New lactic acid bacteria

Abstract

The present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain. The strain carries one or more mutations in one or more genes of the sucrose regulon and, optionally, one or more further mutations affecting a glucose transporter, e.g. glcU and / or its expression. The strain of the invention may further carry mutations in genes encoding proteins of the mannose-glucose specific PTS and glucokinase, proteins of the mannose-glucose specific PTS and catabolite control protein A (CcpA) and / or proteins of the mannose-glucose specific PTS, glucokinase and CcpA. The strain of the invention, when fermenting milk, may do so without the need to add sucrose or with the need to add reduced amounts of sucrose, optionally providing a low-lactose fermented milk and / or a fermented milk that does not undergo post-acidification when stored at fermentation temperatures. The present invention also relates to a composition comprising at least one lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention and the use of this strain or composition for producing a fermented milk product.

Description

[Technical field]

[0001] The present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain. The strain carries one or more mutations in one or more genes of the sucrose regulon and, optionally, one or more further mutations affecting a glucose transporter (non-PTS glucose permease), e.g. glcU and / or affecting its expression. The strain of the invention may further carry mutations in genes encoding proteins of the mannose-glucose specific PTS and glucokinase, proteins of the mannose-glucose specific PTS and catabolite control protein A (CcpA) and / or proteins of the mannose-glucose specific PTS, glucokinase and CcpA.

[0002] The strains of the invention, when fermenting milk containing any sucrose, may do so without consuming this sucrose and thus reduce the need for sucrose addition or provide the need to add reduced amounts of sucrose while still obtaining the expected sweetness level from sucrose. Optionally, the strains of the invention provide low-lactose fermented milk and / or fermented milk that does not undergo post-acidification when stored at fermentation temperatures. The invention also relates to compositions comprising at least one lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention and the use of this strain or composition for producing fermented milk products. [Background technology]

[0003] The food industry uses bacteria to improve the taste and texture of food or feed products. In the case of the dairy industry, lactic acid bacteria are commonly used, for example, to bring about the acidification of milk (by fermentation of lactose) and to give texture to the products in which they are incorporated. For example, lactic acid bacteria of the species Streptococcus thermophilus (S. thermophilus) are widely used, alone or in combination with other bacteria, in the production of fresh fermented dairy products such as cheese or yogurt.

[0004] One of the challenges in the use of lactic acid bacteria in dairy technology is post-acidification, i.e. the production of lactic acid by lactic acid bacteria after the target pH (pH required by the technology) has been obtained [end of fermentation]. The phenomenon of post-acidification is therefore a problem not only for dairy manufacturers (who want to have a flexible production process that does not necessarily have to have a quenching step immediately after the target pH has been obtained) but also for consumers (production of lactic acid leading to increased acidity and reduced quality of the fermented product).

[0005] Another challenge in the dairy industry is that the cultures may consume sucrose added during fermentation or sucrose already present. To achieve the expected sweetness of the fermented product, it is necessary to add sucrose to the milk before, during or at the end of fermentation. Another challenge in the dairy industry is that the cultures may consume sucrose added during fermentation. Therefore, it may be necessary to add excess sucrose to the milk to achieve the expected sweetness. Furthermore, it can be envisaged to further reduce the amount of sucrose added by releasing some or all of the glucose portion resulting from lactose hydrolysis by the culture.

[0006] Furthermore, dairy consumers tend to have fermented products with reduced or low lactose content (lactose intolerance). Summary of the Invention [Problem to be solved by the invention]

[0007] There is therefore a need for improved methods for producing fermented milk products that are not subjected to post-acidification, have a reduced need for added sucrose and have a reduced lactose content, which are satisfactory for both manufacturers and consumers. [Brief description of the drawings]

[0008] [Figure 1] Figure 1: DNA sequence alignment of the chromosomal loci of strain DGCC8368 carrying the ST1 scrA829 FS and pscrB829 mutations. Nucleotides identical in both sequences are indicated by a dot in the DGCC8368 sequence. Differences are indicated and named as bold nucleotides in the DGCC8368 sequence. Difference pscrB829 corresponds to the replacement of a T nucleotide at position 36 of the sequence with a G nucleotide, and difference scrA829 FS corresponds to the insertion of an A nucleotide at position 384 of the sequence. The start codon is underlined in italics, the open reading frame is italicized, and the putative minus 10 box is underlined in bold. [Diagram 2] DNA sequence alignment of the promoter region of the glcU gene of the ST1 strain with the promoter regions of the various sucrose-negative glucose-positive mutants described in Example 5. Nucleotides identical in all sequences are indicated by dots in the mutant sequences. Sequence differences are named and indicated by bold nucleotides in the mutant sequences or by black triangles for IS1191 insertions. The mutation pglcUSNP_C corresponds to the replacement of the T nucleotide at position 96 of the sequence by a C nucleotide, the differences pglcUIN_T and pglcUIN_C correspond to the insertion of a T or C nucleotide at position 102 of the sequence. The mutation pglcUIS corresponds to an IS1191 insertion at positions 111-112 of the sequence (or positions 110-111 of the sequences for example ST1 and ST1-ABU4). The start codon is underlined in italics and the open reading frame is in italics. [Diagram 3]Comparison of μglucose / μmaxlactose ratios calculated for: wild-type (parent) strains (black), their respective scrA829 FS-pscrB829 mutants (white) and their glucose-positive derivatives carrying a mutation in pglcU (grey). [Figure 4] Graphs depicting the change in pH over time (A) and rate as a function of pH (B) for normal milk (1) and milk supplemented with 7% (w / v) sucrose (2) fermented with ST1 (FIG. 4), ST1-GM (FIG. 5), ST1-GM-AB (FIG. 6), ST1-GM-ABU1 (FIG. 7), ST1-CM-ABU1 (FIG. 8). [Diagram 5] Graphs depicting the change in pH over time (A) and rate as a function of pH (B) for normal milk (1) and milk supplemented with 7% (w / v) sucrose (2) fermented with ST1 (FIG. 4), ST1-GM (FIG. 5), ST1-GM-AB (FIG. 6), ST1-GM-ABU1 (FIG. 7), ST1-CM-ABU1 (FIG. 8). [Figure 6] Graphs depicting the change in pH over time (A) and rate as a function of pH (B) for normal milk (1) and milk supplemented with 7% (w / v) sucrose (2) fermented with ST1 (FIG. 4), ST1-GM (FIG. 5), ST1-GM-AB (FIG. 6), ST1-GM-ABU1 (FIG. 7), ST1-CM-ABU1 (FIG. 8). [Figure 7] Graphs depicting the change in pH over time (A) and rate as a function of pH (B) for normal milk (1) and milk supplemented with 7% (w / v) sucrose (2) fermented with ST1 (FIG. 4), ST1-GM (FIG. 5), ST1-GM-AB (FIG. 6), ST1-GM-ABU1 (FIG. 7), ST1-CM-ABU1 (FIG. 8). [Figure 8] Graphs depicting the change in pH over time (A) and rate as a function of pH (B) for normal milk (1) and milk supplemented with 7% (w / v) sucrose (2) fermented with ST1 (FIG. 4), ST1-GM (FIG. 5), ST1-GM-AB (FIG. 6), ST1-GM-ABU1 (FIG. 7), ST1-CM-ABU1 (FIG. 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The starter cultures of Streptococcus thermophilus used in the art consume any amount of sucrose that is normally added to milk for sweetening purposes during yogurt fermentation, with the following results: a) Because the strain uses sucrose (instead of using lactose), more sucrose needs to be added than (theoretically) required to achieve a certain level of sweetness. b) The functionality of the pH control phenotype resulting from fermentation with S. thermophilus mutated in manM (and / or manL, and / or manN) and glcK and / or ccpA (as described in the Examples of WO2019197051) will be reduced or lost in sweet milk. c) Glucose release resulting from fermentation of S. thermophilus mutated in glcK only and / or ccpA or glcK, and / or ccpA and manM (and / or manL, and / or manN) (as described in the Examples of WO2019122365 and WO2019197051) may be reduced or absent in sweet milk. d) The ability of S. thermophilus mutated with glcK, ccpA, glck / ccpA, glcK / manM(manL / manN), ccpA / manM(manL / manN) and glcK / ccpA / manM(manL / manN) (as described in the Examples of WO2019122365 and WO2019197051) to consume excessive lactose by fermentation is reduced or absent in sweet milk.

[0010] The present invention addresses each of these problems in the art and provides a solution in terms of specific mutations that modify sugar metabolism, which can be used to engineer improved Streptococcus thermophilus strains that can also be used to obtain low-lactose fermented milk products and / or to produce fermented milk containing sucrose that does not undergo post-acidification without the above-mentioned challenges, even when stored at fermentation temperatures.

[0011] Thus, in a first aspect, the present invention provides a lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations, the one or more mutations being 1) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or to transport sucrose; and i) scrA gene (phosphoenolpyruvate-sugar phosphotransferase system (PTS) sucrose specific EIIABC スクロース one or more genes of the sucrose regulon, such as the scrB gene (encoding a sucrose-6-phosphate hydrolase component), and / or the scrR gene (encoding a putative sucrose operon regulator); ii) optionally, further mutations in the promoter regions of the sucrose regulon regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to the sucrose-negative phenotype. and / or 2) Optionally, one or more further mutations affecting a glucose transporter, e.g., glcU and / or its expression, which restore or improve glucose consumption of the strain. The present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of:

[0012] It will be appreciated that in some embodiments, lactose-positive, sucrose-negative Streptococcus thermophilus strains according to the invention have essential mutations in one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene. In some embodiments, lactose-positive, sucrose-negative Streptococcus thermophilus strains according to the invention have essential mutations in one or more of the scrA gene, the scrB gene, and / or the scrR gene, such as a mutation that protects the strain from reversion to the sucrose-negative phenotype, or b) one or more further mutations affecting a glucose transporter, e.g. affecting glcU and / or its expression, which restore or improve glucose consumption of the strain when grown on glucose as the sole source of carbohydrate. or both a) and b) have an additional mutation.

[0013] In a second aspect, the present invention relates to a composition comprising at least one, in particular one lactose positive, sucrose negative Streptococcus thermophilus strain according to the invention, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of a strain of the genus Lactobacillus, such as a Lactobacillus delbrueckii subsp. bulgaricus strain, a strain of the genus Lactococcus, such as a Lactococcus lactis strain or a strain of the genus Bifidobacterium.

[0014] In a third aspect, the present invention relates to a method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention or with a composition according to the invention and fermenting the inoculated milk to obtain a fermented milk product.

[0015] In a further aspect, the present invention relates to the use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention or of the composition according to the invention as described above for producing a fermented milk product.

[0016] In a further aspect, the present invention relates to a fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention or obtainable by the method according to the invention as described above.

[0017] In a further aspect, the present invention relates to a Streptococcus thermophilus strain, such as a lactose-positive, sucrose-negative Streptococcus thermophilus strain, selected from the group consisting of: - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34132, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34134, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34138, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34139, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34140 in the name of DuPont Nutrition Biosciences ApS, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34146, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34147, - Strain deposited in accordance with the Budapest Treaty on February 16, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34172.

[0018] In a further aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited according to the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA is the truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. ManA mutant (manM 12997 ) that generates a frameshift in the open reading frame of a gene encoding a protein, and optionally further comprises a ccpA sequence of SEQ ID NO: 71 or a ccpA mutant sequence having at least 95% identity with SEQ ID NO: 71, a strain. - A strain of Streptococcus thermophilus, for example, the strain deposited under the Budapest Treaty on January 14, 2014, at Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM28255 [referred to herein as the ST4 or DSM28255 strain], having the following characteristics: 1) The sequence of scrA is replaced by the corresponding sequence shown in SEQ ID NO: 212 or a mutant sequence that is at least about 90%, for example, at least about 95% identical thereto, which results in a frameshift that causes translation in the truncated EIIABC スクロース (scrA 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example, at least about 95% identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217 - 220 or a mutant sequence that is at least about 90%, for example, at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example, at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ) 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following:

[0019] In a further aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25, wherein the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 1299771, or a variant ccpA sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71. - a strain of Streptococcus thermophilus, such as the strain deposited in accordance with the Budapest Treaty on 18 January 2022 at the Leibniz-Institut DSMZ under the number DSM 34132 in the name of DuPont Nutrition Biosciences ApS (herein referred to as the ST2 or DSM 34132 strain), having the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ) 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following:

[0020] In a further aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217 to 220, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 71, or a variant ccpA sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71. - a strain of Streptococcus thermophilus deposited on 29 September 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), which has the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ) 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following:

[0021] In a further aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 34172 (herein referred to as ST1 or DSM 34172), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and a strain in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ), - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217 to 220, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. ManMutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 71, or a variant ccpA sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71. - a strain of Streptococcus thermophilus deposited on 29 September 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 34172 (herein referred to as ST1 or DSM 34172), which has the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ) 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following:

[0022] In a further aspect, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain selected from the group consisting of: - The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), Streptococcus thermophilus strains, - the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), strains of Streptococcus thermophilus, - the locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto, or the promoter is replaced by the sequence of SEQ ID NO: 221 (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS Streptococcus thermophilus strains, including - The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), strains of Streptococcus thermophilus, - The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), strains of Streptococcus thermophilus, - The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ), and optionally further comprising a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71. - a strain of Streptococcus thermophilus having the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Mutations that generate frameshifts in the open reading frame of protein-coding genes (manM 12997 ) 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following:

[0023] In a further aspect, the present invention provides a method for selecting a lactose-positive, sucrose-negative Streptococcus thermophilus strain, comprising the steps of: 1) A strain of Streptococcus thermophilus, a) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or to transport sucrose, and i) one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene; ii) optionally, further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to the sucrose-negative phenotype; One or more mutations, including at least Providing a strain of Streptococcus thermophilus comprising: 2) screening for strains of Streptococcus thermophilus from step 1) that have a significantly reduced ability of said strains to grow on sucrose and / or to transport sucrose; 3) optionally screening, optionally before, simultaneously with or after any one of steps 1 and 2, for Streptococcus thermophilus strains with restored or improved glucose consumption resulting from one or more additional mutations affecting a glucose transporter, e.g. affecting glcU and / or its expression; The present invention relates to a method comprising the steps of:

[0024] In certain embodiments, a "variant sequence" of a strain as defined herein has at least 90% or at least 95% sequence identity with respect to the particular SEQ ID NO referred to, or with or without a particular mutation or insertion of the strain, with the genomic sequence of the parent strain from which the variant sequence is derived, and in particular has at least 90%, at least 91%, at least 95%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96%, at least 99.98% or at least 99.99% identity with the genomic sequence of the parent strain from which the particular SEQ ID NO or variant referred to is derived. The identity is stated when comparing two sequences over their entire length (global alignment) and can be calculated using any program based on the Needleman-Wunsch algorithm.

[0025] In dairy applications, the Streptococcus thermophilus strains of the invention are expected to not consume sucrose, i.e. to have a sucrose-negative phenotype. Thus, as used herein, "sucrose-negative" when referring to the phenotype of strains within the scope of the present invention means strains that have very limited or no ability to grow on media containing sucrose as the only source of carbohydrate, as measured by Test A described herein. In this context, Streptococcus thermophilus strains that have very limited or no ability to grow on media containing sucrose as the only source of carbohydrate are those that, when tested by Test A described herein, have a ratio of colony formation (cfu) on sucrose (sucrose positive colonies) compared to colony formation (cfu) on lactose of 10 or less. -2 Less than, for example, 10 -3 , 10 -4 , 10 -5 , 10 -6 or 10 -7 As used herein, a mutation that "results in a significant reduction in the ability to grow on sucrose and / or transport sucrose" is a mutation in the strain that induces or helps induce the strain to obtain a "sucrose negative phenotype."

[0026] Streptococcus thermophilus strains, when tested in Test A described herein, have a ratio of colony formation (cfu) on sucrose compared to colony formation (cfu) on lactose (sucrose positive colonies) close to 1 or 10 -2 If it is higher, it is considered a "sucrose positive phenotype."

[0027] Alternatively, enzymatic measures of the ability of a strain to grow on sucrose can be taken, for example by measuring the ability of resting cells of a scrA mutant to transport sucrose or the ability of intracellular extracts of a scrB mutant to hydrolyze phosphorylated sucrose (sucrose-6-phosphate).

