Bacteria

Abstract

The present invention relates to a bacterial cell with texturizing property, starter cultures comprising the cell, and dairy products manufactured using the bacterial cell.

Claims

1. A method for increasing viscosity in a fermented milk product, comprising inoculating and fermenting a milk substrate with a mutant lactic acid bacterium (LAB) exhibiting one or more properties selected from: (a) containing from 0 to less than 10 parts per million (ppm, mg/kg dry weight) of Fe.sup.2+ ions; (b) containing from 0 to less than 6 ppm of Mn.sup.2+ ions; (c) containing from 0 to less than 16 ppm in total of Fe.sup.2+ ions and Mn.sup.2+ ions; (d) carrying a mutation in a gene related to the uptake of a divalent metal ion; (e) having a changed fur gene expression caused by one or more of: a mutation that causes partial or full inactivation of the fur gene, a mutation that causes partial or full deletion of the fur gene, and a mutation that causes insertion of DNA into the fur gene; (f) having reduced mnth gene expression caused by one or more of: a mutation that causes partial or full inactivation of the mnth gene, a mutation that causes partial or full deletion of the mnth gene, and a mutation that causes insertion of DNA into the mnth gene; (g) having reduced fatc gene expression caused by one or more of: a mutation that causes partial or full inactivation of the fatc gene, a mutation that causes partial or full deletion of the fatc gene, and a mutation that causes insertion of DNA into the fatc gene; and (h) being resistant to tellurite, as determined by an ability to form a colony on M17 agar plates containing 0.1 mM K.sub.2TeO.sub.3, wherein the mutant LAB generates a viscosity in milk greater than about 50 Pa.Math.s, measured as shear stress, after inoculating 9.5% reconstituted skim milk with 10.sup.8 CFU/ml milk of the mutant LAB and fermenting for 16 hours at 37° C.

2. The method of claim 1, wherein the mutant LAB exhibits one or more properties selected from: (a) containing from 0 to less than 10 parts per million (ppm, mg/kg dry weight) of Fe.sup.2+ ions; (b) containing from 0 to less than 6 ppm of Mn.sup.2+ ions; and (c) containing from 0 to less than 16 ppm in total of Fe.sup.2+ ions and Mn.sup.2+ ions.

3. The method of claim 1, wherein the mutant LAB exhibits one or more properties selected from: (d) carrying a mutation in a gene related to the uptake of a divalent metal ion; (e) having a changed fur gene expression caused by one or more of: a mutation that causes partial or full inactivation of the fur gene, a mutation that causes partial or full deletion of the fur gene, and a mutation that causes insertion of DNA into the fur gene; (f) having reduced mnth gene expression caused by one or more of: a mutation that causes partial or full inactivation of the mnth gene, a mutation that causes partial or full deletion of the mnth gene, and a mutation that causes insertion of DNA into the mnth gene; (g) having reduced fatc gene expression caused by one or more of: a mutation that causes partial or full inactivation of the fatc gene, a mutation that causes partial or full deletion of the fatc gene, and a mutation that causes insertion of DNA into the fatc gene; and (h) being resistant to tellurite, as determined by an ability to form a colony on M17 agar plates containing 0.1 mM K.sub.2TeO.sub.3.

4. The method of claim 1, wherein the mutant LAB has a perturbed divalent metal ion metabolism (DMIM) as compared to its mother strain.

5. The method of claim 4, wherein the divalent metal ion is selected from one or more of Fe.sup.2+, Mg.sup.2+, and Mn.sup.2+.

6. The method of claim 1, wherein the mutant LAB has been obtained by mutagenesis.

7. The method of claim 1, wherein the mutant LAB has been obtained by genetic engineering.

8. The method of claim 1, wherein the mutant LAB has been obtained by a process comprising growth in a medium having a concentration of one or both of Fe.sup.2+ and Mn.sup.2+ of from 0 to below 0.25 μg/g.

