NEW BACTERIA

Abstract

The present invention relates to novel S. thermophillus strains that are not attacked by common phagesand novel Streptococcus strains with improved stability against phage attacks, methods to obtain Streptococcus strains with improved phage resistance, their use in the manufacture of fermented milk-based products, and novel milk based products containing the strain.

Claims

1. A method for the manufacture of a phage resistant mutant of a strain of the species Streptococcus thermophilus, comprising: mutating a culture of a mother strain of Streptococcus thermophiles to obtain mutant strains of Streptococcus thermophilus; optionally, exposing the mutant strains to a phage that attacks the mother strain; selecting a phage resistant mutant strain comprising a mutation in a gene involved in glycan synthesis.

2. A method for manufacture of a cell count stabilized mutant of a strain of the species Streptococcus thermophilus, comprising: subjecting a culture of a mother strain of Streptococcus thermophilus to mutagenesis to obtain mutant strains of Streptococcus thermophilus; optionally, exposing the mutant strains to a phage that attacks the mother strain; selecting a phage resistant mutant strain comprising a mutation in a gene involved in glycan synthesis.

3. A method according to claim 1, wherein the mother strain from which the mutant is derived is selected from the group consisting of STCH_09 (DSM19243), STCH_12 (DSM32826), STCH_13 (DSM32841), STCH_14 (DSM21408), and STCH_15 (DSM32842), and mutants or variants of any of these.

4. A method according to claim 1, wherein the gene involved in glycan synthesis is a glycosyltransferase gene.

5. A method according to claim 1 wherein the gene involved in glycan synthesis has at least 98%, sequence identity to one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

6. A method according to claim 1 wherein the mutation comprises a substitution, deletion or insertion of one or more nucleotides in one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

7. A method according to claim 1 further comprising deselecting mutants comprising a mutation in one or more of the CRISPR-region and a R/M region.

8. A method for providing a phage resistant and/or cell count stabilized mutant of a strain of the species Streptococcus thermophilus, comprising: introducing by means of genetic engineering a mutation in one or more of the proteins encoded by one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, wherein said mutation results in a change or impairment in glycan synthesis.

9. A mutant strain of the species Streptococcus thermophilus obtained by the method of claim 1.

10. A mutant strain of the species Streptococcus thermophilus, wherein the mutant strains carries a mutation such that expression of a protein encoded by a sequence having at least 98% sequence identity to one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 is impaired and wherein said mutation results in a change in glycan synthesis.

11. A strain of claim 9, wherein said strain shows increased robustness against phage attacks.

12. A strain according to claim 11 wherein the phage is of the cos-, pac- or 987 type.

13. A method of fermenting a milk substrate, comprising adding a strain of claim 9 to the milk substrate.

14. A bacterial culture which contains at least 10E8 CFU per gram of a mutant of a strain of claim 9.

15. The bacterial culture of claim 14, which is in a freeze dried or spray dried form.

16. A kit comprising the strain of claim 9 and one or more of cryoprotectants and germination booster components.

17. A food product containing a strain of claim 9, which is a fermented milk product.

18. A method according to claim 1, wherein the mutant strain comprises a mutation in a gene involved in glycan synthesis selected from extracellular polysaccharide (EPS) synthesis, capsular polysaccharide (CPS) synthesis, and rhamnose-containing cell wall polysaccharides (RGP) synthesis.

19. A method according to claim 2, wherein the mutant strain comprises a mutation in a gene involved in glycan synthesis selected from extracellular polysaccharide (EPS) synthesis, capsular polysaccharide (CPS) synthesis, and rhamnose-containing cell wall polysaccharides (RGP) synthesis.

20. A method according to claim 8, wherein said mutation results in a change or impairment in glycan synthesis selected from extracellular polysaccharide (EPS) synthesis, capsular polysaccharide (CPS) synthesis, and rhamnose-containing cell wall polysaccharides (RGP) synthesis.

Description

DRAWINGS

[0110] FIG. 1: Changes in phage binding to S. thermophilus wild type strains and their phage-resistant mutants.