[0028] The inventors have shown that by mutating selected genes of the sucrose regulon in Streptococcus thermophilus, optionally in combination with one or more mutations affecting a glucose transporter, e.g. glcU and / or its expression, Streptococcus thermophilus strains exhibit a "sucrose-negative phenotype" as defined above and optionally a glucose-positive phenotype.

[0029] In some embodiments, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain further carrying a mutation in a gene selected from the group consisting of: 1) at least one gene encoding a protein of a mannose-glucose specific PTS and the glcK gene, 2) at least one gene encoding a protein of a mannose-glucose specific PTS and the ccpA gene, and 3) a gene encoding a protein of the mannose-glucose specific PTS, the glcK gene, and the ccpA gene, as described in the Examples of WO2019122365 and WO2019197051.

[0030] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries a mutation in one gene encoding a protein of the mannose-glucose specific PTS and in the glcK gene. In one embodiment, the gene encoding a protein of the mannose-glucose specific PTS is selected from the group consisting of the manL gene, the manM gene, the manN gene, and the manO gene. In one embodiment, the 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. In one embodiment, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries a mutation in one gene selected from the group consisting of the manL gene, the manM gene, and the manN gene. In one embodiment, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries mutations in two genes selected from the group consisting of the manL gene, the manM gene, and the manN gene. In one embodiment, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries mutations in the manL, manM and manN genes. In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries a mutation in only one gene encoding a protein of the mannose-glucose specific PTS, the manM gene, as described in the Examples of WO2019122365 and WO2019197051.

[0031] Another aspect of the lactose-positive, sucrose-negative Streptococcus thermophilus strains according to the invention is the release of glucose during milk fermentation. As described herein, the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention can be further characterized by their ability to release glucose when used to ferment milk. This ability is defined herein by the concentration of glucose released in milk when the strains of the invention are used to ferment milk. In one embodiment, the fermentation conditions are according to Example 6 and the concentration of glucose released is determined as described in Example 6. Thus, in some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention releases a concentration of glucose, assayed as described in Example 6, that is at least 50 mM, such as at least 60, 70, 80 or 90 mM, or releases a glucose concentration that is increased by at least 150% or at least 200%, such as 300%, 400%, 500%, compared to the glucose concentration released by a corresponding parent strain not carrying a mutation as described in the invention, both when assayed as described in Example 6. In some embodiments, the mutation affecting the glucose transporter affects glcU, e.g. in the promoter region of glcU, resulting in an increase in the release of this concentration of glucose, assayed as described in Example 6, that is at least 50 mM, such as at least 60, 70, 80 or 90 mM. In some embodiments, the mutation in the promoter region of glcU is as described herein.

[0032] In some embodiments, the present invention relates to a lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying mutations in two or three genes selected from the group consisting of: 1) a gene encoding a protein of a mannose-glucose specific PTS and the glcK gene, 2) a gene encoding a protein of a mannose-glucose specific PTS and the ccpA gene, and 3) a gene encoding a protein of a mannose-glucose specific PTS, the glcK gene, and the ccpA gene (as described in the Examples of WO2019122365 and WO2019197051).

[0033] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention does not carry any mutation in a gene selected from a gene encoding a protein of the mannose-glucose specific PTS, the glcK gene, and the ccpA gene.

[0034] For the avoidance of doubt, the species Streptococcus thermophilus is to be understood as a strain of Streptococcus salivarius subsp. thermophilus.

[0035] The term "lactose positive" refers to a Streptococcus thermophilus strain capable of growing on lactose as the sole source of carbohydrate, in particular on M17 medium supplemented with 3% lactose as described in Test A. In one embodiment, the "lactose positive" phenotype is assayed by inoculating M17 broth containing 3% lactose at a rate of 1% with an overnight culture of the S. thermophilus strain to be tested and incubating at 37°C for 20 hours or alternatively 24 hours, with a pH of 5.5 or less at the end of the incubation indicating a lactose positive phenotype.

[0036] The term "galactose negative" refers to a Streptococcus thermophilus strain that is unable to grow on galactose as the sole source of carbohydrate, specifically on M17 medium supplemented with 2% galactose. In a particular embodiment, the "galactose negative" phenotype is assayed by inoculating M17 broth containing 2% galactose with 1% of an overnight culture of the S. thermophilus strain to be tested and incubating at 37°C for 20 hours, with a pH of 6 or higher at the end of incubation indicating a galactose negative phenotype.

[0037] With respect to a parent strain, the expression "derivative" (e.g. a DSM28255 derivative) means a strain obtained from the original strain (e.g. from a DSM28255 strain) by replacing one of its genes (e.g. scrA, glcK, manM, ...) with another allele of the same gene (especially an allele carrying some specific mutation) or by replacing one gene with a mutated gene. In one embodiment, a derivative is obtained by replacing a gene and its promoter of the original strain with another allele of that gene and its promoter. In one embodiment, a derivative is obtained by replacing all or part of the coding sequence of a gene of the original strain with another allele of that gene or part of it.

[0038] For embodiments involving either glcK sequencing, transferring the glcK allele of a suitable strain into the genome of other S. thermophilus strains, screening and selecting Streptococcus thermophilus strains for glucose excretion properties, identifying relevant mutations in the glcK gene, identifying and using additional glcK mutations, such as the non-conservative amino acid difference G144S, or identifying and using specific mutations in the glcK gene, such as the non-conservative amino acid difference E275K in the GlcK sequence, see co-pending WO 2019197051 A1.

[0039] A derivative of strain DSM28255 was designed in which the glcK gene encodes glucokinase and in which the glutamic acid (E) at position 275 is replaced by the amino acid lysine (K). This derivative (DGCC12534) was deposited at DSMZ under the accession number DSM32587 on August 15, 2017. The sequence of the GlcK protein is as defined in SEQ ID NO:22.

[0040] Lactic acidification performance The acidification properties of S. thermophilus strains can be evaluated by recording the pH over time during milk fermentation as described in Example 5. The pH is monitored for 24 h using a CINAC system (Alliance Instruments, France; pH electrode Mettler 405 DPAS SC, Toledo, Spain). The pH is measured and recorded every 5 or 25 min. The slope (UpH / min) from pH 6.0 to pH 5.5 [gradient pH 6-5.5] is calculated using CINAC v2.07 software.

[0041] The present invention therefore relates to a sucrose-negative strain of Streptococcus thermophilus. This phenotype of the Streptococcus thermophilus strain is achieved by any one or more of the following mutations: 1) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or to transport sucrose, in one or more genes of the sucrose regulon, such as the scrA, scrB and / or scrR genes, optionally with further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, and 2) Optionally, one or more further mutations affecting a glucose transporter, e.g. affecting glcU and / or its expression, that restore or improve glucose consumption of the strain.

[0042] The sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS) and the sucrose regulon The term "sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS)" refers to the sucrose phosphoenolpyruvate-sugar phosphotransferase system (PTS) in its usual sense of a system used by bacteria for sugar uptake where the energy source is derived from phosphoenolpyruvate (PEP). It consists of two non-specific energy coupling components involved in all PTSs, namely enzyme I (EI) and thermostable protein (HPr), and one sucrose-specific multidomain permease EIIABC encoded by scrA, which internalizes sucrose as sucrose-6-phosphate. スクロースThe other three genes are genetically related to scrA (at the same locus) on the sucrose regulon, namely scrB (encoding sucrose-6-phosphate hydrolase), scrR (encoding a GalR-LacI type transcriptional regulator) and scrK (encoding fructokinase). They are located on two operons (scrAK on one side and scrBR on the other side) that are divergently transcribed with an operon operator in between. Thus, as used herein, the term "sucrose regulon" refers to the scrA gene and the genes genetically related to scrA, namely scrB, scrR and scrK.

[0043] In some embodiments, the sucrose-negative phenotype is conferred by a mutation in the scrA gene. We have characterized a single mutation (scrA 829 ) where the mutation is a single base pair (bp) insertion at position 133, which results in a 45 aa truncated EIIABC of the native protein of 539 aa. スクロース It is an effective gene disruption that causes a frameshift of the open reading frame (ORF) resulting in a protein, making the truncated protein product inactive. Therefore, most frameshifts (insertions or deletions (indels) except for 3 bp and multiples of 3 bp insertions or deletions) within a gene can be expected to have the same effect of gene disruption that inactivates the protein product. SNPs (single nucleotide polymorphisms), especially SNPs that generate stop codons or SNPs that affect amino acids involved in the catalytic site of a protein, are expected to have a similar effect of affecting the enzyme active site and inhibiting activity.

[0044] In some embodiments, the sucrose negative phenotype is conferred by a mutation in the scrB gene. In some embodiments, the sucrose negative phenotype is conferred by a mutation in the scrR gene. In some embodiments, one or more genes of the sucrose regulon include one or more mutations in one or more of the genes scrA, scrB, and scrR that disrupt the function of one or more of these genes.

[0045] Mutations in the promoter regions regulating expression of the scrA, scrB and / or scrR genes. The pscrB region is an operator region that drives the expression of the divergently transcribed genes of the sucrose regulon. This promoter region drives the transcription of scrA and scrK on the one hand, and scrB and scrR on the other hand. 829 They characterized one single mutation, termed "SNP 1. This particular mutation was transferred to multiple strains with the same phenotypic outcome, promoting sucrose-negative strains. The mutation is a SNP located in the likely promoter region of scrB (a region likely to contain the -10 and -35 sequences of the promoter), specifically a SNP in the promoter region of scrAB that results in a T to G transition at position -38 of the scrB promoter.

[0046] In some embodiments, the sucrose-negative phenotype is conferred by a mutation in the pscrB region.

[0047] Mutations that affect glucose transporters, e.g. glcU and / or its expression. The gene glcU encodes a non-PTS transporter of glucose, also called "non-PTS glucose permease" or simply "glucose transporter". The gene glcU has its own promoter region (pglcU). Some mutations in pglcU are related to the scrA 829 and / or pscR 829The mutation allows the strain with the mutation to recover its growth rate on glucose similar to that of the parent strain. We characterized four different mutations. One is a SNP, two are insertions at the same position but at different nucleotides, and the third is an IS insertion. All allowed full recovery of growth on glucose. We characterized the mutations in three independent genetic backgrounds. All three strains fully recover the ability to grow on glucose. Thus, such a mutation in the glucose transporter may restore or improve glucose consumption by the strain. As used herein, "recover or improve glucose consumption" in relation to a mutation affecting a glucose transporter by a strain refers to a strain that recovers after this mutation, for example from a mutation in the sucrose regulon, to a growth rate similar to or higher than that of a parent strain without such a mutation in the sucrose regulon, or alternatively, a strain where a mutation affecting the glucose transporter of the strain simply improves the ability of the strain to grow on glucose. In some embodiments, the term "recover or improve glucose consumption" means that such a mutation confers a glucose positive phenotype on the strain and is considered glucose positive as defined herein.

[0048] We performed reverse genetics on one pglcU mutant. The pglcU mutation in one of the mutants was reverted to the original wild-type (WT, parent) sequence. This reversion resulted in a glucose slow-growing strain, demonstrating that the pglcU mutation itself is responsible for the glucose-positive phenotype of the triple mutant (scrA, pscrB, pglcU). Furthermore, the pglcU mutation, when introduced via genetic engineering into a sucrose-negative and glucose-slow-growing strain, renders the strain glucose-positive.

[0049] In one particular embodiment, the mutation affecting the glucose transporter is pglcU IN_T and insertion of a T nucleotide into the polyT at a position corresponding to −75 of the glcU promoter restores / improves glucose uptake and consumption.

[0050] In another particular embodiment, the mutation affecting the glucose transporter is pglcU IN_C and insertion of a C nucleotide in the polyT tract at a position corresponding to −75 of the glcU promoter restores / improves glucose uptake and consumption.

[0051] In another particular embodiment, the mutation affecting the glucose transporter is pglcU SNP_C and a T to C nucleotide mutation in the polyT tract at a position corresponding to −81 of the glcU promoter restores / improves glucose uptake and consumption.

[0052] In another particular embodiment, the mutation affecting the glucose transporter is pglcU IS and a sequence is inserted at a position corresponding to nucleotide positions 110-111 relative to the sequence of the ST1-ABU4 strain identified herein or the sequence of ST1 identified by SEQ ID NO: 216 of the glcU promoter, which insertion restores and / or improves glucose uptake and consumption.

[0053] scrA 829 It is expected that the mutation may have pleiotropic effects on the expression of other genes involved in carbohydrate utilization. This effect is likely to occur at the transcriptional level (as suggested by the location of the mutation in pglcU and the common knowledge on the regulation of carbohydrate catabolism). Thus, scrA 829 Mutations are likely to downregulate glcU expression, and mutations in pglcU may reverse this downregulation, making expression constitutive.

[0054] Thus, in some embodiments, lactose-positive, sucrose-negative Streptococcus thermophilus strains according to the present invention are glucose-positive or glucose slow-growing, and thus, glucose-positive strains can be propagated and produced using glucose but not lactose.

[0055] As used herein, a "glucose positive" strain refers to a Streptococcus thermophilus strain that is capable of growing on glucose as the sole source of carbohydrate, e.g., the rate at which it grows on glucose as the sole source of carbohydrate (μg). グルコース ) (the maximum growth rate of the same strain on lactose (μmax ラクトース ) at a time corresponding to μmax of ラクトース Ratio to (μ グルコース / μmax ラクトース The ratio of μ to glucose (μ = 0.25 or more, e.g., higher than 0.3, e.g., 0.35, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9) indicates that the strain grows well on glucose. Alternatively, μ defined and measured according to Test B described herein グルコース A Streptococcus thermophilus strain is considered "glucose positive" if the value is greater than 0.20, e.g., greater than 0.25, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8.

[0056] As used herein, a "glucose slow-growing" strain refers to a Streptococcus thermophilus strain that does not completely lose its ability to grow on glucose as the sole source of carbohydrate, but that grows significantly slower than a glucose-positive strain, e.g., the rate at which it grows on glucose as the sole source of carbohydrate (μ グルコース ) (the maximum growth rate of the same strain on lactose (μmax ラクトース) at a time corresponding to μmax of ラクトース Ratio to (μ グルコース / μmax ラクトース The μ ratio (μ = 0.25, e.g., 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, or 0.17) is less than 0.25, e.g., 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, or 0.17, but still greater than 0.05, indicating that the strain has the ability to grow on glucose, but grows slowly. Alternatively, the Streptococcus thermophilus strain has a μ ratio (μ = 0.25, μ = 0.25, グルコース Values ​​above 0.01 but below 0.2, for example below 0.18, 0.17, 0.16, 0.15, 0.14, 0.13 or 0.12, are considered "glucose slow growth."

[0057] For dairy applications, it is expected that the strains of the invention will not consume sucrose or at least will consume significantly less sucrose than the parent strain. To assess the sucrose-negative phenotype, the strains of the invention are tested for their ability to grow on a medium containing sucrose as the only source of carbohydrate, for example by kinetic measurement of the OD of a culture of the strain in a synthetic medium (M17) containing sucrose (30 g / L) as the only source of carbohydrate at an appropriate temperature (37° C.). Alternatively, enzymatic means may be considered, for example by measuring the ability of resting cells of a scrA mutant to transport sucrose or the ability of intracellular extracts of a scrB mutant to hydrolyze phosphorylated sucrose (sucrose-6-phosphate). In a preferred assay, the sucrose-negative phenotype of the strains of the invention is tested as described in Test A defined herein.

[0058] In some embodiments, the strain according to the invention comprises a mutation in a gene encoding a protein of the mannose-glucose specific PTS that, alone or in combination with a mutation in another gene, can reduce or eliminate the import of glucose from the medium into the bacterium. In some embodiments, the mutated glcK gene encodes glucokinase, and glucokinase activity in the strain is significantly reduced, but not zero. In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries a mutation in a gene encoding a protein of the mannose-glucose specific PTS that reduces or eliminates the import of glucose from the medium into the bacterium, and carries a mutation in the glcK gene encoding glucokinase, such that glucokinase activity in the strain is significantly reduced, but not zero. In any of these embodiments, the gene encoding a protein of the mannose-glucose specific PTS can be selected from the group consisting of the manL gene, the manM gene, and the manN gene.

