. Typically, the absence of viable cells can be shown as follows: no visible colony on agar plates or no increasing turbidity in liquid growth medium after inoculation with different concentrations of bacterial preparations ('non replicating' samples) and incubation under appropriate conditions (aerobic and/or anaerobic atmosphere for at least 24h).
  • composition may also comprise the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention in an amount of 0,005 mg - 5000 mg, for example 0.5 mg to 50 mg, per daily dose.
  • Figure 1 shows the 'survival curve' for Lactobacillus johnsonii CNCM 1-1225 treated with ethyl methane sulfonate.
  • Figure 2 shows the molecular process of natural mutations from the oxidation of a G base to the segregation of the DNA strands resulting in a mixture of parent and modified DNA types and a mixed parent and modified colony.
  • Figure 3 shows the gene sequence of the Lactobacillus johnsonii CNCM 1-1225 D-lactate dehydrogenase gene (SEQ I D N O : 1) and the changes identified in the corresponding gene in Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM I- 4440, Lactobacillus johnsonii CNCM 1-4442 and Lactobacillus johnsonii CNCM 1-4443 being circled.
  • the translated D-lactate dehydrogenase enzyme (SEQ ID NO: 2) is also shown with the corresponding changes boxed.
  • Lactobacillus johnsonii CNCM 1-4439 contains a larger deletion that was not amplified with the primers used here.
  • Figure 4A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4432 and CN CM 1-4433 D-lactate dehydrogenase genes (SEQ I D N O : 6) with the base at position 464 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (G).
  • Figure 4B shows the protein sequence of Lactobacillus johnsonii CNCM 1-4432 and CNCM 1-4432 D-lactate dehydrogenase enzyme (SEQ I D NO: 10) with the amino acid at position 155 boxed and altered com pared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (G).
  • Figure 5A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4438 D-lactate dehydrogenase gene (SEQ I D N O: 7) with the base at position 704 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (C).
  • Figure 5B shows the protein sequence of Lactobacillus johnsonii CNCM 1-4438 D-lactate dehydrogenase enzyme (SEQ ID NO: 11) with the amino acid at position 235 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (S).
  • Figure 6A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4440 and CN CM 1-4442 D-lactate dehydrogenase genes (SEQ I D N O : 8) with the base at position 706 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (C).
  • Figure 6B shows the protein sequence of Lactobacillus johnsonii CNCM 1-4440 and CNCM 1-4442 D-lactate dehydrogenase enzyme (SEQ I D NO: 12) with the amino acid at position 236 boxed and altered com pared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (R).
  • Figure 7A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4443 D-lactate dehydrogenase gene (SEQ I D N O: 9) with the base at position 458 boxed and deleted compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (C).
  • Figure 7B shows the protein sequence of Lactobacillus johnsonii CNCM 1-4443 D-lactate dehydrogenase enzyme (S EQ I D N O : 13) which is truncated and altered from amino acid 107 compared to the parent strain Lactobacillus johnsonii CNCM I- 1225 (R). The sequence generated from the alternative reading frame until stop is indicated in lower case letters.
  • Example 1 Ethyl methane su lfonate treatment of Lactobacillus johnsonii CN CM 1-1225 cu ltu res. Sa mples of 100 ⁇ containing approximately 10 8 colony forming units of a 16 hr Lactobacillus johnsonii CNCM 1-1225 culture were washed 3 times with Dulbecco's phosphate buffered saline. The cells were finally suspended in 1 ml PBS a nd 0 or 10 ⁇ ethyl methane su lfonate added a nd incu bated at 37°C without shaking.
  • the treated cells were washed twice in PBS, the CFU of treated and not treated cultures determined and plotted as survivors to give the 'survival curve' shown in Figure 1.
  • the conditions producing 1% survivors were initially targeted and bracketed with time points before and after, the cells diluted and plated as single colonies on MRS plates for enumeration.
  • the remaining treated cells were then used to inoculate 10 ml of MRS broth and incubated for 16 hr growth at 37°C.
  • the culture was then dil uted a nd spread on M RS plates to prod uce individ ua l colonies for screening.
  • M utations occu r natu ra l ly i n bacteria mai n ly th rough the oxidation of guanosine (G) residues in double stranded DNA and typically produces single base- pair changes when resolved by DNA replication as shown in Figure 2.
  • G guanosine
  • C cytidine
  • T thymidine
  • Ethyl methane sulfonate acts by chemical oxidation of G bases and results in the same mutation profile as natural mutations, namely G to A and C to T, depending on which DNA strand is sequenced. This was confirmed later by DNA sequencing of the D-lactate dehydrogenase genes and genome sequences where the vast majority of the observed changes were either G to A or C to T.