[0111] Phage DNA was labeled with SYBR Gold and visualized under green fluorescence. (a) Phage CHPC1057 adsorbs to its host STCH_09 (1) and it does not adsorb to STCH_09_BIM (2). (b) Phage CHPC926 adsorbs to its host STCH_15 (1), it has reduced adsorption with STCH_15_BIM_2 (2), and it does not adsorb to STCH_15_BIM_1, STCH_15_BIM_3, STCH_15_BIM_4, STCH_15_BIM_5 (panels no. 3-6, respectively). Scale bars: 1 μm.

[0112] FIG. 2: Fluorescence imaging of phage binding to S. thermophilus strains. (a) Adsorption of phages to their hosts was visualized with a conventional fluorescence microscope, after labeling phage DNA with SYBR Gold. (b) Super-resolution structured illumination microscopy (SRSIM) images of bacterial cells stained with Nile Red (red) and mixed with SYBR Gold DNA-labeled phages (green). Panels with phages and their host strains: (1) CHPC926 and STCH_15, (2) CHPC951 and STCH_12, (3) CHPC1057 and STCH_09, (4) CHPC1014 and STCH_13, (5) CHPC1046 and STCH_14. Two binding patterns are observed: spotty (panels no. 1, 2, 3) or diffused (panels no. 4, 5). Scale bars: 1 μm.

[0113] FIG. 3: Milk acidification of S. thermophilus STCH_15 and its derivatives in the presence and absence of phage CHPC926. (1) STCH_15_ΔepsE_1; (2) STCH_15_ΔepsE_1 +CHPC926; (3) STCH_15_ΔepsE_2; (4) STCH_15_ΔepsE_2+CHPC926; (5) STCH_15; (6) STCH_15 +CHPC926; (7) STCH_15. Samples 1, 2, 3, 4, 7 were supplemented with erythromycin (3 μg/ml). NC—negative control (milk).

DEPOSITS AND EXPERT SOLUTION

[0114]

TABLE-US-00003 Numbering herein Deposit number STCH_14 DSM21408 STCH_09 DSM19243 STCH_15 DSM32842 STCH_12 DSM32826 STCH_13 DSM32841

[0115] The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

[0116] The Applicant requests that a sample of the deposited microorganism should be made available only to an expert approved by the Applicant.