[0059] In some embodiments, a mutation in a gene encoding a protein of the mannose-glucose specific PTS, alone or in combination with a mutation in another gene, reduces or eliminates glucose import from the medium into the bacterium. In some embodiments, a mutation in the ccpA gene results in a lactose-positive, sucrose-negative Streptococcus thermophilus strain with a β-glucose ratio of at least 4.10. -6 , at least 5.10 -6 , at least 6.10 -6 , at least 7.10 -6 Or at least 8.10 -6In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carries a mutation in a gene encoding a protein of a mannose-glucose specific PTS that reduces or eliminates glucose import from the medium into the bacterium, and carries a mutation in the ccpA gene, such that the ratio of the β-galactosidase activity of the strain, as assayed by the β-galactosidase activity assay described herein, to the glucokinase activity of the strain, as assayed by the glucokinase activity assay, is at least 4.10. -6 It is.

[0060] In some embodiments, a mutation in a gene encoding a protein of the mannose-glucose specific PTS, alone or in combination with mutations in other genes, reduces or eliminates glucose import from the medium into the bacterium. In some embodiments, the mutated glcK gene encodes glucokinase and glucokinase activity in the strain is significantly reduced, but not zero, in the strain. In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying a mutation in the glcK gene also carries a mutation in the ccpA gene.

[0061] The following sections describe mutations in the glcK gene, mutations in genes encoding proteins of the mannose-glucose specific PTS (such as mutations in the manL, manM and manN genes) and mutations in the ccpA gene, respectively.

[0062] Although these mutations are disclosed separately herein (for clarity), any embodiment of one moiety can be combined with any embodiment of another moiety or with any embodiment of two other moieties to design a lactose-positive, sucrose-negative Streptococcus thermophilus strain as defined herein.

[0063] For the avoidance of doubt, in some embodiments the present invention relates to a lactose positive, sucrose negative Streptococcus thermophilus strain as defined herein, which possesses: 1) a mutation in at least one, in particular one, gene encoding a protein of the mannose-glucose specific PTS and a mutation in the glcK gene thereof, or 2) a mutation in at least one, in particular one, gene encoding a protein of the mannose-glucose specific PTS and a mutation in its ccpA gene, or 3) A mutation in at least one, in particular in one, gene encoding a protein of the mannose-glucose specific PTS, a mutation in the glcK gene and a mutation in the ccpA gene.

[0064] Mutation in the glcK gene This section describes, in the context of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention, mutations in one or more genes of the sucrose regulon, mutations affecting a glucose transporter, mutations in the glcK gene that may be used in combination with mutations in genes encoding proteins of the mannose-glucose specific PTS and / or in combination with mutations in genes encoding proteins of the mannose-glucose specific PTS defined herein and mutations in the ccpA gene defined herein.

[0065] In some embodiments, the mutant glcK gene of the strain of the invention encodes a glucokinase, and the glucokinase activity in said strain is significantly reduced, but not zero. Indeed, the inventors have presented evidence that several mutant alleles of the glcK gene encode glucokinase (GlcK), and that the glucokinase activity is significantly reduced, but not zero, when said mutant glcK gene is present in lactose-positive Streptococcus thermophilus strains.

[0066] The expression "glcK gene encoding glucokinase" refers to any DNA sequence of a Streptococcus thermophilus strain that encodes the glucokinase enzyme, which catalyzes the conversion of glucose and ATP to glucose-6-phosphate (G6P) and ADP. Non-limiting examples of Streptococcus thermophilus glucokinase sequences are disclosed as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.

[0067] In some embodiments within the present invention, glucokinase activity in a Streptococcus thermophilus strain is significantly reduced, but not zero, as a result of a mutation in its glcK gene, i.e., the allele of the glcK gene carried by the strain is such that glucokinase activity in the strain is significantly reduced, but not zero.

[0068] The phrase "the glucokinase activity in said strain is significantly reduced, but not zero" refers to a strain in which the glucokinase activity is both: - the glucokinase activity in said strain is significantly reduced, in particular compared to that in a strain carrying a non-mutated glcK gene, and - is non-zero, ie activity is detectable by the glucokinase activity assay defined herein.

[0069] According to an embodiment of the present invention, the characteristic that "the glucokinase activity in said strain is significantly reduced, but not zero" can be determined by methods known in the art. Thus, methods for measuring glucokinase activity in Streptococcus thermophilus strains are known and include enzyme assays using commercially available reactants. Reference is made herein to paragraph 2.4 of Pool et al. (2006. Metabolic Engineering 8(5);456-464), which is incorporated herein by reference. In a particular embodiment, the glucokinase activity in the Streptococcus thermophilus strains of the present invention is assayed by a glucokinase activity assay (i.e., a glucokinase activity assay is performed using the Streptococcus thermophilus strains of the present invention).

[0070] Glucokinase activity assay: Obtain a fresh overnight culture of the Streptococcus thermophilus strain to be assayed in M17 containing 30 g / L lactose and use this at 1% (vol / vol) to inoculate 10 ml of fresh M17 30 g / L lactose. Cells are harvested by centrifugation (6000 g, 10 min, 4°C) at an optical density at 600 nm (OD600) of 0.8 + / - 0.2, washed with 5 ml of cold GLCK buffer (5 mM MgCl2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]), and resuspended in 500 μl of cold GLCK buffer. The EDTA-free protease inhibitor "cOmplete™" (Roche, supplier reference number) is added to the GLCK buffer as described by the supplier. 100 mg of glass beads (150-212 μm, Sigma G1145) are added to 200 μl of resuspended cells and cells are disrupted by shaking for 6 min at a frequency of 30 cycles / s in a MM200 vibration mill (Retsch, Haan, Germany). Cell debris and glass beads are removed by centrifugation (14000g, 15 min, 4°C) and the supernatant is transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content is determined using the FLUKA Protein Quantification Kit-Rapid (ref51254). Glucokinase activity in cell extracts was essentially determined by measuring glucose-6-phosphate dehydrogenase (G-6PDH, EC 1.1.1.49): Determined spectrophotometrically by NADPH binding assay (Porter et al., 1982). Each sample (5, 10 and 20 μL) was added to a final volume of 250 μL of assay buffer (10 mM K2HPO4 / KH2PO4 [pH 7.2], 5 mM MgCl2, 1 mM ATP, 20 mM glucose, 1 mM NADP, 1 U G-6PDH) and the mixture was left at 30°C for 5 min. The optical density at 340 nm was measured for 5 min using a Synergy HT multidetection microplate reader (BIO-TEK).One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 μmole of D-glucose to D-glucose 6-phosphate per minute under assay conditions. Glucokinase activity is calculated as follows: Glucokinase activity (U / g of total protein extract) = dOD x V / [dt x l x ε x Qprot] (In the formula, - dOD is the variation of optical density (OD) at 340 nm; - V is the volume of the reaction (here 250 μL), dt = measurement time (min), l = optical path length (here 0.73 cm), ε = molar extinction coefficient of NADPH; H + (Here, 6220 cm2 / μmol) Qprot = amount of protein in the cuvette (g) Measurements were performed in triplicate for each sample, and the glucokinase specific activity values ​​presented herein under Glucokinase Activity Assay are the average of three independent experiments.

[0071] In a first particular embodiment of the feature "the glucokinase activity in said strain is significantly reduced but not zero", the glucokinase activity of the Streptococcus thermophilus strain of the invention is between 200 and 1500 U / g of total protein extract, as assayed by a glucokinase activity assay. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain of the invention is between 300 and 1200 U / g of total protein extract, as assayed by a glucokinase activity assay. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain of the invention is between 400 and 1000 U / g of total protein extract, as assayed by a glucokinase activity assay. In certain embodiments, the glucokinase activity in the Streptococcus thermophilus strains of the invention, as assayed by a glucokinase activity assay, is between a minimum value selected from the group consisting of 200, 300 and 400 U / g of total protein extract and a maximum value selected from the group consisting of 1000, 1200 and 1500 U / g of total protein extract. As mentioned in the glucokinase activity assay, it is worth noting that the glucokinase activity values ​​disclosed herein are the average of three independent experiments (triplicates).

[0072] In a second particular embodiment of the feature "the glucokinase activity in said strain is significantly reduced but not zero", the glucokinase activity of the Streptococcus thermophilus strain of the invention is 5-60% of the activity of the glucokinase activity of strain DGCC7710 deposited at DSMZ on January 14, 2014 under accession number DSM28255. By "glucokinase activity of strain DSM28255" is meant the activity of glucokinase (i.e. SEQ ID NO: 2) of strain DSM28255 as assayed by a glucokinase activity assay in strain DSM28255 [i.e., the glucokinase activity assay is performed using strain DSM28255]. The percentage value is calculated based on the glucokinase activity in the strain of the invention and the glucokinase activity of strain DSM28255, both assayed by a glucokinase activity assay. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain of the invention is between 10 and 50% of the glucokinase activity of the DSM28255 strain. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain of the invention is between 15 and 40% of the glucokinase activity of the DSM28255 strain. In a particular embodiment, the glucokinase activity of the Streptococcus thermophilus strain of the invention is between a minimum percentage selected from the group consisting of 5, 10 and 15% of the glucokinase activity of the DSM28255 strain and a maximum percentage selected from the group consisting of 40, 50 and 60% of the glucokinase activity of the DSM28255 strain. Regardless of the particular embodiment and the percentage range, the activity of the glucokinase activity is assayed by a glucokinase activity assay as described herein. It is worth noting that the percentage values ​​disclosed herein are calculated based on glucokinase activity values ​​that are the average of three independent experiments (triplicates) assayed by the glucokinase activity assay.

[0073] In the first and second particular embodiments, the following strains can be used as controls for the glucokinase activity assay: - as a positive control (i.e. a Streptococcus thermophilus strain representative of strains carrying a non-mutated glcK gene): strain DGCC7710, deposited at the DSMZ on 14 January 2014 under the accession number DSM28255.

[0074] The feature "the glucokinase activity in said strain is significantly reduced, but not zero" can also be characterized by the maximum process velocity of glucokinase (referred to herein as Vmax, defined as the rate of conversion of glucose + ATP to G6P + ADP) or the inverse of the affinity of glucokinase for one or two of its substrates, i.e., glucose and ATP (referred to as Km). In some embodiments, the feature "the glucokinase activity in said strain is significantly reduced, but not zero" for a strain of the invention is further characterized by the maximum process velocity (Vmax) of its glucokinase in said strain.

[0075] Thus, in combination with the first or second particular embodiment of the feature "the glucokinase activity in said strain is significantly reduced but not zero" defined herein, the maximum forward velocity (Vmax) of glucokinase of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is significantly reduced but not zero. The feature "the glucokinase Vmax of said strain is significantly reduced but not zero" can be defined by one or two of these parameters: - When assayed by glucokinase Vmax assay, Vmax is 200-1500 U / g of total protein extract. - When both are assayed by the glucokinase Vmax assay, the Vmax is 5-60% of the Vmax of glucokinase of strain DSM28255 deposited at DSMZ on January 14, 2014 under accession number DSM28255.

[0076] In certain embodiments, the mutated glcK gene of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention encodes glucokinase, and glucokinase activity in said strain (as defined herein) is significantly reduced, but not zero, and the maximum processive velocity (Vmax) of glucokinase in said strain is significantly reduced, but not zero, as defined by one or two of these parameters. - When assayed by glucokinase Vmax assay, Vmax is 200-1500 U / g of total protein extract. - When both are assayed by the glucokinase Vmax assay, the Vmax is 5-60% of the Vmax of glucokinase of strain DSM28255 deposited at DSMZ on January 14, 2014 under accession number DSM28255.

[0077] The glucokinase maximum processive velocity (Vmax) in the Streptococcus thermophilus of the invention is assayed by a glucokinase Vmax assay (performed using the Streptococcus thermophilus strains of the invention).

[0078] Glucokinase Vmax Assay: The maximum forward velocity (Vmax) is determined by using various concentrations of glucose (0, 5, 10, 15, 20 mM) on the crude extract prepared as described in the glucokinase activity assay. Measurements were performed in triplicate for each sample and the Vmax values ​​obtained under the glucokinase Vmax assay are the average of three independent experiments. A linear regression expressing the inverse of the specific velocity as a function of the inverse of the glucose concentration gives the inverse of the maximum forward velocity at the intersection with the Y-axis of the graph.

[0079] In certain embodiments of the maximum processive velocity of glucokinase in the Streptococcus thermophilus strain of the invention, the Vmax is between 200 and 1500 U / g of total protein extract when assayed by a glucokinase Vmax assay. In certain embodiments, the Vmax is between 300 and 1200 U / g of total protein extract when assayed by a glucokinase Vmax assay. In certain embodiments, the Vmax is between 400 U / g and 1000 U / g of total protein extract. In certain embodiments, the Vmax of glucokinase in the Streptococcus thermophilus strain of the invention, when assayed by a glucokinase Vmax assay, is between a minimum value selected from the group consisting of 200, 300 and 400 U / g of total protein extract and a maximum value selected from the group consisting of 1000, 1200 and 1500 U / g of total protein extract.

[0080] In a particular embodiment of the maximum processive velocity of glucokinase in a Streptococcus thermophilus strain of the invention, the Vmax is 5-60% of the Vmax of glucokinase in strain DSM28255. By "Vmax of glucokinase in strain DSM28255" is meant the Vmax of glucokinase in strain DSM28255 (i.e. SEQ ID NO: 2) as assayed by a glucokinase Vmax assay in strain DSM28255 [i.e., a glucokinase Vmax assay is performed using strain DSM28255]. The percentage value is calculated based on the Vmax of glucokinase in the strain of the invention and the Vmax of strain DSM28255, both as assayed by a glucokinase Vmax assay. In a particular embodiment, the glucokinase Vmax of the Streptococcus thermophilus strain of the invention is 10-50% of the Vmax of the glucokinase of the strain DSM28255, when both are assayed by a glucokinase Vmax assay. In a particular embodiment, the glucokinase Vmax of the Streptococcus thermophilus strain of the invention is 15-40% of the Vmax of the glucokinase of the strain DSM28255. In a particular embodiment, the Vmax of the glucokinase of the Streptococcus thermophilus strain of the invention is between a minimum percentage selected from the group consisting of 5, 10 and 15% of the Vmax of the glucokinase activity of the strain DSM28255 and a maximum percentage selected from the group consisting of 40, 50 and 60% of the Vmax of the glucokinase activity of the strain DSM28255.

[0081] A lactose-positive, sucrose-positive Streptococcus thermophilus strain of the invention can carry a mutation in the glcK gene encoding glucokinase, such that glucokinase activity in said strain is significantly reduced, as defined herein, but not zero, and optionally, the maximum processive velocity of glucokinase in said strain is significantly reduced, as defined herein, but not zero.

[0082] By "mutation in the glcK gene" within the present invention is meant any nucleotide mutation in the glcK gene, said mutation at the nucleotide level resulting in a significantly reduced, but non-zero glucokinase activity, as defined herein, in a strain carrying this mutated glcK gene (as the only glcK gene), and optionally resulting in a significantly reduced, but non-zero maximum process velocity of glucokinase, as defined herein, in said strain. In a particular embodiment, by "mutation in the glcK gene" within the present invention is meant any nucleotide mutation in the open reading frame of the glcK gene, said mutation at the nucleotide level resulting in a significantly reduced, but non-zero glucokinase activity, as defined herein, in a strain carrying this mutated glcK gene (as the only glcK gene), and optionally resulting in a significantly reduced, but non-zero maximum process velocity of glucokinase, as defined herein, in said strain.

[0083] Thus, two Streptococcus thermophilus strains may differ by the sequence of their respective glcK genes, but this does not necessarily mean that one of these two glcK genes is mutated within the meaning of the present invention. Indeed, the following are not considered to be mutations within the present invention: - mutations at the nucleotide level that do not result in any change at the protein level (silent mutations) and do not affect the translation of glcK RNA, and - a mutation at the nucleotide level, which leads to a change at the protein level, as defined herein, but which does not affect the glucokinase activity of the resulting GlcK protein and, optionally, the maximum processive velocity of the resulting GlcK protein. Indeed, such a mutation can be observed at the level of the glcK gene of the Streptococcus thermophilus of the invention, without affecting the extent of protection.

