WO2012130970A1 - Dérivé naturel de la souche de lactobacillus johnsonii cncm i-1225, déficient en production d'acide d-lactique - Google Patents

Dérivé naturel de la souche de lactobacillus johnsonii cncm i-1225, déficient en production d'acide d-lactique Download PDF

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WO2012130970A1
WO2012130970A1 PCT/EP2012/055678 EP2012055678W WO2012130970A1 WO 2012130970 A1 WO2012130970 A1 WO 2012130970A1 EP 2012055678 W EP2012055678 W EP 2012055678W WO 2012130970 A1 WO2012130970 A1 WO 2012130970A1
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Prior art keywords
lactobacillus johnsonii
cncm
composition
strain cncm
lactic acid
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PCT/EP2012/055678
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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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Priority to MX2013011233A priority Critical patent/MX2013011233A/es
Priority to EP12711869.3A priority patent/EP2691549A1/fr
Priority to CN201280025966.XA priority patent/CN103582698B/zh
Priority to BR112013025216-2A priority patent/BR112013025216A2/pt
Priority to SG2013070933A priority patent/SG193566A1/en
Priority to AU2012234195A priority patent/AU2012234195A1/en
Publication of WO2012130970A1 publication Critical patent/WO2012130970A1/fr

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    • 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)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
    • 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.
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/10—Anthelmintics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
    • 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 natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 that are deficient in D-lactic acid production.
  • 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 infants, is the p rod u ction of predo m i n a ntly the D-lactic a ci d iso m e r fro m th e 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 Infant 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.
  • G MO genetically 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 natural 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 predominantly point mutations i n 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 produces less than 5%, preferably less than 4%, 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 q ua ntity of lactic acid prod uced by the natu ral derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 is initia lly related to cel l growth .
  • 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 G MO.
  • Such a natural derivative may for example be obtained by screening colonies with are subject to changes in the genome sequence that occu r naturally, e.g., d ue to m is-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 shall be defined according to the DI RECTIVE 2001/18/EC OF TH E EU ROPEAN PARLIAMENT AN D OF TH E COUNCIL of 12 Ma rch 2001 on the deliberate release into the environ ment of genetical ly modified orga nisms an d repea li ng Cou nci l Directive 90/220/EEC. Accordingly, a 'genetica l ly modified organism (G MO)' mea ns an orga nism, with the exception of human beings, in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination.
  • a strain is considered a "derivative" of the Lactobacillus johnsonii strain CNCM 1-1225, if it has a n ucleic acid identity of at least 99.95%, for example of at least 99.99%, prefera bly of at least 99.995%.
  • a strai n is conside red 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.
  • the present inventors have found that the natu ra l derivatives of the 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.
  • CNCM 1-1225 may have an altered the D-lactate dehydrogenase enzyme sequence.
  • the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 may comprise a glycine to aspartic acid amino acid change at amino acid position 153 and/or a serine to leucine amino acid change at amino acid position 204 of the D- lactate dehydrogenase enzyme sequence.
  • the cha nges in the D-lactate dehydrogenase enzyme protein sequence are based on changes in the nucleic acid sequence of the D-lactate dehydrogenase gene.
  • 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 nvention a lso relates to a natural derivative of the 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 G to A transition at nucleic acid position 498 and/or a C to T transition at nucleic acid position 651.
  • 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.
  • This repair frequency may even be higher if the inactivated gene imparts a growth disadvantage to the variant.
  • the inactivated gene imparts a growth disadvantage to the variant.
  • Lactobacillus johnsonii strain CN CM 1-1225 that may be selected from the group consisting of Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM I- 4441.
  • Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM I- 4441 were deposited on February 8 th , 2011, with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 Rue du Dondel 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 CNCM, under the Budapest Treaty.
  • Lactobacillus johnsonii C N C M 1-4434 contains a total of 37 nucleic acid changes, including a single base-pair deletion. This includes the expected change in t h e D-lactate dehydrogenase gene sequence, presumably responsible for the deficiency in the production of D-lactic acid by this strain plus two changes in genes in prophage Lj965 that may be considered as 'selfish' DNA with no likely impact on the physiology of the bacterium. The remaining genes/predicted proteins are mainly only loosely annotated, but several changes affect genes that are more important to bacterial growth.
  • Lactobacillus johnsonii CNCM 1-4441 contains a total of 27 nucleic acid changes, including the expected change in the D lactic acid dehydrogenase gene responsible for the deficiency in the production of D-lactic acid by this strain plus one change each in prophage Lj965 and Lj928, plus a change in the transposase of insertion sequence ISLjo5. There are changes in three genes whose function is predicted to be important for growth, namely LJ1009 - elongation factor Tu, LJ1516 - signal recognition particle protein and U1600 - peptide chain release factor 3. Despite these individual and combined changes CNCM 1-4441 achieves similar final CFU's in MRS and industrial media compared to Lai.
  • the present invention also extends to the natural derivatives of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention, wherein the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 is present as biologically pure culture.
  • the natural 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 further extends to the cell-free culture supernatant isolated from a biologically pure culture of a natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 in accordance with the present invention.
  • 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 hu man 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 CN CM 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 cel l-free culture supernatant thereof in accordance with the present invention provide the same health benefits.
  • the present invention relates to a composition comprising the natural derivative of the Lactobacillus johnsonii strain CNCM 1-1225 and/or the cell- free cultu re su pernatant in accordance with the present invention for use in 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 examples of disorders linked to a weakened imm u ne system may be selected from the group consisting of fl u, rhinitis, common cold, and combinations thereof.
  • T h e 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 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, EHEC, EPEC, 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. They 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.
  • the composition of the present invention is to be administered during the cold season, e.g., from autumn to spring. It 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 immu ne 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.
  • compositions for external topical administration they may be aqueous, aqueous-alcoholic or oily solutions, solutions or dispersions of the lotion or serum type, emulsions of liquid 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 restructu ring, 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 .