TABLE-US-00004 SEQUENCE LISTING >STCH_09_epsH ATGACAATCAGCATAGTAATCCCAGTTTATAATGTTCAAGATTACATAAA AAAGTGTCTAGATTCTATATTAAGCCAGACATTTTCAGATTTAGAAATTA TTCTTGTTGATGATGGTTCTACTGACTTGAGTGGAAGAATTTGTGATTAT TATTCCGAAAATGATAAACGTATTAAAGTAATCCACACAGCAAATGGGGG ACAGTCGGAAGCAAGGAACGTTGGAATCAAAAATGCCACATCAGAATGGA TAACATTTATTGATTCTGATGACTACGTTTCTTCTGATTATATAGAGTAT TTATATAATTTGATTCAAGTACACAATGCAGATATTTCAATAGCTAGTTT TACCTATATCACACCTAAAAAGATAATTAAGCACGGTAACGGTGAAGTAG CTCTTATGGATGCAAAAACTGCAATTCGGAGAATGTTACTGAATGAAGGT TTCGATATGGGAGTTTGGGGGAAAATGTATCGAACGGAGTATTTTAATAA ATATAAATTCGTTTCAGGAAAACTATTTGAAGATTCTTTAATTACATACC AGATATTTTCAGAAGCTTCAACAATTGTTTTTGGAGCAAAGGATATTTAT TTTTATGTTAACAGGAAAAATTCTACTGTTAATGGTACTTTTAATATAAA AAAGTTTGATCTTATTGAAATGAATGAAGAAGCAAATAAGTTTATTAAAC ATAAATTTCCAGATCTTTCATCTGAAGCACATCGTCGAATGATATGGGCA TATTTTAGTACACTAAATCAAGTTTTATCATCAACTAATGAACACGATAT TGATTTATATGCGCCACAATTAGTAGCTTATCTCCTTAAACAGGATAAAT TCATAAAAAGGAATACTTTTATTCCCAAAAGAGATAAGATTGCATTTTTT ATTTTAAAAAATTTTGGTTTAAAGACATATCGTAATGTTTGGAATTTATA TTTAAAAATGACAAGATAA >STCH_09_glycoslytransferase ATGTCAGATTTACTCATTATTATTCCAGCCTATAACGAAGAAGGATCGAT TGAAAATGTTGTTAACAACATCATTCAAAACTATCCTCAATATGACTATG TTATCATCAATGATGGCTCTCGTGATAAAACTTCACAAATTTGCCATGAG AATCATTACAATATTGTAGATTTACCAGTGAATCTCGGTCTTGCTGGGGC TTTCCAAACAGGTTTAAGATATGCGTATGAGCATGGTTATAAAAAAGCGG TGCAATTTGATGCCGATGGTCAACACTTACCAGAATATATTCAAAGTTTG GAAGAAAAGATCGATGATGGTTTTGATCTAGTGATTGGTTCTCGATTTGT CACAGAAAAACGACCAAATTCTTTACGAATGTTAGGCAATATCTTAATTA GTTCAGCTATTAAACTCACTACTGGTAAGACAATTAAGGACCCAACTTCA GGAATGAGAATGTTCTCAGAAGAGTTAATAAAAGAGTTTGCACTCAATAT TAACTATGGTCCAGAGCCAGATACTGTTTCTTATCTCATCCGTAATGGTG TAAAAGTTGCGGAAACACAAGTTAGAATGGAAGATAGACAGGCAGGAGAA AGCTACTTGACTCTTTCTCGTTCGATTAAATATATGACACATATGTTTGT GTCAATCCTACTCATTCAAAACTTTAGAAAGCGAGGCTAG >STCH_13_epsD ATGCCTTTATTAAAGTTAGTTAAATCAAAAGTAGACTTTGCTAAAAAGAC GGAAGAGTATTATAACGCTATTCGCACAAATATTCAATTTTCTGGTGCTC AGATTAAAGTGATTGCGATTAGCTCTGTTGAAGCTGGTGAAGGAAAATCA ACGACATCTGTTAACTTGGCGATTTCATTTGCTAGTGTTGGGCTCCGAAC ACTTCTGATTGATGCGGATACGCGTAACTCTGTTTTGTCAGGTACATTTA AATCAAATGAGCCTTATAAAGGTCTTTCAAATTTCCTTTCAGGAAATGCC GATCTAAATGAAACGATTTGCCAAACTGATATTTCTGGTTTAGATGTTAT TTCATCTGGTCCTGTACCACCTAATCCAACAAGTCTTTTGCAAAATGACA ATTTTAGACATTTGATGGAAGTTGCTCGTAGTCGTTATGATTATGTTATC ATTGATACACCACCAATTGGTCTGGTCATTGATGCTGGTATTATTGCCCA TCAGGCTGATGCTAGTCTTTTGGTTACAGCAGCTGGAAAAATTAAACGTC