[0084] Non-limiting examples of glcK genes that are not considered mutated within the meaning of the present invention are: a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 2, in particular as defined in SEQ ID NO: 1. This GlcK type is one of the strains DGCC7710 deposited at the DSMZ on 14 January 2014 under the accession number DSM28255, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 4, in particular as defined in SEQ ID NO: 3, a polynucleotide encoding a glucokinase as defined in SEQ ID NO:6, in particular as defined in SEQ ID NO:5, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 8, in particular as defined in SEQ ID NO: 7, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 10, in particular as defined in SEQ ID NO: 9, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 12, in particular as defined in SEQ ID NO: 11, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 14, in particular as defined in SEQ ID NO: 13, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 16, in particular as defined in SEQ ID NO: 15, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 18, in particular as defined in SEQ ID NO: 17, a polynucleotide encoding a glucokinase as defined in SEQ ID NO: 20, in particular a polynucleotide as defined in SEQ ID NO: 19.

[0085] Furthermore, some nucleotide mutations in the glcK gene are not considered suitable for the present invention because they result in a glucokinase whose activity, when assayed by a glucokinase activity assay, is zero or below the minimum value defined herein. In some embodiments, the Streptococcus thermophilus of the present invention does not possess a mutation selected from the group consisting of a mutation that results in a knockout of the glcK gene and a large deletion in the glcK gene.

[0086] In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention carries a mutation in the open reading frame of the glcK gene that results in a substitution of an amino acid in the GlcK protein, and whose glucokinase activity is significantly reduced (as defined herein) in said strain carrying a mutant glcK gene, but not zero, and optionally, the maximum processive rate of glucokinase in said strain is significantly reduced (as defined herein) in said strain carrying a mutant glcK gene, but not zero, and optionally, the maximum processive rate of glucokinase in said strain is significantly reduced (as defined herein) in said strain carrying a mutant glcK gene, but not zero. In certain embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention carries a mutation in the glcK gene that results in a substitution of an amino acid in the GlcK protein, and whose glucokinase activity is significantly reduced (as defined herein) in said strain carrying a mutant glcK gene, but not zero, and optionally, the maximum processive rate of glucokinase in said strain is significantly reduced (as defined herein) in said strain carrying a mutant glcK gene, but not zero. In certain embodiments, a Streptococcus thermophilus strain of the invention harbors a mutation in the glcK gene such that the GlcK protein is 322 amino acids in length, and glucokinase activity in said strain is significantly reduced, as defined herein, but not zero, and optionally the maximum processive velocity of glucokinase in said strain is significantly reduced, as defined herein, but not zero.

[0087] As mentioned above, several DNA modifications can be observed at the level of the glcK gene of the Streptococcus thermophilus of the invention that do not affect the glucokinase activity of the strain. Based on the glucokinase activity assay defined herein, together with the control strain defined herein, the skilled artisan will know how to identify 1) a glcK gene encoding a glucokinase in which the glucokinase activity (as defined herein) is significantly reduced, but not zero, in a strain carrying this glcK gene, and optionally, the maximum processive velocity of glucokinase (as defined herein) in a strain carrying this mutated glcK gene is significantly reduced, but not zero, 2) a glcK gene with a modification that does not affect the glucokinase activity in a strain carrying this modification, or 3) a glcK gene encoding a glucokinase in which the glucokinase activity (as defined herein) is zero in a strain carrying this glcK gene.

[0088] The inventors have identified two positions in glucokinase that show that the nature of the amino acids affect the activity of glucokinase such that the glucokinase activity is significantly reduced, but not zero, as defined herein, and that affect the Vmax of glucokinase such that the Vmax is significantly reduced, but not zero, as defined herein: positions 144 and 275 of glucokinase (i.e., codons 144 and 275 of the glcK gene). It is worth noting that based on the glucokinase activity assay and the glucokinase Vmax assay defined herein, together with the control strain, a person skilled in the art would know how to identify other positions and suitable amino acids in glucokinase to obtain a significantly reduced, but not zero glucokinase activity (as defined herein) and optionally a significantly reduced, but not zero maximum processive velocity, and therefore the corresponding glcK gene.

[0089] In some embodiments, the amino acid at position 275 of glucokinase (encoded by the glcK gene of a Streptococcus thermophilus strain of the invention) is not glutamic acid (i.e., any amino acid except glutamic acid). Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is not GAA or GAG. In particular embodiments, the amino acid at position 275 of glucokinase is not an acidic amino acid (i.e., any amino acid except acidic amino acids). Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is a codon encoding a non-acidic amino acid. In particular embodiments, the amino acid at position 275 of glucokinase is selected from the group consisting of lysine and any of its conservative amino acids. Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is a codon encoding an amino acid selected from the group consisting of lysine and any of its conserved amino acids. In particular embodiments, the amino acid at position 275 of glucokinase is lysine. Thus, in some embodiments, codon 275 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is either AAA or AAG. In particular embodiments, nucleotides 823-825 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention are AAA or AAG.

[0090] In certain embodiments, the sequence of the GlcK protein of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of: a) a sequence as defined in SEQ ID NO: 25, in which the amino acid at position 275 is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine; and b) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In a particular embodiment, the GlcK variant sequence is 322 amino acids in length.

[0091] In another embodiment, the amino acid at position 144 of glucokinase (encoded by the glcK gene of a Streptococcus thermophilus strain of the invention) is not glycine (i.e., any amino acid except glycine). Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is not GGT, GGC, GGA, or GGG. In particular embodiments, the amino acid at position 144 of glucokinase is not an aliphatic amino acid (i.e., any amino acid except an aliphatic amino acid). In particular embodiments, the amino acid at position 144 of glucokinase is selected from the group consisting of serine and any of its conserved amino acids. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is a codon encoding an amino acid selected from the group consisting of serine and any of its conserved amino acids. In certain embodiments, the amino acid at position 144 of glucokinase is serine. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is AGT, AGC, TCT, TCC, TCA, or TCG. In certain embodiments, nucleotides 430-432 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention are AGT, AGC, TCT, TCC, TCA, or TCG.

[0092] In certain embodiments, the sequence of the GlcK protein of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine, and b) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except an aliphatic amino acid, in particular serine. In a particular embodiment, the GlcK variant sequence is 322 amino acids in length.

[0093] For the definition of a GlcK variant having at least 90% similarity or identity with SEQ ID NO:25, the similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portions of the sequences]. Position 275 as defined in SEQ ID NO:25 is not considered for the calculation of similarity or identity. In certain embodiments, the GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO:25, and the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO:25, and the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO:25, and the amino acid corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine.

[0094] In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by between 1 and 30 amino acid substitutions, and the amino acid at position 275 of said GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, in particular lysine (position 275 is not considered for calculating the number of substitutions). In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by between 1 and 20 amino acid substitutions, and the amino acid at position 275 of said GlcK mutant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by between 1 and 15 amino acid substitutions, and the amino acid at position 275 of said GlcK mutant is any amino acid except glutamic acid, in particular any amino acid except acidic amino acids, in particular lysine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by 1 to 10 amino acid substitutions, and the amino acid at position 275 of said GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 25 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, and the amino acid at position 275 of said GlcK mutant is any amino acid except glutamic acid, particularly any amino acid except acidic amino acids, and particularly lysine.

[0095] For the definition of a GlcK variant having at least 90% similarity or identity with SEQ ID NO: 46, the similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portions of the sequences]. Position 144 as defined in SEQ ID NO: 46 is not considered for the calculation of similarity or identity. In certain embodiments, the GlcK variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity with SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In some embodiments, the GlcK variant sequence has at least 95% similarity or identity to SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine. In some embodiments, the GlcK variant sequence has at least 97% similarity or identity to SEQ ID NO: 46, and the amino acid corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, and particularly serine.

[0096] In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 46 by between 1 and 30 amino acid substitutions, and the amino acid at position 144 of said GlcK mutant is any amino acid except glycine, particularly any amino acid except aliphatic amino acids, in particular serine (position 144 is not considered for calculating the number of substitutions). In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 46 by between 1 and 20 amino acid substitutions, and the amino acid at position 144 of said GlcK mutant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 46 by between 1 and 15 amino acid substitutions, and the amino acid at position 144 of said GlcK mutant is any amino acid except glycine, in particular any amino acid except aliphatic amino acids, in particular serine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 46 by between 1 and 10 amino acid substitutions, and the amino acid at position 144 of said GlcK mutant is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, particularly an amino acid that is serine. In certain embodiments, the GlcK mutant sequence differs from SEQ ID NO: 46 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, and the amino acid at position 144 of said GlcK mutant is any amino acid except glycine, particularly any amino acid except an aliphatic amino acid, particularly an amino acid that is serine.

[0097] In some embodiments, the sequence of the GlcK protein of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, and the amino acid at position 275 of said variant is any amino acid except glutamic acid, in particular any amino acid except an acidic amino acid, in particular lysine. Thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain of the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 33 and 34, and the amino acid at position 275 of glucokinase is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine, respectively.

[0098] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase), as either SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), is not glutamic acid. Thus, in some embodiments, codon 275 of the glcK gene possessed by the Streptococcus thermophilus strain of the invention is not GAA or GAG. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not glutamic acid.

[0099] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase), as either SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), is not an acidic amino acid. Thus, in some embodiments, codon 275 of the glcK gene carried by the Streptococcus thermophilus strain of the invention is a codon that does not encode an acidic amino acid. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is not an acidic amino acid.

[0100] In certain embodiments, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase), as either SEQ ID NO:25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 defined herein (particularly SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), is selected from the group consisting of lysine and any of its conserved amino acids. Thus, in some embodiments, codon 275 of the glcK gene possessed by the Streptococcus thermophilus strains of the invention is a codon encoding an amino acid selected from the group consisting of lysine and any of its conserved amino acids. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO:25 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO:25 as defined herein (in particular SEQ ID NOs:26, 27, 28, 29, 30, 31, 32, 33 or 34), wherein the amino acid of glucokinase corresponding to position 275 of SEQ ID NO:25 (or the amino acid at position 275 of glucokinase) is either lysine or a conserved amino acid thereof.

[0101] In a particular embodiment, the amino acid of glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of glucokinase) as either SEQ ID NO: 25 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25 defined herein (in particular SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33 or 34) is lysine. Thus, in some embodiments, codon 275 of the glcK gene carried by the Streptococcus thermophilus strain of the invention is a codon encoding lysine, in particular AAA or AAG, respectively. Thus, in a particular embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NOs: 22, 35, 36, 37, 38, 39, 40, 41, 42 and 43. Thus, in some embodiments, the glcK gene possessed by a Streptococcus thermophilus strain of the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 22, 35, 36, 37, 38, 39, 40, 41, 42, and 43. In a particular embodiment, the glcK gene possessed by a Streptococcus thermophilus strain of the invention is as defined in SEQ ID NO: 21.

[0102] In another embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, and the amino acid at position 144 of said variant is any amino acid except glycine, in particular any amino acid except an aliphatic amino acid, in particular serine. Thus, in some embodiments, the glcK gene carried by the Streptococcus thermophilus strain of the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55, and the amino acid at position 144 of glucokinase is not glycine, in particular is not an aliphatic amino acid, in particular serine.

[0103] In certain embodiments, the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase), as either SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), is not glycine. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is not GGT, GGC, GGA or GGG. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is not glycine.

[0104] In certain embodiments, the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase), as either SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), is not an aliphatic amino acid. Thus, in some embodiments, codon 144 of the glcK gene carried by a Streptococcus thermophilus strain of the invention is a codon that does not encode an aliphatic amino acid. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is not an aliphatic amino acid.

[0105] In certain embodiments, the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase), as either SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (particularly SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), is selected from the group consisting of serine and any of its conserved amino acids. Thus, in some embodiments, codon 144 of the glcK gene possessed by a Streptococcus thermophilus strain of the invention is a codon encoding an amino acid selected from the group consisting of serine and any of its conserved amino acids. Thus, in some embodiments, the glcK gene carried by a Streptococcus thermophilus strain of the invention encodes a GlcK protein selected from the group consisting of SEQ ID NO: 46 and any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 as defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55), wherein the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) is either serine or a conserved amino acid thereof.

[0106] In a particular embodiment, the amino acid of glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of glucokinase) as either SEQ ID NO: 46 or any GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46 defined herein (in particular SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54 or 55) is serine. Thus, in some embodiments, codon 144 of the glcK gene carried by the Streptococcus thermophilus strain of the invention is a codon encoding serine, in particular AAA or AAG. Thus, in a particular embodiment, the sequence of the GlcK protein of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NOs: 45, 56, 57, 58, 59, 60, 61, 62, 63 and 64. Thus, in some embodiments, the glcK gene possessed by a Streptococcus thermophilus strain of the invention encodes a GlcK protein whose sequence is selected from the group consisting of SEQ ID NOs: 45, 56, 57, 58, 59, 60, 61, 62, 63, and 64. In a particular embodiment, the glcK gene possessed by a Streptococcus thermophilus strain of the invention is as defined in SEQ ID NO:44.

[0107] When the sequence of the GlcK protein of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention is defined, in accordance with the teachings of the present application, the glucokinase activity in a strain expressing this GlcK protein is significantly reduced, as defined herein, but not zero, and optionally, the Vmax of glucokinase in this strain is significantly reduced, as defined herein, but not zero.

[0108] Mutations in genes encoding proteins of the mannose-glucose specific PTS, in particular mutations in the manL, manM and manN genes. This section describes, in the context of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention, mutations in the genes encoding proteins of the mannose-glucose specific PTS, in particular mutations in the manL, manM and manN genes that may be used in combination with a mutation in the glcK gene defined herein, or in combination with a mutation in the ccpA gene defined herein, or in combination with both a mutation in the glcK gene and a mutation in the ccpA gene defined herein.

[0109] In lactose-positive, sucrose-negative Streptococcus thermophilus strains, any mutation in a gene encoding a protein of the mannose-glucose specific PTS is suitable, and may be combined with a mutated glcK gene as defined herein, or may be combined with a mutation in the ccpA gene as defined herein, or may be combined with both a mutated glcK gene and a mutated ccpA gene as defined herein.

[0110] The inventors have shown that mutations in genes encoding proteins of the mannose-glucose specific PTS in lactose-positive, sucrose-negative Streptococcus thermophilus strains, in particular mutated manL genes, mutated manM genes or mutated manN genes that reduce or eliminate glucose import from the medium, are particularly advantageous in the present invention. In some embodiments, a mutation in a gene encoding a protein of the mannose-glucose specific PTS leads to a reduction or elimination of the glucose import activity of the protein encoded by this gene. In some embodiments, the mutated gene is the manL gene and the mutation in the manL gene is EIIABMan In some embodiments, the mutated gene is a manM gene, and the mutation in the manM gene results in a reduction or loss of glucose import activity of the protein. Man In some embodiments, the mutated gene is a manN gene, and the mutation in the manN gene results in a reduction or loss of glucose import activity of the protein. Man This results in a reduction or abolishment of the glucose import activity of the protein.

[0111] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, is a mutation that results in the knockout (ie complete disruption) of the gene.

[0112] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, is a mutation in the promoter of the gene, in particular a mutation in the promoter of the gene that reduces or inhibits transcription of the gene.

[0113] In some embodiments, a mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, is a mutation introduced into the coding sequence of the gene, in particular a reduction or abolishment of the glucose import activity of the protein encoded by the mutated gene, in particular the EIIAB Man Protein, EIIC Man Protein or EIID Man These are mutations that result in a reduction or abolishment of the glucose import activity of the protein.

[0114] In some embodiments, the mutation in a gene encoding a protein of the mannose-glucose specific PTS, in particular the manL gene, the manM gene or the manN gene, results in a truncated protein, in particular the truncated EIIAB Man Protein, truncated EIIC ManProtein or truncated EIID Man Proteins, particularly truncated proteins with reduced or eliminated glucose import activity (e.g., truncated EIIAB Man Protein, truncated EIIC Man Protein or truncated EIID Man In some embodiments, the mutation introduced into the gene is a mutation in the coding sequence of the gene that results in a stop codon or a deletion, insertion, or deletion / insertion that results in a frameshift of the open reading frame and a premature stop codon. In some embodiments, the mutation introduced into the gene is a nucleotide substitution that results in a stop codon. In some embodiments, the mutation introduced into the gene is a deletion, insertion, or deletion / insertion that results in a frameshift of the open reading frame and a premature stop codon.