  • these adjuvants may be introduced into the fatty phase and/or into the aque
  • fatty substances that may be used in the invention, mention may be made of m i nera l oi ls such as, for exa m ple, hyd rogenated 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.
  • 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 such as, for example, per
  • 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 m a de of glyce ryl stea rate, po lyso rbate 60, th e m ixtu re of cetylstea ryl 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 stea rate, o r oxyethyle n ated sorbita n monostearate (20 EO).
  • 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
  • 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.
  • oral supplements in dry form and oral supplements in liq uid form are suita ble for use as dietetic or pharmaceutica l supports or food carriers.
  • the composition may be, for example, a food su pplement, 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 example 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 components, humectants, thickeners, preservatives, texturing agents, flavour enhancers and/or coating agents, antioxidants, preservatives and dyes that are customary in the food sector.
  • excipients and components for such oral compositions or food supplements i.e. in particular fatty and/or aqueous components, humectants, thickeners, preservatives, texturing agents, flavour enhancers and/or coating agents, antioxidants, preservatives and dyes that are customary in the food sector.
  • compositions are administered in an amount sufficient to at least partially cure or arrest the symptoms of a disease and its complications.
  • An amount 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 amount that is sufficient to at least partially reduce the risk of developing a disease.
  • 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.
  • 1-1225 in accordance with the present invention may also be present in a non- replicating form.
  • Non-replicating natural derivatives of the Lactobacillus johnsonii strain 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.
  • Figu re 1 shows the 'su rvival cu rve' for Lactobacillus johnsonii CN CM 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 (S EQ I D N O : 1 ) and the changes identified in the correspon ding genes in Lactobacillus johnsonii C N C M 1-4434 and Lactobacillus johnsonii CNCM 1-4441 being circled.
  • the translated D-lactate dehydrogenase enzyme (SEQ I D NO: 2) is also shown with the corresponding changes for Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM 1-4441.
  • Figure 4A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4434 D-lactate dehydrogenase gene (SEQ I D N O: 6) with the base at position 611 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (C).
  • Figure 4B shows the protein sequence of the Lactobacillus johnsonii CNCM I-
  • FIG. 4434 D-lactate dehydrogenase enzyme (SEQ ID NO: 8) with the amino acid at position 204 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM I- 1225 (S).
  • Figure 5A shows the gene sequence of the Lactobacillus johnsonii CNCM 1-4441 D-lactate dehydrogenase gene (SEQ I D NO: 7) with the base at position 458 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM 1-1225 (G).
  • Figure 5B shows the protein sequence of the Lactobacillus johnsonii CNCM I- 4441 D-lactate dehydrogenase enzyme (SEQ ID NO: 9) with the amino acid at position 153 boxed and altered compared to the parent strain Lactobacillus johnsonii CNCM I- 1225 (G).
  • 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 and 0, 10 ⁇ ethyl methane sulfonate added and incubated 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.
  • 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 diluted and spread on MRS plates to produce individual 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.
  • 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 ⁇ M RS 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 sun rise microplate reader.
  • screening individ ua l colon ies for the presence of D-lactic acid it is a lso clea r that 'mixed' cultures containing 50% of a D-lactic acid producer and 50% of a D-lactic acid non- producer will result in a culture that is 'positive' for the presence of D-lactic acid.
  • the D-lactic acid deficient phenotype that we target is therefore considered as 'recessive' to the D-lactic acid production phenotype.
  • Example 3 Determination of D-lactic acid levels in culture medium. Cultures were grown in M RS broth at 37°C for 16 hou rs a nd the bacte ria 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 standa rd curve prepared with dilutio n s of sod i u m 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.
  • the results of this analysis for CNCM 1-4431 and CNCM I- 4434 are shown in Table 1 and include the controls of Lactobacillus johnsonii CNCM I- 1225, Lactobacillus johnsonii N C C9006 wi t h a G M O i n a ct i va te d D-lactate dehyd rogenase gene a nd Lactobacillus paracasei NCC2461 a n d Lactobacillus rhamnosus NCC4007, both considered as L-lactic acid producing strains.
  • Lactobacillus paracasei NCC2461 (accession n u m ber: 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.
  • CGMCC General Microbiological Culture Collection Center
  • Lactobacillus johnsonii NCC9006 is th e stra i n d e rivi ng fro m Lactobacillus johnsonii Lai, as described in the article by Lapierre et al. entitled "D- Lactate Dehydrogenase Gene (IdhD) Inactivation and Resulting Metabolic Effects in the Lactobacillus johnsonii Strains Lai and N312" (Appl. Environ. Microbiol. 1999, 65(9):4002).
  • Table 1 Values of D- and L-lactic acid determined for CNCM 1-1225, CNCM I-
  • Experiments were performed in triplicate and are given as the mean value plus standard deviation in brackets.
  • the results show that the D-lactic acid production values for Lactobacillus johnsonii CNCM 1-1225 are approximately 65% and that the D-lactic acid production values for Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM I- 4441 are greatly reduced.
  • Lactobacillus johnsonii CNCM 1-4434 there is a small biological variation in D-lactic acid production levels that vary between 3 and 4% of total lactic acid, depending on the experiment.
  • the D-lactic acid production levels for Lactobacillus johnsonii CNCM 1-4441 are very low and below the detection levels for this assay.
  • 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 and compiled and analysed in the DNASTAR suite of programs. The results are shown in Figure 3.
  • the strain Lactobacillus johnsonii CNCM 1-4434 contains a C to T change at base pair 651 of Figure 3 and Figure 4A, and which results in a serine to leucine amino acid change at position 204 (S204L) in the signature 3 domain of the D-lactate dehydrogenase enzyme sequence ( Figure 4B).
  • the strain Lactobacillus johnsonii CNCM 1-4441 contains a G to A change at base pair 498 of Figure 3 and Figure 5A, and which results in a glycine to aspartic acid amino acid change at position 153 (G153D) in the signature 1 domain of the D-lactate dehydrogenase enzyme sequence (Figure 5B).
  • the gene sequencing data shows that the D-lactic acid deficient production phenotype in strains Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM 1-4441 is accompanied by a corresponding change in the D-lactate dehydrogenase gene and enzyme sequence.
  • Example 5 Determination of the phenotypic stability of Lactobacillus johnsonii CNCM 1-4434 and Lactobacillus johnsonii CNCM 1-4441. 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. To investigate this we cultivated Lactobacillus johnsonii CNCM I- 4434 and Lactobacillus johnsonii CNCM 1-4441 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 . This a na lysis was a lso performed after pilot-scale production of spray-dried powders and Lcl Drink products with the same results.
  • lactic acid bacteria In lactic acid bacteria a single copy of D- or L-lactate dehydrogenase is essential for the production of lactic acid and 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 and is essential for growth. I n the case of the D-lactic acid deficient strains there is no selective pressure 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.