GTTTCGTAACTAAGGCGGTCGAACAATTGAAACAAAGTGGTTCTCAGTTC TTAGGTGTCGTCCTTAATAAAGTTGACATGACAGTTGATAAATATGGATC ATATGGTTCTTACGGATCATATGGTGAGTACGGGAAAAAAACAGACCAAA AAGAAGGTCATTCAAGAGCACATCGTCGTAGAAAAGGATAG >STCH_14_epsE GTGAAAGAAAAACAAGAAATTCGTCGCATTGAAATTGGTATTATACAGTT GGTTGTGGTTGTTTTCGCAGCCATGGTAGCTAGTAAAATACCATATACAG AGATTACCCAAGGAAGCATTGTCCTTTTAGGTGTCGTACATGTAGTGTCT TACTATATCAGTAGTTATTATGAAAATCTTAAGTATAGAGGCTACTTGGA TGAACTCATTGCAACTGTCAAATATTGTTTCATATTTGCTCTAATTGTAA CATTTCTCTCGTTTTTTGCAGATGGAAGTTTTTCAATCTCACGTCGCGGA CTTCTTTACGTCACCCTGATTTCAGGTGTTCTCTTATACGTTACAAATAC TGTTCTTAAGTATTTCCGCTCATCTATTTATACACGTCGTAAAAGTAACA AGAATATTCTCTTGATTTCTGATCAAGCACGTCTAGAAAATGTTTTGTCT CGTATGAAAGACAATATGGATGGTAGGATTACAGCAGTTTGTGTCTTGGA TAATCCTTATTTTACCGATCCATTTATCAAGAGTGTTAAACCTGAAAATT TGATTGAATATGCGACACACTCAGTAGTAGACCAAGTTTTGATTAATCTG CCAAGTGGGCAGTATAAGATTTGGGATTATGCATCACCTTTTGAGATCAT GGGAATTCCAGTTTCTATTAATTTGAATGCCCTTGAATTTATGAATCAAG GTGAAAAACGTATTCAACAATTGGGTCCTTTCAAAGTTGTTACGTTTTCA ACGTATTTTTATAGCTATGGAGATATCTTGGCGAAACGTTTCCTCGATAT CTGTGGAGCTCTAGTTGGTTTGTTGCTCTGTGGTATTGTAGGAATCTTCC TTTATCCTCTTATTCGTAAAGATGGAGGACCAGCCATTTTTGCTCAAGAC CGTGTGGGAGAAAATGGACGTATCTTCAAGTTTTATAAATTCCGTTCTAT GTGTGTTGATGCGGAAGAAATCAAGAAGAATTTGATGGCACAGAATCAAA TGTCTGGTGGTATGTTTAAGATGGACAATGATCCACGTATTACCAAAATT GGACATTTCATTCGTAAAACGAGTCTTGATGAACTTCCACAATTTTGGAA TGTTCTAAAAGGTGATATGAGCTTGGTTGGGACACGTCCTCCAACAGTTG ATGAGTATGAAAAATATACACCTGAACAGAAACGTCGTTTAAGTTTTAAA CCTGGTATCACTGGTCTTTGGCAAGTAAGCGGTCGAAGTGAAATTACTGA TTTTGATGAAGTTGTAAAACTAGACGTTGCTTATTTGGACGGATGGACAA TCTGGCGTGATATCAAAATCTTATTGAAAACGATTAAAGTAGTAGTAATG AAGGATGGAGCAAAGTGA >STCH_15_epsD_epsE ATGCCTCTATTAAAGTTAGTAAAATCTAAAGTAAACTTTGCCAAACAAAC AGAAGAGAATTACAATGCCATTCGCACAAATATTCAATTTTCTGGTGCTC AGATTAAAGTGATTGCGATTAGCTCTGTTGAAGCTGGTGAAGGAAAATCA ACGACATCTGTTAACTTGGCGATTTCATTTGCTAGTGTTGGGCTCCGAAC ACTTCTGATTGATGCGGATACGCGTAATTCTGTTTTGTCAGGTACATTTA AATCAAATGAGCCTTATAAAGGTCTTTCAAATTTCCTTTCAGGAAATGCC GATCTAAATGAAACGATTTGCCAAACTGATATTTCTGGTTTAGATGTTAT TGCATCTGGTCCTGTTCCACCTAATCCAACAAGTCTTTTGCAAAATGATA ATTTTAGACATTTGATGGAAGTTGCTCGTAGTCGTTATGATTATGTCATC ATCGATACACCACCTGTTGGGGTAGTTATTGATGCAGTTATTATTGCCCA TCAGGCTGATGCTAGTCTTTTGGTTACAGAAGCTGGGAAAATCAAACGTC GTTTCGTAACTAAGGCCGTTGAACAATTGGAACAAAGTGGTTCTCAGTTC TTAGGGGTCGTCCTTAATAAAGTTGACATGACAGTTGATAAATATGGATC GTATGGTTCTTACGGATCATATGGCGAGTATGGAAAAAAATCTGACCAAA