[0115] Two Streptococcus thermophilus strains may differ by the sequence of their respective manL, manM or manN genes, but this does not necessarily mean that one of these genes is mutated within the meaning of the present invention. Indeed, what is not considered as a mutation of the manL, manM or manN genes within the present invention is the following: - mutations at the nucleotide level that do not result in any change at the protein level (silent mutations) and do not affect the translation of manL, manM or manN RNA, and - a mutation at the nucleotide level which results in a change at the protein level in the lactose-positive, sucrose-negative phenotype of a Streptococcus thermophilus strain, but which is not effective when combined with a mutant glcK gene as defined herein, or when combined with a mutation in the ccpA gene as defined herein, or when combined with both a mutant glcK gene and a mutant ccpA gene.

[0116] The manL, manM and manN genes (EIIAB Man Protein, EIIC Man Proteins and EIIDs Man Non-limiting examples of ribozymes (which each encode a protein) are as follows: - EIIAB as defined in SEQ ID NO: 78 Man A polynucleotide encoding a protein, in particular the polynucleotide defined in SEQ ID NO: 77. Man The type is that of strain DSM28255. - EIIAB as defined in SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110 Man A polynucleotide encoding a protein, in particular a polynucleotide as defined in SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109. EIIAB as defined in SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 and 110. Man The protein sequence is 98.4-99.6% identical to SEQ ID NO: 78; - EIIC as defined in SEQ ID NO:130 Man A polynucleotide encoding a protein, in particular the polynucleotide defined in SEQ ID NO: 129. Man The type is DSM28255. EIICs as defined in SEQ ID NOs: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156 Man Polynucleotides encoding proteins, in particular polynucleotides as defined in SEQ ID NOs: 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153 and 155. EIICs as defined in SEQ ID NOs: 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154 and 156. Man The protein sequence is 98.5-99.6% identical to SEQ ID NO: 130; - EIID defined in SEQ ID NO:167 Man A polynucleotide encoding a protein, in particular a polynucleotide as defined in SEQ ID NO: 166; Man The type is that of strain DSM28255. - EIIDs defined by SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205 Man Polynucleotides encoding proteins, in particular polynucleotides as defined in SEQ ID NOs: 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204. EIIDs as defined in SEQ ID NOs: 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 and 205. Man The protein sequence is 97.3-99.6% identical to SEQ ID NO:167.

[0117] The inventors have identified at least one mutation in the manL gene that, when inserted into the manL gene of a parent lactose-positive, sucrose-negative Streptococcus thermophilus strain (mutated in the glcK gene, the ccpA gene or both the glcK gene and the ccpA gene as defined herein), enables an interesting phenotype within the present invention.

[0118] In some embodiments, the mutation in the manL gene is EIIAB at position 305. Man In some embodiments, the mutation in the manL gene is a substitution of nucleotide G for nucleotide T at position 916 (resulting in a stop codon at position 306). ManThe protein is referred to herein as IIAB Man 305 It is called.

[0119] In some embodiments, the EIIAB Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 112, and b) EIIAB, which has at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 112, and is in particular 305 amino acids long. Man Mutant sequences.

[0120] EIIAB having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO:112. Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences]. Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:112.

[0121] In certain embodiments, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1-30 amino acid substitutions. In certain embodiments, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1-20 amino acid substitutions. In certain embodiments, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1-15 amino acid substitutions. In certain embodiments, EIIAB Man The variant sequence differs from SEQ ID NO: 112 by 1-10 amino acid substitutions. In certain embodiments, EIIAB ManThe variant sequence differs from SEQ ID NO: 112 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIIAB sequence of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention Man The protein sequence is selected from the group consisting of SEQ ID NOs: 112-128.

[0122] In some embodiments, the manL gene carried by the Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NO:112 and any EIIAB gene having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO:112 as defined herein. Man EIIAB selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 113 to 128) Man In some embodiments, the manL gene carried by a Streptococcus thermophilus strain of the invention is as defined in SEQ ID NO:111.

[0123] The inventors have identified at least one mutation in the manM gene that, when inserted into the manM gene of original lactose-positive, sucrose-negative Streptococcus thermophilus strains (mutated in the glcK gene, the ccpA gene or both the glcK gene and the ccpA gene as defined herein), enables several interesting properties of these strains.

[0124] In some embodiments, the mutation in the manM gene is an EIIC at position 208. ManIn some embodiments, the mutation in the manM gene is a substitution of nucleotide G for nucleotide T at position 625 (resulting in a stop codon at position 209). Man Proteins are referred to herein as EIIC Man208 It is called.

[0125] In some embodiments, the EIIC truncated at position 208 Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 158, and b) an EIIC having at least 90% similarity or identity to SEQ ID NO: 158, in particular an EIIC having a length of 208 amino acids; Man Mutant sequences.

[0126] EIIC with at least 90% similarity or identity to SEQ ID NO: 158 Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences]. In certain embodiments, the EIIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:158.

[0127] In certain embodiments, the EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1-30 amino acid substitutions. In certain embodiments, the EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1-20 amino acid substitutions. In certain embodiments, the EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1-15 amino acid substitutions. In certain embodiments, the EIIC Man The variant sequence differs from SEQ ID NO: 158 by 1-10 amino acid substitutions. In certain embodiments, the EIIC ManThe variant sequence differs from SEQ ID NO: 158 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIIC of the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention Man The protein sequence is selected from the group consisting of SEQ ID NOs: 158-165.

[0128] In some embodiments, the manM gene carried by the Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NO: 158 and any EIICs whose sequence has at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 158 as defined herein. Man EIIC selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 159 to 165) Man In some embodiments, the manM gene carried by a Streptococcus thermophilus strain of the invention is as defined in SEQ ID NO:157.

[0129] In some embodiments, the mutation in the manM gene generates a frameshift in manM and is represented by SEQ ID NO: 130 (manM 12997 The insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 129 results in a stop codon at position 180 of the resulting Streptococcus thermophilus IIC truncated at position 179. Man Proteins are referred to herein as IIC Man179 It is called.

[0130] In some embodiments, the IIC truncated at position 179 Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 223, and b) IIC having at least 90% similarity or identity to SEQ ID NO: 223, in particular an IIC having a length of 179 amino acids Man Mutant sequences.

[0131] IIC having at least 90% similarity or identity to SEQ ID NO: 223 Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portions of the sequences]. In certain embodiments, the IIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:223.

[0132] In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1-30 amino acid substitutions. In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1-20 amino acid substitutions. In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1-15 amino acid substitutions. In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1-10 amino acid substitutions. In certain embodiments, IIC Man The variant sequence differs from SEQ ID NO: 223 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. In some embodiments, the IIC of lactose-positive Streptococcus thermophilus strains used according to the invention Man The sequence of the protein is selected from the group consisting of SEQ ID NO:223.

[0133] In some embodiments, the manM gene carried by the Streptococcus thermophilus strain used according to the invention is selected from the group consisting of SEQ ID NO: 223 and any IIC sequences having at least about 90%, such as at least 95%, similarity or identity to SEQ ID NO: 223 as defined herein.Man IIC selected from the group consisting of mutant sequences Man Encodes proteins.

[0134] The present inventors have identified at least one mutation in the manN gene that, when inserted into the manN gene of the original lactose-positive, sucrose-negative Streptococcus thermophilus strains (mutated in the glcK gene, the ccpA gene or both the glcK gene and the ccpA gene as defined herein), enables several interesting properties of these strains of Streptococcus thermophilus.

[0135] In some embodiments, the mutation in the manN gene is an EIID Man In some embodiments, the mutation in the manN gene is an insertion of a nucleotide A in a stretch of five nucleotide A at positions 37-41 (resulting in a stretch of six nucleotide A, a frameshift of the open reading frame and an EIID at position 28). Man This Streptococcus thermophilus EIID truncated at position 28 Man Proteins are referred to herein as IIDs. Man 28 It is called.

[0136] In some embodiments, the EIID is truncated at position 28. Man The protein sequence is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 207, and b) an EIID having at least 90% similarity or identity to SEQ ID NO: 207, in particular an EIID having a length of 28 amino acids; Man Mutant sequences.

[0137] EIIDs having at least 90% similarity or identity to SEQ ID NO: 207Man For the definition of variants, similarity or identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e., the number of similar or identical amino acid residues in the aligned portion of the sequences]. In certain embodiments, the EIIC Man The variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% similarity or identity to SEQ ID NO:207.

[0138] In certain embodiments, the EIID Man The variant sequence differs from SEQ ID NO: 207 by 1-10 amino acid substitutions. Man The variant sequence differs from SEQ ID NO: 207 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the EIID of the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is Man The protein sequence is selected from the group consisting of SEQ ID NOs: 207-211.

[0139] In some embodiments, the manN gene carried by the Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NO:207 and any EIID gene having at least 90% similarity or identity to SEQ ID NO:207 as defined herein. Man EIID selected from the group consisting of mutant sequences (particularly SEQ ID NOs: 208 to 211) Man In some embodiments, the manN gene carried by a Streptococcus thermophilus strain of the invention is as defined in SEQ ID NO:206.

[0140] Regardless of the mutation in the glcK gene as defined herein and the mutation in the ccpA gene as defined herein (present alone or in combination in the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention), at least one gene encoding a protein of the mannose-glucose specific PTS is mutated as defined herein. Regardless of the embodiment, the invention encompasses lactose-positive, sucrose-negative Streptococcus thermophilus strains carrying a mutation in one, two or three genes selected from the group consisting of the manL gene, the manM gene and the manN gene. In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention carry a mutation in manL. In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention carry a mutation in manM. In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention possesses a mutation in manN. In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention possesses a mutation in manL and a mutation in manM. In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention possesses a mutation in manL and a mutation in manN. In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention possesses a mutation in manM and a mutation in manN.In some embodiments, a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention harbors a mutation in manL, a mutation in manM, and a mutation in manN.

[0141] Any method can be used to identify mutations in genes encoding proteins of a mannose-glucose specific PTS, particularly in the manL gene, manM gene or manN gene suitable within the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention.

[0142] By way of example, to identify suitable mutations in the manL, manM or manN genes, one skilled in the art can proceed by the following method: a) providing strain DSM32587, which is mutated in its glcK gene; b) carrying out mutagenesis, for example by random or directed mutagenesis, on the manL, manM or manN gene of the strain of a) to obtain a manL, manM or manN gene whose sequence differs from the sequence of the manL, manM or manN gene of DSM 32587, thereby obtaining a man mutant DSM 32587 strain; c) pH STOP To determine whether the phenotype has been achieved.

[0143] Alternatively, the skilled person can proceed by the following method: a) a mutated ccpA gene (ccpA Δ1A114-120 ), as used herein, DGCC7710-ccpA Δ1A114-120 providing a parent strain, such as strain DSM28255, b) performing mutagenesis, e.g., by random or directed mutagenesis, on the manL, manM or manN genes of the strain of a) to determine whether the sequence is DGCC7710-ccpAΔ1A114-120 and obtaining a manL, manM, or manN gene different from the sequence of the manL, manM, or manN gene of the strain DGCC7710-ccpA. Δ1A114-120 Obtaining shares, c) pH STOP To determine whether the phenotype has been achieved.

[0144] Once identified, the mutated manL, manM or manN gene according to the present invention can be introduced in place of manL, manM or manN of a lactose-positive, sucrose-negative Streptococcus thermophilus strain to obtain a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention.

[0145] Mutations in the ccpA gene This section describes mutations in the ccpA gene that may be used in combination with a mutation in a gene encoding a protein of the mannose-glucose specific PTS defined herein, or in combination with a mutation in a gene encoding a protein of the mannose-glucose specific PTS defined herein and a mutation in the glcK gene defined herein in the context of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention.

[0146] In a lactose-positive, sucrose-negative Streptococcus thermophilus strain, when combined with a mutated gene encoding a protein of the mannose-glucose specific PTS as defined herein, or when combined with both a mutated glcK gene as defined herein and a mutated gene encoding a protein of the mannose-glucose specific PTS as defined herein, the strain is used to ferment milk, thereby increasing the pH STOP Any mutation in the ccpA gene is suitable as long as the phenotype is obtained.

[0147] To determine the ratio of β-galactosidase activity to glucokinase activity in strains of the invention, a β-galactosidase activity assay as described herein and a glucokinase activity assay as described herein are used.

[0148] β-galactosidase activity assay: During milk fermentation, lactose contained in milk (as the main carbohydrate source in milk) is transferred to a Streptococcus thermophilus strain. The intracellular lactose is then cleaved into glucose and galactose by the β-galactosidase enzyme (so that 1 mole of lactose gives 1 mole of glucose and 1 mole of galactose), this activity is also called β-galactosidase activity.

[0149] β-galactosidase activity in a Streptococcus thermophilus strain of the invention is assayed by a β-galactosidase activity assay (performed using a Streptococcus thermophilus strain of the invention).

[0150] A fresh overnight culture of the Streptococcus thermophilus strain to be assayed in M17 containing 30 g / L lactose is obtained and used at 1% (vol / vol) to inoculate 10 ml of fresh M17 30 g / L lactose. After 3 h of growth on M17 + 30 g / L lactose at 42°C, the cells are harvested by centrifugation (6000 g, 10 min, 4°C), washed with 1.5 ml of cold lysis buffer (KPO4 0.1 M) and resuspended in 300 μl of cold lysis buffer. The EDTA-free protease inhibitor "cOmplete™" (Roche, supplier reference number 04693132001) is added to the lysis buffer as described by the supplier. Cells are disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μl of resuspended cells and shaking for 6 min at a frequency of 30 cycles / s in a MM200 vibration mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4 °C) and the supernatant was transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content is determined using the FLUKA Protein Quantification Kit-Rapid (ref 51254). β-galactosidase activity in cell extracts is determined spectrophotometrically by monitoring the hydrolysis of O-nitro-phenol-β-galactoside (ONPG) to galactose and O-nitro-phenol (ONP). 20 μL of bacterial extract is mixed with 135 μL of reaction buffer (NaPO4 0.1M + KCl 0.01M + MgSO4 0.001M + ONPG 3mM + β-mercaptoethanol 60mM, pH=6). ONP production results in a yellow color in the tube. When this color appears, the reaction is stopped by adding 250 μL of stop buffer (Na2CO3 1M). The optical density at 420 nm is measured using a Synergy HT multidetection microplate reader (BIO-TEK). One unit of galactosidase corresponds to the amount of enzyme that catalyzes the production of 1 μmole of ONP per minute under the assay conditions. β-galactosidase activity is calculated as follows: β-galactosidase activity (U / g of total protein extract) = dOD × V / [dt × l × ε × Qprot] (In the formula, - dOD is the variation of optical density (OD) at 420 nm between the blank and the test sample; - V is the volume of the reaction in which the optical density is measured (here 250 μL), - dt = the duration in minutes between the addition of 20 μL of bacterial extract and the addition of 250 μL of stop buffer, - l = optical path length (here 0.73 cm), - ε = molar extinction coefficient of ONP (here 4500 cm 2 / μmol), - Qprot = amount of protein in the cuvette (g) Measurements were performed at least in triplicate for each sample, and the β-galactosidase specific activity values ​​reported herein under the β-galactosidase activity assays presented herein are the average of three independent experiments.

[0151] Glucokinase activity assay: Glucokinase activity in a Streptococcus thermophilus strain of the invention is assayed as follows using a Streptococcus thermophilus strain of the invention for ratio determination.

