DK2860267T3 - Kulturer med forbedret fagresistens - Google Patents

Kulturer med forbedret fagresistens Download PDF

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DK2860267T3
DK2860267T3 DK14195132.7T DK14195132T DK2860267T3 DK 2860267 T3 DK2860267 T3 DK 2860267T3 DK 14195132 T DK14195132 T DK 14195132T DK 2860267 T3 DK2860267 T3 DK 2860267T3
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crispr
seq
sequence
bacteriophage
crispr locus
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Christophe Fremaux
Philippe Horvath
Dennis Romero
Patrick Boyaval
Rodolphe Barrangou
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Dupont Nutrition Biosci Aps
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • 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; PREPARATION 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
    • 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; PREPARATION 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
    • A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
    • 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; 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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; PREPARATION THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/202—Genetic engineering of microorganisms used in dairy technology
    • 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
    • A23V2400/21—Streptococcus, lactococcus
    • A23V2400/249—Thermophilus
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
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  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Fodder In General (AREA)
  • Food Preservation Except Freezing, Refrigeration, And Drying (AREA)

Claims (40)

1. Fremgangsmåde til frembringelse af en starterkultur, der omfatter mindst to forskellige bakteriofagresistente variantstammer, der omfatter trinene: (a) eksponering af en parental bakteriestamme, der omfatter mindst en del af et CRISPR-locus, for mindst én bakteriofag til frembringelse af en blanding af bakterier, der omfatter mindst én bakteriofagresistent variantstamme, der omfatter et modificeret CRISPR-locus, der omfatter mindst én yderligere spacer i det modificerede CRISPR-locus; (b) eksponering af en parental bakteriestamme, der omfatter mindst en del af et CRISPR-locus, for mindst én bakteriofag til frembringelse af en blanding af bakterier, der omfatter mindst en anden bakteriofagresistent variantstamme, der omfatter et modificeret CRISPR-locus, der omfatter mindst én yderligere spacer i det modificerede CRISPR-locus; (c) selektion af de bakteriofagresistente variantstammer fra blandingerne af bakterier; (d) selektion af de bakteriofagresistente variantstammer, der omfatter en yderligere spacer i det modificerede CRISPR-locus, fra de bakteriofagresistente stammer, der er selekteret i trin (c); og (e) isolering af de bakteriofagresistente variantstammer, hvor stammerne omfatter en yderligere spacer i det modificerede CRISPR-locus.
2. Fremgangsmåde ifølge krav 1, hvor fremgangsmåden omfatter trinene: (a) eksponering af en parental bakteriestamme, der omfatter mindst en del af et CRISPR-locus, for en blanding af to eller flere bakteriofager til frembringelse af en blanding af bakterier, der omfatter mi ndst én bakteriofagresistent variantstamme, der omfatter et modificeret CRISPR-locus, der omfatter mindst én yderligere spacer i det modificerede CRISPR-locus; (b) eksponering af en parental bakteriestamme, der omfatter mindst en del af et CRISPR-locus, for en blanding af to eller flere bakteriofager til frembringelse af en blanding af bakterier, der omfatter mindst en anden bakteriofagresistent variantstamme, der omfatter et modificeret CRISPR-locus, der omfatter mindst én yderligere spacer i det modificerede CRISPR-locus; (c) selektion af de bakteriofagresistente variantstammer fra blandingerne af bakterier; (d) selektion af de bakteriofagresistente variantstammer, der omfatter en yderligere spacer i det modificerede CRISPR-locus, fra de bakteriofagresistente stammer, der er selekteret i trin (c); og (e) isolering af de bakteriofagresistente variantstammer, hvor stammerne omfatter en yderligere spacer i det modificerede CRISPR-locus.