AAGAAGGTCATTCAAGAGCACATCGTCGTAGAAAAGGATGGCATTAATGG GGATGATGCGGTTCCTTATAACCTTAACAGATTAAAAAGGGGTTTAGAGT GAAAGAAAAACAAGAAATTCATCGCATTGAAATTGGTATTATACAGTTGG TTGTGGTTGTTTTTGCAGCCATGATAGCTAGTAAAATACCTTATACAGAG ATTACCCAAGGAAGCATTGTCCTTTTAGGTGTCATACATGTAGTGTCTTT CTATATCAGTAGTTATTATGAAAATCTTAAGTATAGAGGCTACTTGGATG AACTCATTGCAACTGTCAAATATTGTTTCATATTTGCTCTCATTGCAACA TTTCTCTCGTTTTTTGCAGATGGAAGTTTTTCAATCTCACGTCGCGGACT TCTTTACGTCACCCTGATTTCAGGTGTTCTCTTATATGTTACAAATACTG TTCTTAAGTATTTCCGCTCATCTATTTATACACGTCGTAAAAGTAACAAG AATATTCTCTTGATTTCTGATCAAGCACGTCTAGAAAATGTTTTGTCTCG TATGAAAGACAATATGGATGGTAGGATTACAGCAGTTTGTGTCTTGGATA ATCCTTATTTTACCGATCCATTTATCAAGAGTGTTAAACCTGAAAATTTG ATTGAATATGCGACACACTCAGTAGTAGACCAAGTTTTGATTAATCTGCC AAGTGGGCAGTATAAGATTTGGGATTATGCATCACCTTTTGAGATCATGG GAATTCCAGTTTCTATTAATTTGAATGCCCTTGAATTTATGAGTCAAGGT GAAAAACGTATTCAACAATTGGGTCCTTTCAAAGTTGTTACGTTTTCAAC GCAATTTTATAGCTATGGAGATATCTTGGCGAAACGTTTCCTCGATATCT GTGGAGCCCTAGTTGGTTTGGTGCTCTGTGGGATTGTTGGAATCTTCCTT TATCCACTTATTCGTAAGGATGGTGGGCCAGCCATTTTTGCTCAAGACCG TGTGGGAGAAAATGGACGTATCTTCAAGTTTTATAAATTCCGTTCTATGT GTGTTGATGCGGAAGAAATCAAGAAGGATTTGATGGCACAGAATCAAATG TCTGGTGGTATGTTTAAGATGGACAATGATCCACGTATTACCAAAATTGG ACATTTCATTCGTAAAACGAGTCTTGATGAACTTCCACAATTTTGGAATG TTCTAAAAGGTGATATGAGCTTGGTAGGAACACGTCCACCAACATTGGAT GAGTACGAATCTTATACACCGGAACAAAAACGTCGCCTCAGCTTTAAACC AGGTATTACTGGTCTTTGGCAAGTAAGCGGTCGAAGTGAAATTACTGATT TTGATGAAGTTGTAAAACTAGACGTTGCTTATTTGGACGGATGGACAATC TGGCGCGATATCAAAATCTTATTGAAAACAATTAAAGTAGTAGTAATGAA GGATGGAGCAAAGTGATGGCTTTCACCATTTCTTTTAATGGTGATTAAAT GACAAAAACAGTTTATAGCGTTTGTTCTAAGGGGATTCCAGCAAAATATG GTGGATTTGAGACCTTTGTTGAGAAGTTGACAGAGTTCCAACAAGACAAA GATATCCAATATTATGTAGCTTGTATGCGGGAAAACTCTGCAAAATCAGA CATTACAGCAGATGTTTTTGAAGGTTCTGTTGCGAAGTTTGAAAATCAAA CGCGCCCGCTCTGA >STCH_15_epsK ATGAAAATAGCATTAGTAGGTTCCAGCGGTGGCCATTTGACACACATGTA TTTATTAAAAAAGTTTTGGGAAAATGAAGATAGATTTTGGGTCACATTTG ATAAAACAGATGCAAAATCTATATTGAAAGAAGAAAGATTTTATCCTTGT TATTATCCCACAAATAGAAATGTAAAAAACACGATAAAAAATACCATTCT TGCATTTAAAATACTTAGAAAAGAAAAACCAGATTTGATTATTTCGAGTG GTGCTGCGGTAGCCGTTCCTTTTTTTTGGTTAGGTAAACTATTCGGTGCA AAGACAGTCTATATTGAAATATTTGACCGGATCGATAAACCAACCTTAAC AGGAAAATTAGTTTATCCAGTTACTGATAAGTTTATAGTTCAATGGGAAG AGTTAAAAAAAGTTTACCCTAAAGCGATTAATCTAGGAGGGATTTTCTAA

REFERENCES

[0117] All references cited in this patent document are hereby incorporated herein in their entirety by reference.