[0152] A fresh overnight culture of the Streptococcus thermophilus strain to be assayed in M17 containing 30 g / L lactose is obtained and used at 1% (vol / vol) to inoculate 10 ml of fresh M17 30 g / L lactose. After 3 h of growth on M17 + 30 g / L lactose at 42°C, the cells are harvested by centrifugation (6000 g, 10 min, 4°C), washed with 1.5 ml of cold GLCK buffer (5 mM MgCl2, 10 mM K2HPO4 / KH2PO4 [pH 7.2]) and resuspended in 300 μl of cold GLCK buffer. The EDTA-free protease inhibitor "cOmplete™" (Roche, supplier reference number 04693132001) is added to the GLCK buffer as described by the supplier. Cells are disrupted by adding 100 mg of glass beads (150-212 μm, Sigma G1145) to 250 μl of resuspended cells and shaking for 6 min at a frequency of 30 cycles / s in a MM200 vibration mill (Retsch, Haan, Germany). Cell debris and glass beads were removed by centrifugation (14000 g, 15 min, 4 °C) and the supernatant was transferred to a clean 1.5 mL centrifuge tube kept on ice. Total protein content is determined using the FLUKA Protein Quantification Kit-Rapid (ref 51254). Glucokinase activity in cell extracts is determined spectrophotometrically by the glucose-6-phosphate dehydrogenase (G-6PDH, EC 1.1.1.49):NADPH binding assay (Porter et al., 1982), essentially as described by Pool et al (2006). Each sample (5, 10 and 20 μL) was added to a final volume of 250 μL of assay buffer (10 mM K2HPO4 / KH2PO4 [pH 7.2], 5 mM MgCl2, 1 mM ATP, 20 mM glucose, 1 mM NADP, 1 U G-6PDH) and the mixture was left for 5 min at 30° C. The optical density at 340 nm was measured for 5 min using a Synergy HT multidetection microplate reader (BIO-TEK).One unit of glucokinase corresponds to the amount of enzyme that catalyzes the phosphorylation of 1 μmole of D-glucose to D-glucose 6-phosphate per minute under assay conditions. Glucokinase activity is calculated as follows: Glucokinase activity (U / g of total protein extract) = dOD x V / [dt x l x ε x Qprot] (In the formula, - dOD is the variation of optical density (OD) at 340 nm; - V is the volume of the reaction (here 250 μL), dt = measurement time (min), l = optical path length (here 0.73 cm), ε = molar extinction coefficient of NADPH; H + (Here, 6220 cm2 / μmol) Qprot = amount of protein in the cuvette (g) Measurements were performed in triplicate for each sample, and the glucokinase specific activity values ​​presented herein under Glucokinase Activity Assay are the average of three independent experiments.

[0153] In some embodiments, the ccpA gene mutation is not a mutation that results in a knockout (ie, complete disruption) of the gene.

[0154] In some embodiments, the ccpA gene mutation is a mutation in the coding sequence of the ccpA gene, in particular in the first 270 nucleotides of the coding sequence of the ccpA gene. In some embodiments, the mutation is a mutation selected from the group consisting of: a) a nonsense mutation located at nucleotide 1 to nucleotide 270 of the coding sequence of the ccpA gene (i.e. resulting in a stop codon), and b) a mutation located in the first quarter of the coding sequence of the ccpA gene (i.e., nucleotide 1 to nucleotide 250) that results in a frameshift in the open reading frame of the ccpA gene.

[0155] In some embodiments, the mutation resulting in a frameshift in the open reading frame of the ccpA gene is located in the coding sequence of the ccpA gene from nucleotide 50 to nucleotide 200. In some embodiments, the mutation resulting in a frameshift in the open reading frame of the ccpA gene is located in the coding sequence of the ccpA gene from nucleotide 100 to nucleotide 150. Regardless of the location of the mutation resulting in a frameshift, the mutation is selected from the group consisting of a deletion, an insertion, or a deletion / insertion (all of which are not a multiple of 3).

[0156] Two Streptococcus thermophilus strains may differ by the sequence of their respective ccpA genes, but this does not necessarily mean that one of these two ccpA genes is mutated within the meaning of the present invention. Indeed, what is not considered as a mutation of the ccpA gene within the present invention are mutations at the nucleotide level that result in a change at the protein level.

[0157] Non-limiting examples of ccpA genes that are not considered to be mutated within the meaning of the present invention are: - a polynucleotide defined in SEQ ID NO: 65 (ccpA type ST1), said ccpA type being that of the strain DSM28255, - a polynucleotide defined in SEQ ID NO: 66 (ccpA type ST2), which has 99.8% identity with SEQ ID NO: 65; - a polynucleotide defined in SEQ ID NO: 67 (ccpA type ST3), which has 99.8% identity with SEQ ID NO: 65; - a polynucleotide defined in SEQ ID NO: 68 (ccpA type ST4), which has 99.7% identity to SEQ ID NO: 65; - a polynucleotide defined in SEQ ID NO: 69 (ccpA type ST5), which has 99.8% identity with SEQ ID NO: 65, and - the polynucleotide defined in SEQ ID NO: 70 (ccpA type ST6), which has 99.7% identity with SEQ ID NO: 65.

[0158] The present invention may, in some embodiments, relate to a lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying a mutation in the ccpA gene selected from the group consisting of a nonsense mutation located between nucleotides 1 and 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, resulting in a frameshift of the open reading frame of the ccpA gene.

[0159] In some embodiments, the mutation in the ccpA gene is a deletion of nucleotide A in a stretch of seven nucleotide A from positions 114 to 120 (resulting in a frameshift in the open reading frame of the ccpA gene). Such a Streptococcus thermophilus mutant ccpA gene is referred to herein as ccpA Δ1A114-120 It is called.

[0160] In some embodiments, the sequence of the ccpA gene having a stop codon at codon 66 is selected from the group consisting of: a) the sequence defined in SEQ ID NO: 71, and b) A ccpA mutant sequence having at least 90% identity with SEQ ID NO: 71. A ccpA mutant as defined herein carries a mutation as defined above, i.e. selected from the group consisting of a nonsense mutation located from nucleotide 1 to nucleotide 270 of the coding sequence of the ccpA gene and a mutation located in the first quarter of the coding sequence of ccpA, resulting in a frameshift of the open reading frame of the ccpA gene.

[0161] For the definition of a ccpA variant having at least 90% identity to SEQ ID NO:71, identity is calculated herein over the entire length of the two sequences after optimal alignment [i.e. the number of identical nucleotides in the aligned portions of the sequences]. In certain embodiments, the ccpA variant sequence has at least 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:71. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1-30 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1-20 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1-15 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1-10 nucleotide substitutions. In certain embodiments, the ccpA variant sequence differs from SEQ ID NO:71 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide substitutions. In some embodiments, the sequence of the ccpA gene of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is selected from the group consisting of SEQ ID NOs: 71, 72, 73, 74, 75, and 76.

[0162] Those skilled in the art are provided with guidance as to how to identify mutations in the ccpA gene other than those specifically disclosed in this section of the application.

[0163] Thus, the skilled person can proceed in the following way: a) the manL gene is a mutated manL gene as defined in SEQ ID NO: 111 (IIAB Man 305 The manL gene encoding the DGCC7710-IIAB protein is substituted for the DGCC7710-IIAB gene described herein. Man 305 providing a parent strain, such as strain DSM28255, b) mutagenesis of the ccpA gene of the strain of a) by, for example, random or directed mutagenesis, to identify the sequence DGCC7710-IIAB Man 305 The ccpA gene sequence of the strain was obtained by the ccpA mutation DGCC7710-IIAB. Man 305 To obtain shares.

[0164] Alternatively, the skilled person can proceed by the following method: a) a mutated manM gene (EIIC) as defined in SEQ ID NO: 157; Man 208 The manM gene encoding the .DGCC7710-IIC protein is replaced by the DGCC7710-IIC gene. Man 208 providing a parent strain, such as strain DSM28255, b) mutagenesis of the ccpA gene of the strain of a) is carried out, for example by random or directed mutagenesis, to identify the sequence DGCC7710-IIC Man 208 The ccpA gene sequence of the strain was obtained by the ccpA mutation DGCC7710-IIC Man 208 To obtain shares.

[0165] Alternatively, the skilled person can proceed by the following method: a) the manM gene is replaced by a mutated manM gene, the mutation resulting in a stop codon at position 180 of SEQ ID NO: 222, for example by the insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene; EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), providing parent strains such as strain DSM28255, b) mutagenesis of the ccpA gene of the strain of a) is carried out, for example by random or directed mutagenesis, to identify the sequence DGCC7710-IIC Man 208 The ccpA gene sequence of the strain was obtained by the ccpA mutation DGCC7710-IIC Man 208 To obtain shares.

[0166] The skilled person can also proceed in the following way: a) a mutated manN gene, the manN gene being defined in SEQ ID NO: 206 (IID Man 28 The manN gene encoding the . Man 28 providing a parent strain, such as strain DSM28255, b) mutagenesis of the ccpA gene of the strain of a) is carried out, for example by random or directed mutagenesis, to identify the sequence DGCC7710-IID Man 28 The ccpA gene sequence of the strain was obtained by the ccpA mutation DGCC7710-IID Man 28 To obtain shares.

[0167] Once identified, the mutated ccpA gene identified herein can be introduced in place of the ccpA gene of a lactose-positive, sucrose-negative Streptococcus thermophilus strain to obtain a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention.

[0168] Further characterization of the strains of the present invention The inventors have shown that a strain carrying a mutation in a gene encoding a protein of the mannose-glucose specific PTS and carrying a mutation in the glcK gene and / or in the ccpA gene, when used to ferment milk (both normal milk and milk with added sucrose), allows: 1) obtaining low-lactose fermented milk, and / or 2) To obtain fermented milk that does not undergo post-acidification even when stored at fermentation temperatures.

[0169] In some embodiments, the lactose positive, sucrose negative Streptococcus thermophilus strain of the present invention is characterized by the fact that the strain, when used to ferment milk, results in a low-lactose fermented milk.

[0170] In some embodiments, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention is characterized by the fact that the strain results in fermented milk that does not undergo post-acidification when stored at fermentation temperatures.

[0171] In some embodiments, the lactose positive, sucrose negative Streptococcus thermophilus strain of the invention is characterized by the fact that the strain results in a low-lactose fermented milk that does not undergo post-acidification when stored at fermentation temperatures (when used to ferment milk).

[0172] The expression "low-lactose fermented milk" means fermented milk having a residual amount of lactose in the fermented milk of less than 60 mM, less than 50 mM, less than 45 mM, less than 40 mM, less than 35 mM or less than 30 mM at the end of the fermentation described in Example 5. It is worth noting that the amount of lactose in the milk, measured according to Example 6, is about 50 g / L before fermentation.

[0173] The expression "not undergoing post-acidification" refers to a specific pH value at which, when inoculated with a strain of the invention and fermented as described in Example 5, the pH drops significantly to a value at which the rate of acidification is less than 0.1 mUpH / min (i.e. pH STOP value) and STOP The value is between 4.4 and 5.3, and optionally the gradient from pH 6 to pH 5.5 is at least -0.008 UpH / min.

[0174] As used herein, pH STOP The term "phenotype" refers to a strain of Streptococcus thermophilus that, when used in the fermentation of milk as described in Example 5, produces a milk product that is "not subject to post-acidification" as defined herein.

[0175] The absence of post-acidification is therefore characterized by the fact that the pH of the fermented milk stops at 4.4 to 5.3. The pH is said to have stopped (pH STOP "Being certain" means that pH STOP This means that after the acidification rate is reached, the rate of acidification remains below 0.1 mUpH / min for the remainder of the fermentation as described in Example 5 (i.e. up to 24 hours at the fermentation temperature).

[0176] In some embodiments, the pH obtained using the strains of the invention STOP In some embodiments, the pH obtained using the strains of the present invention is 4.7 to 5.2. STOP In some embodiments, the pH obtained using the strains of the present invention is 4.8 to 5.1. STOP is between a minimum value selected from the group consisting of 4.4, 4.5, 4.6, 4.7, and 4.8 and a maximum value selected from the group consisting of 5.1, 5.2, and 5.3.

[0177] In some embodiments, the fermented milk that is not subjected to post-acidification is also characterized by a slope between pH 6 and pH 5.5. The slope represents the inverse of the rate (how fast the acidification occurs). In some embodiments, the slope is at least -0.009 UpH / min. In some embodiments, the slope is at least -0.01 UpH / min.

[0178] Compositions, methods and uses with lactose-positive, sucrose-negative Streptococcus thermophilus strains of the present invention The present invention also relates to a bacterial composition comprising or consisting of at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention. In a particular embodiment, the bacterial composition is a pure culture, i.e. comprising or consisting of a single bacterial strain. In another embodiment, the bacterial composition is a mixed culture, i.e. comprising or consisting of a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention and at least one other bacterial strain. "At least" (with respect to strains or bacteria) means one or more, in particular one, two, three, four or five strains.

[0179] Thus, in some embodiments, the bacterial composition of the invention comprises or consists of at least one lactic acid bacterium of a species selected from the group consisting of lactose positive, sucrose negative Streptococcus thermophilus strains of the invention, as well as Lactococcus species, Streptococcus species, Lactobacillus species including Lactobacillus acidophilus, Enterococcus species, Pediococcus species, Leuconostoc species, Bifidobacterium species and Oenococcus species, or any combination thereof. Lactococcus species include Lactobacillus acidophilus and Lactococcus lactis, including Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar diacetylactis. Bifidobacterium species include Bifidobacterium animalis, especially Bifidobacterium animalis subsp. lactis.Other lactic acid bacteria species include Leuconostoc species, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus helveticus.

[0180] In some embodiments, the bacterial composition comprises or consists of at least one Streptococcus thermophilus strain different from the lactose-positive, sucrose-negative Streptococcus thermophilus strains of the invention and the S. thermophilus strains of the invention, and / or at least one strain of Lactobacillus species, and / or any combination thereof. In a particular embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, one or several strains of Lactobacillus delbrueckii subsp. bulgaricus and / or one or several strains of Lactobacillus helveticus species and / or any combination thereof, optionally with at least one Streptococcus thermophilus strain different from the S. thermophilus strain of the invention. In a particular embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, at least one strain of the Streptococcus thermophilus species different from the S. thermophilus strain of the invention, and a strain of Lactobacillus delbrueckii species subsp. bulgaricus. In another particular embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention and a strain of Lactobacillus delbrueckii species subsp. bulgaricus.

[0181] In some embodiments, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, Lactococcus lactis subsp. lactis and / or Lactococcus lactis subsp. cremoris.

[0182] In certain embodiments of any bacterial composition defined herein, either as a pure culture or a mixed culture, the bacterial composition further comprises at least one probiotic strain, such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paracasei, or Lactobacillus casei.

[0183] In a particular embodiment, the bacterial composition is in a frozen, dried or lyophilized liquid or solid format, in the form of a pellet or frozen pellet, or in the form of a powder or dry powder, either as a pure culture or mixed culture as defined above. In a particular embodiment, the bacterial composition of the invention is in a frozen format or in the form of a pellet or frozen pellet, particularly contained in one or more boxes or sachets. In another embodiment, the bacterial composition defined herein is in the form of a powder, such as a dry powder or lyophilized powder, particularly contained in one or more boxes or sachets.

[0184] In a particular embodiment, the bacterial composition of the invention, either as a pure culture or mixed culture as defined above, and in any format (frozen, dried, lyophilized liquid or solid format, pellet or frozen pellet form, or powder or dry powder), contains a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention at a concentration of 10 per gram of bacterial composition. 5 ~10 12 In a particular embodiment, the concentration of lactose positive, sucrose negative Streptococcus thermophilus strains in the bacterial composition of the present invention is in the range of 10 per gram of bacterial composition. 7 ~10 12 cfu, particularly at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 Or at least 10 11 In a particular embodiment, when in the form of a frozen concentrate or a dry concentrate, the concentration of lactose-positive, sucrose-negative Streptococcus thermophilus strains as a pure culture or as a mixed culture in the bacterial composition is in the range of 10 CFU / g of the frozen concentrate or the dry concentrate. 8 ~10 12 cfu / g, more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 Or at least 10 12 cfu / g of frozen concentrate or dry concentrate.