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Abstract

L'invention se rapporte généralement au domaine des bactéries probiotiques et, en particulier, à des dérivés naturels de la souche de Lactobacillus johnsonii CNCM I-1225, déficients en production d'acide D-lactique
PCT/EP2012/055678 2011-03-29 2012-03-29 Dérivé naturel de la souche de lactobacillus johnsonii cncm i-1225, déficient en production d'acide d-lactique WO2012130970A1 (fr)

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MX2013011233A MX2013011233A (es) 2011-03-29 2012-03-29 Derivado natural de la cepa de lactobacillus johnsonii cncm i-1225, deficiente en la produccion de acido d-lactico.
EP12711869.3A EP2691549A1 (fr) 2011-03-29 2012-03-29 Dérivé naturel de la souche cncm i-1225 de lactobacillus johnsonii déficient en production d'acide d-lactique
CN201280025966.XA CN103582698B (zh) 2011-03-29 2012-03-29 缺失d-乳酸产生的约氏乳杆菌菌株cncm i-1225的天然衍生物
BR112013025216-2A BR112013025216A2 (pt) 2011-03-29 2012-03-29 derivado natural da cepa de lactobacillus johnsonii cncm i-1225 deficiente na produção de ácido d-láctico
SG2013070933A SG193566A1 (en) 2011-03-29 2012-03-29 Natural derivative of the lactobacillus johnsonii strain cncm i-1225 deficient in d-lactic acid production
AU2012234195A AU2012234195A1 (en) 2011-03-29 2012-03-29 Natural derivative of the Lactobacillus johnsonii strain CNCM I-1225 deficient in D-lactic acid production

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SG193566A1 (en) 2013-10-30
BR112013025216A2 (pt) 2018-09-04
MX2013011233A (es) 2013-10-17
AU2012234195A1 (en) 2013-10-10
EP2691549A1 (fr) 2014-02-05

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