  • Example 2 Screening of individual colonies for strains deficient in D-lactic acid production. Individual ethyl methane sulfonate treated colonies were picked into 96- well plates containing 200 ⁇ MRS broth and incubated at 37°C for 24 hr to form mini cultures. The growth of the cultures was estimated by absorbance at 620 nm using a Tecan sunrise microplate reader.
  • Example 3 Determination of D-lactic acid levels in culture medium. Cultures were grown in MRS broth at 37°C for 16 hours and the bacteria removed by centrifugation. To determine D-lactic acid concentrations, the cell-free culture supernatants were diluted in water, analysed as described above and compared to a standard curve prepared with dilutions of sodium D-lactic acid. L-lactic acid concentrations were determined in the same way by exchanging the enzyme D- lactate dehydrogenase with rabbit muscle L-lactic dehydrogenase and using sodium L-lactic acid as standard.
  • Lactobacillus paracasei NCC2461 (accession number: CNCM 1-2116) was deposited under the Budapest Treaty on 12 January 1999, with the CNCM (address already mentioned).
  • Lactobacillus rhamnosus NCC4007 (accession number: CGMCC 1.3724) was deposited under the Budapest Treaty in October 2004, with the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No.1, West Beichen Road, Chaoyang District, Beijing 100101, China.
  • Lactobacillus johnsonii NCC9006 is the strain deriving from Lactobacillus johnsonii Lai, as described in the article by Lapierre et al.
  • CNCM 1-1225 Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM I- 4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii CNCM 1-4440, Lactobacillus johnsonii CNCM 1-4442 and Lactobacillus johnsonii CNCM 1-4443 from cell-free cultures.
  • Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM 1-4439 and Lactobacillus johnsonii CNCM 1-4443 are similar to the results obtained for the Lactobacillus johnsonii NCC9006 containing an inactivated D-lactate dehydrogenase gene created using gene technology methods.
  • the D-lactic acid levels for Lactobacillus johnsonii CNCM 1-4440 are also reduced to less than 3% of total lactic acid while the D-lactic acid levels for Lactobacillus johnsonii CNCM 1-4442 is reduced to approximately 1.2% of total lactic acid.
  • the control strains Lactobacillus paracasei NCC2461 and Lactobacillus rhamnosus NCC4007 both considered as L-lactic acid producers, and which produce 3 and 3.5% D-lactic acid under these conditions, respectively.
  • Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii CNCM I- 4440, Lactobacillus johnsonii CNCM 1-4442 and Lactobacillus johnsonii CNCM 1-4443 can be considered as L-lactic acid producers can be considered as L-lactic acid producers and phenotypically distinct from Lactobacillus johnsonii CNCM 1-1225.
  • Example 4 Identification of changes in the D-lactate dehydrogenase genes.
  • the region was amplified from one ⁇ of bacterial culture using the primers PI TCAGCACATAACCAGCAGCT (SEQ ID NO: 3) plus P2 GCAATAATACTGTCGCCGGT (SEQ ID NO: 4).
  • the amplicons were purified and sequenced with primers PI, P3 GTGTATAATAAAAGACGGTC (SEQ ID NO: 5) plus P2, compiled and analysed in the DNASTAR suite of programs. The results are shown in Figure 3.
  • the strain Lactobacillus johnsonii CNCM 1-4432 contains a G to A change at base pair 504 of Figure 3 and Figure 4A, and which results in a glycine to aspartic acid amino acid change at position 155 (G155D) positioned within the conserved signature domain 1 of the D-lactate dehydrogenase enzyme sequence ( Figure 4B).
  • the strain Lactobacillus johnsonii CNCM 1-4433 also contains a G to A change at base pair 504 of Figure 3 and Figure 4A, and which results in a glycine to aspartic acid amino acid change at position 155 (G155D) positioned within the conserved signature domain 1 of the D-lactate dehydrogenase enzyme sequence (Figure 4B).
  • Lactobacillus johnsonii CNCM 1-4432 and Lactobacillus johnsonii CNCM 1-4433 were isolated from the same experiment.
  • the strain Lactobacillus johnsonii CNCM 1-4438 contains a C to T change at base pair 744 of Figure 3 and Figure 5A, and which results in a serine to leucine amino acid change at position 235 (S235L) positioned within the conserved signature domain 3 of the D-lactate dehydrogenase enzyme sequence ( Figure 5B).
  • the strain Lactobacillus johnsonii CNCM 1-4439 contains a deletion of all or part of the D-lactate dehydrogenase gene region and hence no amplification product was produced with primers PI and P2.
  • the strain Lactobacillus johnsonii CNCM 1-4440 contains a C to T change at base pair 746 of Figure 3 and Figure 6A, and which results in a arginine to cystine amino acid change at position 236 (R236C) positioned within the conserved signature domain 3 of the D-lactate dehydrogenase enzyme sequence ( Figure 6B).