9. The method of claim 1, wherein the mutant LAB belong to the species Lactobacillus bulgaricus.

10. The method of claim 1, wherein the mutant LAB belong to the species Streptococcus thermophilus.

11. The method claim 1, wherein the mutant LAB is selected from Streptococcus thermophilus strain CHCC15712 deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany, under accession number DSM25955, and mutants thereof wherein less than 0.1% of the nucleotides in the bacterial genome of the mutant have been changed relative to CHCC15712 and wherein the mutant exhibits the same or improved properties with respect to exopolysaccharide (EPS) production as CHCC15712.

12. The method of claim 1, wherein the mutant LAB is provided in a composition comprising a metal ion chelator.

13. The method of claim 1, wherein the milk substrate has a concentration of one or both of Fe.sup.2+ and Mn.sup.2+ of from 0 to below 0.25 μg/g.

14. The method of claim 1, wherein the milk substrate comprises Fe.sup.2+ ions and the method further comprises removing Fe.sup.2+ ions from the milk substrate before, during or after inoculation with the mutant LAB, to obtain a milk substrate having a Fe.sup.2+ concentration from 0 to below 0.25 μg/g.

15. The method of claim 1, wherein the milk substrate comprises Mn.sup.2+ ions and the method further comprises removing Mn.sup.2+ ions from the milk substrate before, during or after inoculation with the mutant LAB, to obtain a milk substrate having a Mn.sup.2+ concentration from 0 to below 0.25 μg/g.

Description

DRAWING

(1) FIG. 1a depicts the alignment of Fur boxes from Bacillus, Streptococcus gordonii and CHCC9844. Nucleotides found in all three strains are coloured red, those shared between Bacillus and CHCC9844 are green, those shared between CHCC9844 and S. gordonii are blue. FIG. 1a discloses SEQ ID NOS 1-3, respectively, in order of appearance.

(2) FIG. 1b depicts the promoter regions and first part of epsA gene in S. thermophilus CHCC9844. The postulated fur box is coloured blue. Other features of the promoter region are indicated on the figure. FIG. 1b discloses SEQ ID NO: 4.

(3) FIG. 2 depicts a viscosity test with texturing mutant CHCC15712. Viscosity was measured by calculating the efflux time from a 25 ml volumetric pipette. The graph shows the average from three measurements.

(4) FIG. 3 depicts the monosaccharide composition of exopolysaccharide excreted in M17 broth. The concentration of the single monosaccharides is indicated in ppm.

(5) FIG. 4 depicts a viscosity test with fur mutant KA 509. Viscosity was measured by calculating the efflux time from a 25 ml volumetric pipette. The graph shows the average from three measurements.

(6) FIG. 5 depicts a viscosity test with four tellurite resistant mutants from CHCC9844. Viscosity was measured by calculating the efflux time in seconds from a 25 ml volumetric pipette. The graph shows the average from three measurements.

(7) FIG. 6 depicts a viscosity test in milk with and without 1 mM EDTA. Viscosity was measured by calculating the efflux time in seconds from a 25 ml volumetric pipette. The graph shows the average from three measurements.

(8) FIGS. 7a, 7b and 7c depict the intracellular concentrations of Fe, Mg, and Mn, resp.

DEPOSITS and EXPERT SOLUTION

(9) The strain Streptococcus thermophilus CHCC15712 was deposited at DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) under the accession number DSM25955, on Apr. 27, 2012.

(10) CHCC4895 was deposited with DSMZ under the accession no. DSM19242, on 29 Mar. 2007

(11) CHCC8833 was deposited with DSMZ under the accession no. DSM17876, on 11 Jan. 2006.

(12) The deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

(13) The Applicant requests that a sample of the deposited microorganism should be made available only to an expert approved by the Applicant.

REFERENCES

(14) Lin et al., Microbiology (2011), 157, 419-429. E. P. Skaar, PLoS Pathog. (2010) August 12; 6(8):e1000949. Baichoo et al., Molecular Microbiology (2002) 45 (6), 1613-1629. Kosikowski, F. V. and Mistry, V. V., “Cheese and Fermented Milk Foods”, 1997, 3rd Ed. F. V. Kosikowski, L.L.C. Westport, Conn. Albert Saavedra et al., 2013, Advanced Materials Research, 825, 115

(15) All references cited in this patent document are hereby incorporated herein in their entirety by reference.