3. Fremgangsmåde ifølge krav 1 eller krav 2, hvor fremgangsmåden yderligere omfatter trinet sammenligning af CRISPR-locuset eller en del deraf i den parentale bakteriestamme og det modificerede CRISPR-locus i de bakteriofagresistente variantstamme til identificering af bakteriofagresistente variantstammer, der omfatter mindst én yderligere spacer i det modificerede CRISPR-locus, der ikke er til stede i CRISPR-locuset i den parentale bakteriestamme.
4. Fremgangsmåde ifølge krav 1, hvor den parentale bakteriestamme eksponeres for to eller flere bakteriofager.
5. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme eksponeres samtidigt for to eller flere bakteriofager.
6. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme eksponeres sekventielt for to eller flere bakteriofager.
7. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme inficeres af bakteriofagen.
8. Fremgangsmåde ifølge krav 7, hvor den mindst ene bakteriofag er valgt fra gruppen af virusfamilier, der består af: Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae og Tectiviridae.
9. Fremgangsmåde ifølge krav 7, hvor den mindst ene bakteriofag er en naturligt forekommende bakteriofag.
10. Fremgangsmåde ifølge krav 7, hvor den mindst ene bakteriofag er en muteret bakteriofag opnået ved hjælp af selektionstryk ved anvendelse af en bakteriofagresistent bakterie.
11. Fremgangsmåde ifølge krav 1 eller krav 2, hvor 5'-enden og/eller 3'-enden af CRISPR-locuset i den parentale bakteriestamme sammenlignes med det modificerede CRISPR-locus i de bakteriofagresistente variantstamme.
12. Fremgangsmåde ifølge krav 1 eller krav 2, hvor 5'-enden og/eller 3'-enden af den første CRISPR-spacer i CRISPR-locuset i den parentale bakteriestamme sammenlignes med det modificerede CRISPR-locus i de bakteriofagresistente variantstamme.
13. Fremgangsmåde ifølge krav 1 eller krav 2, hvor mindst en del af CRISPR-locuset i den parentale bakteriestamme og mindst en del af det modificerede CRISPR-locus i de bakteriofagresistente variantstamme sammenlignes ved hjælp af amplificering af mindst en del af CRISPR-locuset og mindst en del af det modificerede CRISPR-locus til frembringelse af en amplificeret CRISPR-locussekvens og en amplificeret, modificeret CRISPR-locussekvens.
14. Fremgangsmåde ifølge krav 13, hvor amplificeringen udføres ved anvendelse af polymerasekædereaktion.
15. Fremgangsmåde ifølge krav 1 eller krav 2, hvor mindst en del af CRISPR-locuset i den parentale bakteriestamme og mindst en del af det modificerede CRISPR-locus i de bakteriofagresistente variantstamme sammenlignes ved hjælp af sekventering af mindst en del af CRISPR-locuset og mindst en del af det modificerede CRISPR-locus.
16. Fremgangsmåde ifølge krav 13, der yderligere omfatter trinet sekventering af den amplificerede CRISPR-locussekvens og den amplificerede, modificerede CRISPR-locussekvens.
17. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den yderligere spacer i det modificerede CRISPR-locus udgør en del af en gentagelse-spacer-enhed.
18. Fremgangsmåde ifølge krav 17, hvor gentagelses-spacer-enheden omfatter mindst 44 nukleotider.
19. Fremgangsmåde ifølge krav 17, hvor gentagelses-spacer-enheden omfatter mellem 44 og 119 nukleotider.
20. Fremgangsmåde ifølge krav 17, hvor gentagelses-spacer-enheden omfatter mindst én nukleotidsekvens, der har mindst ca. 95 % identitet med en CRISPR-gentagelse i CRISPR-locuset i den parentale bakteriestamme.
