Lactic acid bacteria
The invention relates to a lactose-positive, galactose-negative, Streptococcus thermophilus strain carrying a mutation in 2 or 3 genes selected from the group consisting of 1) a gene encoding a protein of the mannose-glucose-specific PTS and a glcK gene, 2) a gene encoding a protein of the mannose-glucose-specific PTS and a ccpA gene, and 3) a gene encoding a protein of the mannose-glucose-specific PTS, a glcK gene and a ccpA gene, wherein said strain, when used to ferment milk, provides a low lactose fermented milk and/or a fermented milk not undergoing acidification when stored at fermentation temperature.
1. A lactose-positive, galactose-negative, Streptococcus thermophilus strain comprising:
at least one mutated gene encoding a protein of the mannose-glucose-specific phosphotransferase system (mannose-glucose-specific PTS), the glucose import activity of which is decreased or abolished, and
at least one mutated gene selected from the group consisting of:
a) a mutation in a glucokinase (glcK) gene such that the glucokinase activity of said strain is reduced but not null,
wherein the protein sequence of said glucokinase is a glcK variant sequence having at least 90% identity with SEQ ID NO:25 or SEQ ID NO:46,
wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO: 25 is not a glutamic acid, and/or the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 is not a glycine;
b) a mutation in a catabolite control protein A (ccpA) gene, wherein the sequence of said mutated ccpA gene is selected from the group consisting of:
i) a sequence comprising SEQ ID NO:71, and
ii) a ccpA variant sequence having at least 90% identity with SEQ ID NO:71; and
c) a combination thereof.
2. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 , carrying a mutation in at least one gene encoding a protein of the mannose-glucose-specific PTS, the glucose import activity of which is decreased or abolished, and carrying a mutation in a glcK gene such that the glucokinase activity of said strain is reduced but not null,
wherein the protein sequence of said glucokinase is a glcK variant sequence having at least 90% identity with SEQ ID NO:25 or SEQ ID NO:46,
wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO: 25 is not a glutamic acid, and/or the amino acid of the glucokinase corresponding to position 144 of SEQ ID NO:46 is not a glycine.
3. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 2 , wherein the protein sequence of said glucokinase is a glcK variant sequence having at least 90% similarity or identity with SEQ ID NO:25, wherein the amino acid of the glucokinase corresponding to position 275 of SEQ ID NO:25 is not a glutamic acid.
4. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 2 , wherein the protein sequence of said glucokinase is a glcK variant sequence having at least 90% similarity or identity with SEQ ID NO:46, wherein the amino acid of said glucokinase corresponding to position 144 of SEQ ID NO:46 is not a glycine.
5. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 2 , further carrying a mutation in the ccpA gene.
6. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 , carrying a mutation in at least one gene encoding a protein of the mannose-glucose-specific PTS, the glucose import activity of which is decreased or abolished, and carrying a mutation in a catabolite control protein A (ccpA) gene,
wherein the sequence of said mutated ccpA gene is selected from the group consisting of:
i) a sequence comprising SEQ ID NO:71, and
ii) a ccpA variant sequence having at least 90% identity with SEQ ID NO:71.
7. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 6 , wherein the ccpA gene comprises a mutation selected from the group consisting of:
a non-sense mutation located between the nucleotide 1 and the nucleotide 270 of the coding sequence of the ccpA gene, and
a mutation located in the first quarter of the coding sequence of the ccpA gene leading to a frameshift of the open reading frame of the ccpA gene.
8. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 , wherein the at least one gene encoding a protein of the mannose-glucose-specific PTS is selected from the group consisting of the manL gene, the manM gene and the manN gene.
9. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 , wherein the mutated gene encoding a protein of the recite—mannose-glucose-specific PTS, the glucose import activity of which is decreased or abolished, is selected from the group consisting of:
a) a Streptococcus thermophilus IIAB Man protein truncated in position 305 (IIAB Man 305 );
b) a Streptococcus thermophilus IIC Man protein truncated in position 208 (IIC Man 208 ); and
c) a Streptococcus thermophilus IID Man protein truncated in position 28 (IID Man 28 ).
10. The lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 9 , wherein the protein sequence of said IIAB Man 305 protein is selected from the group consisting of:
a) a sequence as defined in SEQ ID NO:112; and
b) a IIAB Man variant sequence having at least 90% sequence similarity or identity with SEQ ID NO:112; wherein the protein sequence of said IIC Man 208 is selected from the group consisting of:
a) a sequence as defined in SEQ ID NO:158; and
b) a IIC Man variant sequence having at least 90% sequence similarity or identity with SEQ ID NO: 158;
wherein the protein sequence of said IID Man 28 protein is selected from the group consisting of:
a) a sequence as defined in SEQ ID NO:207; and
b) a IID Man variant sequence having at least 90% sequence similarity or identity with SEQ ID NO: 207.
11. A composition comprising at least one lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 in combination with one or more strain(s) selected from the group consisting of a strain of the Lactobacillus genus, a strain of the Lactococcus genus, or a strain of the Bifidobacterium genus.
12. A method for manufacturing a fermented dairy product comprising:
inoculating a milk substrate with the lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 or a composition comprising at least one lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 ; and
fermenting said inoculated milk, to obtain a fermented dairy product.
13. A fermented dairy product comprising at least one lactose-positive, galactose-negative, Streptococcus thermophilus strain according to claim 1 .
14. The composition of claim 11 , wherein the strain of the Lactobacillus genus is Lactobacillus delbrueckii subsp bulgaricus strain.
15. The composition of claim 11 , wherein the strain of the Lactococcus genus is a Lactococcus lactis strain.