[0185] The present invention also relates to a method for producing a fermented product, comprising a) inoculating a substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention, and b) fermenting the inoculated substrate to obtain a fermented product. In a particular embodiment, the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the present invention is inoculated as a bacterial composition as defined herein, such as a pure culture or a mixed culture. In some embodiments, the substrate to which the S. thermophilus strain of the present invention or the bacterial composition is added is a milk substrate. By "milk substrate" is meant milk of animal and / or plant origin. In a particular embodiment, the milk substrate is of animal origin, such as cow, goat, sheep, buffalo, zebra, horse, donkey or camel. The milk can be in the natural state, reconstituted milk, skimmed milk or milk supplemented with compounds essential for bacterial growth or for the further processing of the fermented milk. Thus, in a particular embodiment, the present invention also provides a method for producing a fermented milk product, the method comprising: a) inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain or bacterial composition of the present invention; and b) fermenting the inoculated milk substrate to obtain a fermented milk product.

[0186] The present invention also relates to the use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to the invention or a composition according to the invention for producing a fermented milk product.

[0187] The present invention also relates to a fermented milk product obtained using the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention or the bacterial composition of the invention, in particular obtained or obtainable by the method of the invention. Thus, the present invention relates to a fermented milk product comprising the lactose-positive, sucrose-negative Streptococcus thermophilus strain of the invention. In a particular embodiment, the fermented milk food product of the invention is fresh fermented milk.

[0188] Various features and embodiments of the invention will now be described by way of non-limiting examples.

[0189] example Assays used in the following examples 1. Test A: The S. thermophilus strains to be tested were cultured overnight at 37° C. in 10 mL of M17 medium (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4° C.) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. One hundred microliters of serial 10-fold dilutions of the cell suspension were used to plate on the surface of agar plates of M17 medium containing either 30 g / L lactose or sucrose. After 24 h of incubation at 37° C. under anaerobic conditions (in a closed vessel using the Anaerocult® A system (Merck, Darmstadt, Germany)), colonies grown on lactose and on sucrose were counted. The ratio of colony formation (cfu) on sucrose (sucrose positive colonies) compared to lactose (lactose positive colonies) was calculated.

[0190] 2. Test B: The S. thermophilus strains to be tested were cultured overnight at 37° C. in 10 mL of M17 medium (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4° C.) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. Two hundred microliters of the cell suspension was used to inoculate 10 mL of M17 medium containing no sugar or 30 g / L sugar (either lactose or glucose). Two hundred microliters of inoculation medium from each of the three conditions (sugar-free, lactose or glucose) was distributed in triplicate into a 96-well microtiter plate and 100 μL of paraffin oil was added to the top of each well. The microtiter plate was incubated at 37° C. Bacterial growth was monitored for 24 h (readings every 15 min) using a SpectraMax ID5 (Molecular Devices, San Jose) at 600 nm with 5 s of high speed orbital shaking before each reading. To assess the ability of strains to grow on glucose as the sole source of carbohydrate, the rate of growth on glucose (μ グルコース To ensure a proper comparison between strains while limiting the effect of inoculation rate, this value was calculated at the time point corresponding to the maximum growth rate of the strain on lactose (μmax ラクトース ). Furthermore, μmax ラクトース μ for グルコース The ratio corresponding to was determined.

[0191] 3. Test C: The S. thermophilus strains to be tested were grown overnight at 37°C in 10 mL of M17 (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4°C) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. Two hundred microliters of the cell suspension was used to inoculate 10 mL of M17 medium containing either 30 g / L lactose or sucrose. After 24 h of incubation at 37°C, the OD of the cultures was measured using a Biochrom WPA Biowave DNA spectrophotometer (Biochrom Ltd., Cambridge, UK). 600 were measured at 600 nm and compared.

[0192] Numbered embodiments of the present invention: 1. A lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations, wherein the one or more mutations are 1) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or transport sucrose; and i) one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene; ii) optionally, further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to the sucrose-negative phenotype; and one or more mutations including at least 2) Optionally, one or more further mutations affecting a glucose transporter, e.g., glcU and / or its expression, which restore or improve glucose consumption of the strain upon growth on glucose as the sole source of carbohydrate. A lactose positive, sucrose negative Streptococcus thermophilus strain selected from the group consisting of:

[0193] 2. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to embodiment 1, which is glucose positive or glucose slow growing.

[0194] 3. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to embodiment 1 or 2, having one or more mutations in the sucrose regulon in one or more of the scrA and scrB genes.

[0195] 4. Mutations in one or more genes of the sucrose regulon, such as in the scrA gene, effectively disrupt gene function, e.g., EIIABC スクロース A frameshift in the open reading frame of the protein-coding gene results in the truncated EIIABC encoded by the scrA gene. スクロース 4. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 3, which is mutated by an insertion or deletion that results in translation of a protein, such as an insertion or deletion of a nucleotide in the open reading frame (ORF) of the scrA gene, such as at or around position 133 of SEQ ID NO: 212.

[0196] 5. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 4, having a mutation in the promoter region regulating the expression of any one of the scrA, scrB and / or scrR genes.

[0197] 6. The mutation may be a SNP in the promoter region of the scrA and / or scrB genes, such as a SNP in the promoter region of the scrB gene, such as a mutation near the -10 and -35 sequences of the promoter, such as a T to G mutation corresponding to position 38 of SEQ ID NO: 213 upstream of the scrB promoter, such as a mutation comprising the entire sequence identified by SEQ ID NO: 213 or a variant sequence that is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 6. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 5, wherein the lactose-positive, sucrose-negative Streptococcus thermophilus strain is a lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 5.

[0198] 7. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 6, having a mutation affecting a glucose transporter, which mutation restores or improves glucose consumption of said strain when grown on glucose as the sole source of carbohydrate, e.g. completely restoring growth on glucose compared to that of the lactose-positive and sucrose-positive Streptococcus thermophilus parent strain.

[0199] 8. The mutation affecting the glucose transporter affects glcU and / or its expression, for example located within the promoter region of glcU, e.g. a) An insertion of a T nucleotide in the polyT region at a position corresponding to position −75 of the glcU promoter identified as SEQ ID NO: 216 (pglcU IN_T ), such as a mutation containing a T nucleotide at position 102 of SEQ ID NO: 217, such as a mutation comprising the entire sequence identified by SEQ ID NO: 217 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto; b) Insertion of a C nucleotide in the polyT region at a position corresponding to position −75 of the glcU promoter identified as SEQ ID NO: 216 (pglcU IN_C), such as a mutation containing a C nucleotide at position 102 of SEQ ID NO: 218, such as a mutation comprising the entire sequence identified by SEQ ID NO: 218 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto; c) a mutation from T to C nucleotide in the polyT region at a position corresponding to position −81 of the glcU promoter identified as SEQ ID NO: 216 (pglcU SNP_C ), such as a mutation containing a C nucleotide at position 96 of SEQ ID NO: 219, such as a mutation comprising the entire sequence identified by SEQ ID NO: 219 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto; d) a mutation comprising an insertion of a sequence at a position corresponding to nucleotide positions 110 to 111 of the glcU promoter identified as SEQ ID NO: 216, e.g., an insertion of a sequence identified by nucleotides 1 to 1321 of SEQ ID NO: 221, e.g., the entire sequence identified by SEQ ID NO: 220 or a variant sequence which is at least about 90%, e.g., at least about 95%, identical thereto, or SEQ ID NO: 221 or a variant sequence which is at least about 90%, e.g., at least about 95% identical thereto (pglcU IS ) 8. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 7, wherein the mutation is selected from the group consisting of:

[0200] 9. a) a mutation in the glcK gene encoding glucokinase, which mutation significantly reduces, but does not reduce, the glucokinase activity of the glucokinase in said strain, and / or b) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Mannucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 (resulting in a mutant allele), a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41 9. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of embodiments 1 to 8, further comprising one or more mutations selected from:

[0201] 10. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 9, which is galactose-negative, for example by virtue of a mutation in the glcK gene and / or the ccpA gene, optionally combined with a mutation in one of the genes encoding a protein of the mannose-glucose specific PTS.

[0202] 11. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to embodiments 9-10, having a mutation in the glcK gene encoding glucokinase, the mutation providing for a significantly reduced, but not zero, glucokinase activity in said strain.

[0203] 12. The mutated glcK gene is a) a glucokinase having at position 275 an amino acid which is not glutamic acid, in particular which is not an acidic amino acid, in particular which is lysine; b) at position 275, an amino acid which is not glutamic acid, in particular which is not an acidic amino acid, in particular which is lysine (glcK 378 and having an arginine at position 278 and / or a serine at position 279, c) a glucokinase having at its position 144 an amino acid which is not glycine, in particular is not an aliphatic amino acid, in particular is serine; 12. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of embodiments 9 to 11, wherein the strain encodes a glucokinase selected from the group consisting of:

[0204] 13. The mutant glcK gene encodes glucokinase, the sequence of which is a) the sequence defined in SEQ ID NO: 25, in which the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine, and b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of said glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of said glucokinase) is not glutamic acid, in particular is not an acidic amino acid, in particular is a lysine; c) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is not a glycine, in particular is not an aliphatic amino acid, in particular is a serine; and d) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid of said glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of said glucokinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine. 13. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 9 to 12, selected from the group consisting of:

[0205] 14. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 13, further comprising a mutation in the ccpA gene.

[0206] 15. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 14, further comprising a mutation in a gene encoding a protein of a mannose-glucose specific PTS, such as a gene selected from the group consisting of the manL gene, the manM gene and the manN gene.

[0207] 16. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 14 or 15, wherein the ccpA gene harbors a mutation selected from the group consisting of a nonsense mutation located at nucleotide 1 to 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, resulting in a frameshift of the open reading frame of the ccpA gene.

[0208] 17. The sequence of the mutant ccpA gene is a) the sequence defined in SEQ ID NO: 71, and b) a ccpA variant sequence having at least 90%, such as at least 95%, identity to SEQ ID NO: 71 17. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of embodiment 16, selected from the group consisting of:

[0209] 18. During the 24 hour fermentation, the milk can be fermented as described in Example 5; a) the concentration of lactose remaining in the fermented milk is less than 60 mM, and / or b) The pH of milk is adjusted to a pH that ensures that the rate of acidification is less than 0.1 mUpH / min (pH STOP ) and the pH STOP 18. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of the preceding embodiments, wherein the pH is 4.4 to 5.3, and optionally the gradient from pH 6 to pH 5.5 is at least -0.008 UpH / min.

[0210] 19. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 18, having the ability to over-consume lactose compared to a parent strain not having said one or more mutations, e.g. when fermenting sweet milk as described and assayed according to Example 6, the amount of lactose remaining is less than 100 mM, such as less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 8 mM, 6 mM, 4 mM, 2 mM or 1 mM.

[0211] 20. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 19, having the ability to release an increased amount of glucose compared to a parent strain not having said one or more mutations, e.g., glucose release as described and assayed according to Example 6 is greater than 10 mM, such as greater than 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM or 90 mM.

[0212] 21. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 20, which releases a concentration of glucose, assayed as described in Example 6, that is at least 50 mM, such as at least 60, 70, 80 or 90 mM, or that releases a concentration of glucose that is increased by at least 150% or at least 200%, such as 300%, 400%, 500% compared to the glucose concentration released by the parent strain, both when assayed as described in Example 6.

[0213] 22. A composition comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of a strain of the genus Lactobacillus, such as a Lactobacillus delbrueckii subsp. bulgaricus strain, a strain of the genus Lactococcus, such as a Lactococcus lactis strain or a strain of the genus Bifidobacterium.

[0214] 23. A method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or with the composition according to embodiment 19, and fermenting the inoculated milk to obtain a fermented milk product.

[0215] 24. Use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or the composition according to embodiment 22 for producing a fermented milk product.

[0216] 25. A fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of embodiments 1 to 21 or obtainable by the method according to embodiment 23.

[0217] 26. A Streptococcus thermophilus strain, such as a lactose-positive, sucrose-negative Streptococcus thermophilus strain, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34132, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34134, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34138, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34139, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34140 in the name of DuPont Nutrition Biosciences ApS, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34146, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS under number DSM 34147, and - strain deposited in accordance with the Budapest Treaty on February 16, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS A Streptococcus thermophilus strain selected from the group consisting of:

[0218] 27.- A strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on 14 January 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as strain ST1 or DSM 34172), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as strain ST1 or DSM 34172), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as strain ST1 or DSM 34172), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as strain ST1 or DSM 34172), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity, such as at least 95%, to SEQ ID NO: 71; - a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 according to the Budapest Treaty (herein referred to as strain ST1 or DSM 34172), which has the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcUSNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 having a mutation involving an insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity with SEQ ID NO: 71, at least 95% A strain containing one or more of - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited according to the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence which is at least about 90%, such as at least about 95% identical thereto ( SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as strain ST4 or DSM 28255), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as strain ST4 or DSM 28255), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as strain ST4 or DSM 28255), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity, such as at least 95%, to SEQ ID NO: 71; - a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 28255 according to the Budapest Treaty (herein referred to as the ST4 or DSM 28255 strain), which has the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcUSNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 having a mutation involving an insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity with SEQ ID NO: 71, at least 95% A strain containing one or more of - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglc IN_T , pglc IN_C , pglc IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence of scrA is the truncated EIIABC スクロース212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene. Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; - a strain of Streptococcus thermophilus, such as the strain deposited in accordance with the Budapest Treaty on 18 January 2022 at the Leibniz-Institut DSMZ under the number DSM 34132 in the name of DuPont Nutrition Biosciences ApS (herein referred to as the ST2 or DSM 34132 strain), having the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcUIN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 having a mutation involving an insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41, resulting in a mutant allele; 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain containing one or more of the following: - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB 829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB829 ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), and optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK 378 ), and optionally, the strain is, for example, an EIIC, resulting in a stop codon at position 180 of SEQ ID NO:222, due to an insertion of a single nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene.Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN 13741 a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO: 71; - a strain of Streptococcus thermophilus deposited on 29 September 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), which has the following characteristics: 1) The sequence of scrA is a truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in 829 FS ), 2) The sequence upstream of the start codon of the scrB gene is the corresponding sequence shown in SEQ ID NO: 213 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto (pscrB 829 ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU SNP_C , pglcU IN_T , pglcU IN_C , pglcU IS ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO: 25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK 378 ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO: 223 in the manM gene results in a stop codon at position 180 of SEQ ID NO: 222, EIIC Man Generates a frameshift in the open reading frame of a protein-coding gene (manM 12997 ), and / or SEQ ID NO: 80 (IIAB Man nucleotide G to T substitution at position 916 of SEQ ID NO: 79 of the manL gene resulting in a stop codon at position 306 of the protein (manL 13740 ) and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM 16404 ) and / or the IID at position 28 of SEQ ID NO: 167 Man Generates a frameshift in the manN gene, resulting in a truncated protein (manN13741 having a mutation involving an insertion of one nucleotide A in a stretch of five nucleotide A's at positions 37-41, resulting in a mutant allele; 6) Having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 Strains containing one or more of the following: A lactose positive, sucrose negative Streptococcus thermophilus strain selected from the group consisting of:

[0219] 28. A polynucleotide comprising one or more mutations in one or more genes of the sucrose regulon, wherein the one or more mutations are a) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255, wherein said polynucleotide replaces the corresponding sequence in DSM 28255, b) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132, wherein said polynucleotide replaces the corresponding sequence in DSM 34132; c) a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651, wherein the polynucleotide replaces the corresponding sequence in DSM 33651; d) a strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on February 16, 2022, wherein the polynucleotide replaces the corresponding sequence in DSM 34172. A polynucleotide which provides a significant reduction in the ability to take up and consume any sucrose in a strain selected from the group consisting of:

[0220] 29. A polynucleotide comprising one or more mutations affecting a glucose transporter, wherein the one or more mutations are a) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255, wherein said polynucleotide replaces the corresponding sequence in DSM 28255, b) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132, wherein said polynucleotide replaces the corresponding sequence in DSM 34132; c) a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651, wherein the polynucleotide replaces the corresponding sequence in DSM 33651; d) a strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on February 16, 2022, wherein the polynucleotide replaces the corresponding sequence in DSM 34172. A polynucleotide which restores or improves glucose consumption in a strain selected from the group consisting of:

[0221] 30. The polynucleotide according to any one of embodiments 28 or 29, wherein the one or more mutations are as identified in any one of embodiments 3 to 8.