  • the strain Lactobacillus johnsonii CNCM 1-4442 contains the same C to T change at base pair 746 of Figure 3 and Figure 6A, and which results in a arginine to cystine amino acid change at position 236 (R236C) positioned within the conserved signature domain 3 of the D-lactate dehydrogenase enzyme sequence ( Figure 6B).
  • D- lactate dehydrogenase changes are identical for both strains Lactobacillus johnsonii CNCM 1-4440 and Lactobacillus johnsonii CNCM 1-4442 they were isolated from independent experiments and are hence not identical.
  • the strain Lactobacillus johnsonii CNCM 1-4443 contains a single base pair deletion at base pair 363 of Figure 3 and Figure 7 A, and which results in a frame shift and premature termination of translation and an inactive D-lactate dehydrogenase enzyme ( Figure 7B).
  • the gene sequencing data shows that the D-lactic acid deficient production phenotypes in these strains are accompanied by corresponding changes in the D-lactate dehydrogenase gene and enzyme sequence.
  • Example 5 Determination of the phenotypic stability of Lactobacillus johnsonii CNCM 1-4432, Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM I- 4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii CNCM 1-4440, Lactobacillus johnsonii CNCM 1-4442 and Lactobacillus johnsonii CNCM 1-4443. Given the large number of generations from the culture collection to the final product, it is important that the D-lactic acid deficient phenotype is stable and that reversion to production of D-lactic acid is very rare.
  • Lactobacillus johnsonii CNCM 1-4432 Lactobacillus johnsonii CNCM 1-4433, Lactobacillus johnsonii CNCM 1-4438, Lactobacillus johnsonii CNCM 1-4439, Lactobacillus johnsonii CNCM I- 4440, Lactobacillus johnsonii CNCM 1-4442 and Lactobacillus johnsonii CNCM 1-4443 in MRS broth for a total of 100 generations and then tested 300 individual colonies for D-lactic acid production. The results are that none of the colonies tested showed D-lactic acid levels above that determined for each strain.
  • lactic acid bacteria In lactic acid bacteria a single copy of D- or L-lactate dehydrogenase is essential for the prod uction of lactic acid a nd the regeneration of NADH, a co-factor in this reaction, to NAD. In lactic acid bacteria this is the only route to regenerate NAD under anaerobic conditions a nd is essentia l for growth. I n th e case of th e D-lactic acid deficient strai ns there is no selective pressu re for the reversion to D-lactic acid production as the L-lactate dehydrogenase enzyme is sufficient to cover the lack of D- lactate dehydrogenase enzyme activity.

Abstract

La présente invention concerne de manière générale le domaine des bactéries probiotiques. En particulier, la présente invention concerne des bactéries probiotiques déficientes en production d'acide D-lactique. Par exemple, la présente invention concerne des dérivés naturels de la souche probiotique bien connue Lactobacillus johnsonii CNCM I-1225 qui a cette propriété.
PCT/EP2012/055672 2011-03-29 2012-03-29 Dérivé naturel d'une souche probiotique bien connue et efficace déficiente en production d'acide d-lactique WO2012130965A1 (fr)

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WO2014092832A2 (fr) 2012-09-19 2014-06-19 Novozymes, Inc. Procédés pour améliorer la dégradation ou la conversion de matériau cellulosique
WO2014058896A1 (fr) 2012-10-08 2014-04-17 Novozymes A/S Polypeptides ayant une activité améliorant la cellulolyse et polynucléotides codant pour ceux-ci
EP3586610A1 (fr) 2012-10-08 2020-01-01 Novozymes A/S Polypeptides ayant une activité améliorant la cellulolyse et polynucléotides codant pour ceux-ci
WO2014066141A2 (fr) 2012-10-24 2014-05-01 Novozymes A/S Polypeptides ayant une activité d'amélioration cellulolytique et polynucléotides codant pour ceux-ci
WO2014182990A1 (fr) 2013-05-10 2014-11-13 Novozymes A/S Polypeptides présentant une activité xylanase et polynucléotides codant pour ceux-ci
WO2016145350A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique à étages multiple d'une biomasse lignocellulosique
WO2016145363A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique multi-étape d'une biomasse lignocellulosique faisant appel à une oxydoréductase et à un polypeptide aa9
WO2016145358A1 (fr) 2015-03-12 2016-09-15 Novozymes A/S Hydrolyse enzymatique avec des enzymes hémicellulolytiques
WO2016162227A1 (fr) 2015-04-09 2016-10-13 Nestec S.A. Procédé de formation d'un acide dihydroférulique

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