21. Fremgangsmåde ifølge krav 17, hvor gentagelses-spacer-enheden omfatter mindst én nukleotidsekvens, der har mindst ca. 95 % identitet med en nukleotidsekvens i genomet i mindst én bakteriofag.
22. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme er en industrielt anvendelig stamme.
23. Fremgangsmåde ifølge krav 22, hvor den parentale bakteriestamme er modtagelig for infektion af mindst én bakteriofag.
24. Fremgangsmåde ifølge krav 22, hvor den parentale bakteriestamme er egnet til anvendelse som en starterkultur, en probiotisk kultur og/eller en kosttilskudskultur.
25. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme er udvalgt blandt Escherichia, Shigella, Salmonella, Erwinia, Yersinia, Bacillus, Vibrio, Legionella, Pseudomonas, Neisseria, Bordetella, Helicobacter, Listeria, Agrobacterium, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Corynebacterium, Mycobacterium, Treponema, Borrelia, Francisella, Brucella, Campylobacter, Klebsiella, Frankia, Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix, Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus, Lactobacillus, Pediococcus, Leuconostoc og Oenococcus.
26. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme er følsom over for alle bakteriofagerne, som den eksponeres for i blandingen.
27. Fremgangsmåde ifølge krav 1 eller krav 2, hvor den parentale bakteriestamme er følsom over for nogle af bakteriofagerne, som den eksponeres for i blandingen.
28. Fremgangsmåde ifølge krav 1 eller krav 2, hvor to eller flere forskellige spacere hver integreres i to eller flere forskellige CRISPR-loci.
29. Fremgangsmåde ifølge krav 1 eller krav 2, hvor to eller flere forskellige spacere integreres i ét CRISPR-locus.
30. Starterkultur opnået ved hjælp af fremgangsmåden ifølge et hvilket som helst af ovennævnte krav.
31. Starterkultur ifølge krav 30 i form af en tørret starterkultur, en dehydreret starterkultur, en frosset starterkultur eller en koncentreret starterkultur.
32. Starterkultur ifølge krav 31 i form af en tablet, en pellet, en kapsel, et granulat eller et pulver.
33. Starterkultur ifølge krav 31, hvor den tørrede starterkultur er befugtelig, sprøjtetørret, frysetørret eller lyofiliseret.
34. Starterkultur ifølge krav 31 i form af en dybfrosset pellet eller et frysetørret pulver.
35. Starterkultur ifølge krav 31 i form af et koncentrat.
36. Fremgangsmåde til fermentering, der omfater tilsætning af starterkulturen ifølge et hvilket som helst af kravene 30 til 35 til et fermenteringsmedium under betingelser, således at der forekommer fermentering af bestanddelene i fermenteringsmediet.
37. Fremgangsmåde ifølge krav 36, hvor fermenteringen ikke påvirkes af tilstedeværelsen af bakteriofager.
38. Fremgangsmåde ifølge krav 36, hvor fermenteringsmediet er et fødevareprodukt.
39. Fremgangsmåde ifølge krav 38, hvor fødevareproduktet er et mej eriprodukt.
40. Fremgangsmåde ifølge krav 39, hvor mejeriproduktet er mælk.
DK14195132.7T 2007-03-02 2008-02-29 Kulturer med forbedret fagresistens DK2860267T3 (da)

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US90472107P 2007-03-02 2007-03-02
EP08726285.3A EP2126130B1 (en) 2007-03-02 2008-02-29 Cultures with improved phage resistance

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US (2) US20110002889A1 (da)
EP (3) EP2126130B1 (da)
JP (2) JP5932207B2 (da)
CN (2) CN101688241B (da)
AU (1) AU2008223544B2 (da)
BR (1) BRPI0808704B1 (da)
CY (1) CY1116417T1 (da)
DK (2) DK2860267T3 (da)
ES (2) ES2719789T3 (da)
HR (1) HRP20150676T1 (da)
HU (1) HUE025412T2 (da)
MX (1) MX2009009071A (da)
NZ (2) NZ579002A (da)
PL (1) PL2126130T3 (da)
PT (1) PT2126130E (da)
RU (2) RU2531343C2 (da)
SI (1) SI2126130T1 (da)
TR (1) TR201905633T4 (da)
WO (1) WO2008108989A2 (da)

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