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1. WO2006097107 - LACTOSE-POSITIVE RECOMBINANT LEUCONOSTOC STRA

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Claims:
1. An isolated recombinant lactose-positive Leuconostoc carnosum strain comprising one or more genes encoding:
- a lactose transporter and/or
- a β-galactosidase or β-glucosidase.

2. A Leuconostoc carnosum strain according to claim 1 , wherein said strain produces a bacteriosin.

3. A Leuconostoc carnosum strain according to claim 1 or 2, wherein said strain is a mutant and the product of mutagenesis of one or more native gene of the Leuconostoc carnosum strain.

4. A Leuconostoc carnosum strain according to claim 1 , wherein said:
- lactose transporter and/or
- β-galactosidase or β-glucosidase
are encoded by heterologous genes stably inherited in the Leuconostoc carnosum strain.

5. A Leuconostoc carnosum strain according to claim 4, wherein said β- galactosidase is a polypeptide having an amino acid sequence that is at least 60% homologous to the amino acid sequence of SEQ ID NO: 3, or a fragment thereof, conferring functional β-galactosidase activity.

6. The Leuconostoc carnosum strain according to claim 5, wherein said β-galactosidase has the amino acid sequence of SEQ ID NO: 3.

7. A Leuconostoc carnosum strain according to claim 4, wherein said β- galactosidase comprises an L and an M polypeptide having an amino acid sequence that is at least 60% homologous to the amino acid sequence of SEQ ID NO: 13 and 15 respectively, or a fragment thereof, and conferring functional β-galactosidase activity.

8. A Leuconostoc carnosum strain according to claim 4, wherein said β- galactosidase comprises an L and an M polypeptide having the amino acid sequence of SEQ ID NO: 13 and 15 respectively.

9. The Leuconostoc carnosum strain according to claim 5 or 6, wherein said β-galactosidase is encoded by a nucleic acid molecule that hybridises to a nucleic acid molecule with a nucleotide sequence consisting of SEQ ID NO: 1 or 4, under washing stringency conditions of no less than IxSSC at 650C.

10. The Leuconostoc carnosum strain according to claim 9, wherein said said nucleic acid molecule has the nucleotide sequence of SEQ ID

NO: 1 or 4.

11. The Leuconostoc carnosum strain according to claim 7 or 8, wherein said L and M polypeptide are encoded by a nucleic acid molecule that hybridises to a nucleic acid molecule with a nucleotide sequence consisting of SEQ ID NO: 12, under washing stringency conditions of no less than IxSSC at 650C.

12. The Leuconostoc carnosum strain according to claim 11 , wherein said said nucleic acid molecule has the nucleotide sequence of SEQ ID

NO: 12.

13. A Leuconostoc carnosum strain according to claim 4, wherein said lactose transporter is a polypeptide having an amino acid sequence that is at least 60% homologous to the amino acid sequence of SEQ ID NO: 2, or a fragment thereof, conferring functional lactose transporter activity.

14. A Leuconostoc carnosum strain according to claim 13, wherein said lactose transporter has the amino acid sequence of SEQ ID NO: 2.

15. A Leuconostoc carnosum strain according to claim 4, wherein said lactose transporter is a polypeptide having an amino acid sequence that is at least 60% homologous to the amino acid sequence of SEQ ID NO: 11 , or a fragment thereof, conferring functional lactose transporter activity.

16. A Leuconostoc carnosum strain according to claim 15, wherein said lactose transporter has the amino acid sequence of SEQ ID NO: 11.

17. A Leuconostoc carnosum strain according to claim 13 or 14, wherein said lactose transporter is encoded by a nucleic acid molecule that hybridises to a nucleic acid molecule with a nucleotide sequence consisting of SEQ ID NO: 1 or 6, under washing stringency conditions of no less than IxSSC at 650C.

18. A Leuconostoc carnosum strain according to claim 17, wherein said nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1 or 6.

19. A Leuconostoc carnosum strain according to claim 15 or 16, wherein said lactose transporter is encoded by a nucleic acid molecule that hybridises to a nucleic acid molecule with a nucleotide sequence consisting of SEQ ID NO: 10, under washing stringency conditions of no less than 1 xSSC at 650C.

20. A Leuconostoc carnosum strain according to claim 19, wherein said nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 10.

21. A Leuconostoc carnosum strain according to any one of claims 9-12 and 17-20, wherein said nucleic acid molecule is harboured on a self- replicating plasmid.

22. A Leuconostoc carnosum strain according to claim 21 , wherein said plasmid is selected from the group consisting of pCI372, pGhosfδ, and pCI3340.

23. A Leuconostoc carnosum strain according to any one of claims 9-12 and 17-20, wherein said nucleic acid molecule is integrated into the genome of said strain, said genome including a bacterial chromosome and/or a native plasmid.

24. A Leuconostoc carnosum strain according to any one of claims 9-12 and 17-20, wherein said nucleic acid molecule is comprised within an operon.

25. A Leuconostoc carnosum strain according to any one of claims 9-12, 17-20 and 24, wherein said nucleic acid molecule or operon is operably linked to a homologous or heterologous promoter.

26. A Leuconostoc carnosum strain according to any one of claims 1 to

25, wherein said strain is selected on a chemically defined medium, according to the composition of Table 3, and further supplemented with lactose.

27. A Leuconostoc carnosum strain according to any one of claims 21 to

23, wherein said strain is sensitive to an antibiotic selected from the group consisting of penicillin, chloramphenicol, tetracycline, erythromycine and kanamycin.

28. A starter culture for use in the production of milk products, comprising a recombinant Leuconostoc carnosum strain according to any one of claims 1 to 27.

29. Use of a Leuconostoc carnosum strain according to any one of claims 1 to 27 in the production of a milk product.

30. Use of a Leuconostoc carnosum strain according to claim 29, wherein said milk product is selected from the group consisting of cheese, butter milk, sour cream, yoghurt and kefir.

31. Use of a starter culture according to claim 28 in the production of a milk product.

32. Use of a starter culture according to claim 31 , wherein said milk product is selected from the group consisting of cheese, butter milk, sour cream, yoghurt and kefir.

33. A milk product comprising a Leuconostoc carnosum strain according to any one of claims 1 to 27, or a starter culture according to claim 28.

34. A milk product according to claim 33, wherein said milk product is selected from the group consisting of cheese, butter milk, sour cream, yoghurt and kefir.

35. A method for producing a milk product comprising the steps of: a. adding a cell culture comprising cells of a Leuconostoc camosum strain according to any one of claims 1 to 27, or a starter culture according to claim 28, to a volume of milk,
b. incubating the product of (a) to form a cheese milk,
c. coagulating the product of (b) to form milk curds,
d. separating the milk curds from the product of (c).

36. A method of constructing a recombinant Leuconostoc carnosum strain according to any one of claims 1-27, comprising the steps of:
a) transforming Leuconostoc carnosum with at least one nucleic acid molecule encoding a lactose transporter and a β-galactosidase, and
b) selecting transformed cells of Leuconostoc carnosum of step (a) characterised by the ability to grow on lactose as sole carbon source.