[0222] 31. A method for selecting a lactose-positive, sucrose-negative Streptococcus thermophilus strain, comprising: 1) A strain of Streptococcus thermophilus, a) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or to transport sucrose, and i) one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene; ii) optionally, further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to the sucrose-negative phenotype; One or more mutations, including at least Providing a strain of Streptococcus thermophilus comprising: 2) screening for strains of Streptococcus thermophilus from step 1) that have a significantly reduced ability to grow on sucrose and transport sucrose; 3) optionally screening, optionally before, simultaneously with or after any one of steps 1 and 2, for Streptococcus thermophilus strains with restored or improved glucose consumption resulting from one or more additional mutations affecting a glucose transporter, e.g. affecting glcU and / or its expression; The method includes:

[0223] 32. The method according to embodiment 31, wherein the strain is as defined in any one of embodiments 1 to 21. EXAMPLES

[0224] Example 1: Identification of mutations in the promoters of the scrA and scrB genes Streptococcus thermophilus has been described as a species capable of growing both on lactose (lactose-positive phenotype) and on sucrose (sucrose-positive phenotype). A proprietary strain collection was screened for strains of S. thermophilus that are unable to grow on sucrose as the sole source of carbohydrate. The S. thermophilus strains to be tested were cultured overnight at 37°C in M17 (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4°C) and the cell pellets were washed twice with tryptone salts (TS; tryptone 1 g / L, NaCl 8.5 g / L) and then resuspended in 10 mL of fresh sugar-free M17 medium. Washed cells were used to inoculate 200 μL of fresh M17 medium containing 30 g / L sugar (either lactose or sucrose) at 1% (v / v) in triplicate in 96-well microtiter plates, and 100 μL of paraffin oil was added to the top of each well. The microtiter plates were incubated at 37°C. Bacterial growth was monitored for 24 h (readings every 15 min) using a SpectraMax ID5 (Molecular Devices, San Jose) at 620 nm with fast orbital shaking for 5 s before each reading. One lactose-positive strain, DGCC8368, was found to be unable to grow on sucrose as the sole source of carbohydrate. The ability to grow on sucrose was determined when the strains reached an OD of >0.5 upon 24 h of incubation at 37°C. 600 was defined as the ability to reach

[0225] Several genes of DGCC8368 known to be involved in sucrose catabolism in S. thermophilus (scrA, scrB, scrK, and scrR genes) were sequenced and compared with the corresponding gene sequences of other S. thermophilus strains. First, we identified a single A nucleotide insertion in the polyA tract at position 133 of the open reading frame (ORF) of scrA in DGCC8368 (Figure 1; SEQ ID NO: 212). 829 FS This insertion results in a frameshift, resulting in a truncated EIIABC スクロース Furthermore, a substitution of nucleotide A with a C nucleotide was identified at position -38 upstream of the start codon of the scrB gene (position 38 of SEQ ID NO: 213). 829 and is located two nucleotides upstream of the putative −10 box (FIG. 1). Neither of these two mutations was found in the scrA gene or scrB promoter sequences of other S. thermophilus strains from the collection or public databases.

[0226] Example 2. scrA on growth of S. thermophilus strains on sucrose as the sole source of carbohydrate 829 FS Impact. The effect of scrA on the ability of S. thermophilus strains to grow on sucrose 829 FSTo evaluate the effect of the mutation, derivatives of S. thermophilus strains ST1 (DSM34172), ST2 (DSM34132) and ST3 (DSM33651) were constructed in which this mutation replaced the native gene allele. To evaluate the ability of the strains to grow on sucrose as the sole source of carbohydrate, the constructs were analyzed using test A. In test A, the S. thermophilus strains to be tested were cultured overnight at 37° C. in 10 mL of M17 medium (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4° C.) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. One hundred microliters of serial 10-fold dilutions of the cell suspension were used to plate on the surface of agar plates of M17 medium containing either 30 g / L lactose or sucrose. After 24 hours of incubation at 37° C. under anaerobic conditions (in a closed container using the Anaerocult® A system (Merck, Darmstadt, Germany)), colonies grown on lactose and sucrose were counted. The ratio of colony formation (cfu) on sucrose (sucrose positive colonies) compared to lactose (lactose positive colonies) was calculated. The results are summarized in Table 1.

[0227] scrA 829 FS The mutants carrying the mutations (ST1-A, ST2-A, and ST3-A) were barely able to grow on sucrose as the sole source of carbohydrate. In test A, the ratio of sucrose-positive colonies to lactose-positive colonies was 10 for the mutants. -6 The ratio of scrA was less than 1, whereas in the parent strain the ratio was close to 1. Globally, this result 829 FSThe mutation was shown to render the S. thermophilus strain sucrose negative, meaning that less than 0.01% of the cfu growing on lactose were able to form colonies on sucrose as the sole source of carbohydrate.

[0228] [Table 1]

[0229] Example 3. Incorporation of scrA to Stabilize the Sucrose-Negative Phenotype of S. thermophilus Strains 829 FS and pscR 829 Combine with. In test A, three scrA strains were grown on sucrose-containing agar plates. 829 FS A low level of colonies was observed from the cultures of the mutants (ST1-A, ST2-A and ST3-A) (see Example 2, Table 1), suggesting instability of the sucrose-negative phenotype. This instability was clearly observable when these mutants were cultured in medium containing sucrose as the only source of carbohydrate (Test C). In Test C, the S. thermophilus strains to be tested were cultured overnight at 37°C in 10 mL of M17 containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100g, 10 min at 4°C) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. Two hundred microliters of the cell suspension was used to inoculate 10 mL of M17 medium containing either 30 g / L lactose or sucrose. After 24 h of incubation at 37°C, the OD of the cultures was measured using a Biochrom WPA Biowave DNA spectrophotometer (Biochrom Ltd., Cambridge, UK). 600 The OD values ​​of the three scrA strains were measured at 600 nm and compared. The results (Table 2) are shown for the parent strains grown in a range of sucrose-containing M17 media with OD values ​​between 2.8 and 6.0. 829 FSThe values ​​measured for the mutants (OD in the range of 3.7–4.6) 600 However, for strains exhibiting a sucrose-negative phenotype, the OD 600 It should be expected that the value will be close to 0, so scrA 829 FS It is likely that the phenotype associated with was unstable.

[0230] Mutation scrA for stabilization of the sucrose-negative phenotype 829 FS and pscR 829 To evaluate the effect of the combination of both and pscrB 829 Mutations replaced the native gene allele in scrA 829 FS Mutant derivatives were constructed. 829 FS Single mutants and scrA 829 FS -pscrB 829 The double mutant parental strains were compared using test C. scrA 829 FS In contrast to what was observed with the single mutants, scrA 829 FS -pscrB 829 OD of double mutant 600 The OD values ​​remained low over the 24 h of incubation. 600 did not reach values ​​above 0.14. This is because scrA 829 FS and pscR 829 The combination of mutations in scrA 829 FS In test A, the scrA mutant was found to be sucrose-negative and stabilise the sucrose-negative phenotype conferred by the mutant. 829 FS -pscrB 829 No colonies were observed for the double mutant, and reversion of the sucrose-negative phenotype was below the detection threshold (<0.5 10 -8 It is also worth noting that

[0231] [Table 2]

[0232] Example 4. pscR 829 scrA with or without mutations 829 FS The mutation impairs the ability of S. thermophilus strains to grow on glucose, and pglcU restores this ability. Strains of the species S. thermophilus have been described that are able to grow naturally on glucose as the sole source of carbohydrate. This ability is due to the pscRb 829 scrA with or without mutations 829 FS A control was performed for the engineered strains with mutations. For this control, test B was performed. In test B, the S. thermophilus strains to be tested were cultured overnight at 37° C. in 10 mL of M17 medium (Thermo Scientific™ Oxoid™, CM0785) containing 30 g / L lactose. Cells from each culture were harvested by centrifugation (2100 g for 10 min at 4° C.) and the cell pellet was resuspended in 10 mL of fresh sugar-free M17 medium. Inoculation medium from 200 microliters of cell suspension was used to inoculate 10 mL of M17 medium containing no sugar or 30 g / L sugar (either lactose or glucose) at 1% (v / v). 200 micrometers of each condition (sugar-free, lactose or glucose) were distributed in triplicate into a 96-well microtiter plate and 100 μL of paraffin oil was added to the top of each well. Microtiter plates were incubated at 37° C. Bacterial growth was monitored for 24 h (readings every 15 min) using a SpectraMax ID5 (Molecular Devices, San Jose) at 600 nm with high speed orbital shaking for 5 s before each reading.

[0233] To evaluate the ability of strains to grow on glucose as the sole source of carbohydrate, the rate of growth on glucose (μ グルコース To ensure a proper comparison between strains while limiting the effect of inoculation rate, this value was calculated at the time point corresponding to the maximum growth rate of the strain on lactose (μmax ラクトース ). Furthermore, μmax ラクトース μ for グルコース The ratios corresponding to μ were determined. The data are summarized in Table 3 and FIG. グルコース / μmax ラクトース A graphical comparison of the ratios is provided for the lactose-positive and glucose-positive parental S. thermophilus strains. Typically, for the lactose-positive and glucose-positive parental S. thermophilus strains, this ratio was above 0.5, indicating that the strains grew well on glucose, even though at a slower rate than on lactose. Surprisingly, the pscB 829 scrA with or without mutations 829 FS The strains carrying the mutations were affected in their ability to grow on glucose compared to their parents. グルコース / μmax ラクトース The ratio values ​​were significantly lower, less than 0.25, at least for scrA 829 FS We showed that the mutation adversely affected the growth of S. thermophilus on glucose: growth on glucose was not completely abolished but was significantly slowed, and we defined this phenotype as "glucose slow growth."

[0234] Spontaneous mutants showing an improved ability to consume glucose likely arose from glucose-slow growing strains when cultivated on glucose as the sole source of carbohydrate (possibly due to the high OD recorded when these were cultivated for 24 h in M17-glucose medium, not shown). 600 (Explains some scrA 829 FS -pscrB 829Cultures of the strains were plated on M17-agar plates containing 30 g / L glucose as the sole source of carbohydrate. Colonies were picked upon incubation at 37°C and their ability to grow on glucose was investigated as described in test B. Several selected clones (ST1-ABU1, ST1-ABU4, ST2-ABU1, ST2-ABU2, ST2-ABU3, ST3-ABU1 and ST3-ABU2) had μ values ​​in test B similar to or higher than those calculated for the ST1, ST2 and ST3 parent strains. グルコース / μmax ラクトース The ratios of β-glucose to β-glucose were shown (Table 3). These clones were further investigated for mutations in their genome. Comparative genomics of the selected clones with their parents allowed the identification of four different mutations (SEQ ID NOs. 217-220), each of which is likely responsible for the reversion of the glucose slow growth phenotype. Mutations were found for all clones located at a locus corresponding to the promoter region upstream of the glcU gene (pglcU). This glcU gene is predicted to code for a non-PTS transporter of glucose. One mutation was a SNP (pglcU SNP_C ), and the two mutations are single nucleotide (same position but different nucleotide) insertions (pglcU IN_T and pglcU IN_C ) and the last one (pglcU IS ) was an IS insertion (see Figure 2).

[0235] To confirm the pglcU mutation responsible for the reversion of the glucose slow phenotype, ST2-AB was transformed with pglcU in the promoter region of glcU. SNP_C Genetically engineered ST2-ABU3 by inserting a mutation ge The strain ST2-ABU3 was generated. ge was compared with ST2-ABU3 in Test B. The growth rates of the two strains in glucose-containing medium were fast (0.477 and 0.480, respectively) and similar, indicating that pglcU SNP_CIt was confirmed that the mutation was responsible for reversion of the glucose slow growth phenotype. With the same goal in mind, the wild-type allele of pglcU from the parent ST2 was used to mutate pglcU. SNP_C To replace the mutation, we performed reverse genetics on ST2-ABU3. wt The growth rates of the two strains in glucose-containing medium were slow (0.161 and 0.081, respectively) and similar, and therefore pglcU SNP_C It was again confirmed that the mutation was responsible for reversion of the glucose slowing phenotype.

[0236] [Table 3]

[0237] Example 5: glcK E275K and manM 12997 Combination with mutations or ccpA 855 and manM 12997 In addition to any of the combinations with the mutations, scrA 829 FS and pscR 829 pH of sweet fresh fermented products using S. thermophilus strains with and without the pglcU mutation STOP . Lactose-positive, galactose-negative Streptococcus thermophilus strains carrying a mutation in the glcK and / or ccpA genes combined with a mutation in one of the genes encoding the proteins of the mannose-glucose specific PTS were shown to be interesting for controlling the final pH of fresh fermented products such as yogurt (WO2019197051). However, this property was significantly affected in fresh fermented products to which sucrose was added to obtain a sweet product.

[0238] scrA in the S. thermophilus genome 829 FS and pscR 829 To evaluate the effect and benefit of the pglcU mutation and the pglcU mutation on the acidification kinetics of normal and sweet milk, the following strains were engineered: scrA 829 FS and pscR 829 ST1-GM with the mutation (glcK of ST1 378 -manM 12997 ST1-GM-AB, a derivative of ST1 double mutant ·glcK 378 and manM 12997 ST1-GM-ABU1, a derivative of ST1-ABU1 with a mutation ·ccpA 855 and manM 12997 ST1-CM-ABU1, a derivative of ST1-ABU1 with a mutation

[0239] The engineered strains and the appropriate control strains (ST1 and ST1-GM) were used to ferment normal and sweet milk as follows: Fresh precultures of the strains to be tested (in reconstituted milk containing 10% (w / v) skim milk powder pasteurized at 120°C for 20 min) were used to inoculate 100 mL of normal milk ("Candia Grand Lait" pasteurized at 90°C for 10 min and diluted with 93% (v / v) water) and 100 mL of sweet milk ("Candia Grand Lait" pasteurized at 90°C for 10 min and supplemented with 7% (w / v) sucrose) at 2% (v / v). The inoculated milk was incubated statically for 24 h in a water bath at 43°C. The acidification properties of the engineered strains were evaluated by recording the pH during milk fermentation. The pH was monitored for 24 h using a CINAC system (Alliance Instruments, France; p Helectrode Mettler 405 DPAS SC, Toledo, Spain). The pH was recorded every 5 or 25 minutes.

[0240] The change in pH over time is shown in Figures 4A1, 4A2, 5A1, 5A2, 6A1, 6A2, 7A1, 7A2, 8A1 and 8A2. The rate from pH 6 to pH 5.5 was calculated as the slope of a linear model estimated from the change in pH as a function of time for pH values ​​from 6 to 5.5. The slope value is the reciprocal of the rate (Table 4). The change in rate as a function of pH is also shown (Figures 4B1, 4B2, 5B1, 5B2, 6B1, 6B2, 7B1, 7B2, 8B1 and 8B2). The rate is determined as the instantaneous derivative of the pH change as a function of time. pH STOP was characterized as the pH value at which the rate reduction became undetectable (<0.1 mUpH / min) (Table 4). STOP The time corresponding to TpH STOP ) was also determined (Table 4).

[0241] The results are shown in Table 4. The parent strain (ST1) produced a pH of less than 4.60 in both normal and sweet milk under the conditions described above. STOP As expected, glcK 378 -ManM 12997 pH in normal milk fermented with the double mutant (ST1-GM) STOP In contrast, the pH value in sweet milk was above 4.6. STOP The value was less than 4.6 for the parent strain. 829 FS and pscR 829 The engineered strain ST1-GM-AB carrying the mutations produced a high pH of >4.6 in sweet milk. STOP The pH values ​​in normal and sweet milk were also significantly higher than those in ST1-GM-ABU1, because the values ​​were still higher than 4.6 in ST1-GM-ABU1. STOP Similarly, ccpA 855 and manM 12997 In addition to the mutation scrA 829 FS , pscR 829 and the strain carrying the pglcU mutation (ST1-CM-ABU1) produced a high pH above 4.6 in both normal fermented milk and fermented sweet milk. STOP The values ​​were shown.

[0242] Table 4 similarly shows 378 -ManM 12997 TpH within 450 min in normal milk fermented with the double mutant STOP However, the double mutant still acidified sweet milk at 24 hours of incubation. STOP The value exceeded 1425 min. In contrast, scrA with or without the pglcU mutation 829 FS and pscR 829 For strains carrying the mutation, TpH STOP The values ​​were short (<450 min) for both plain and sweet fermented milk. These data are consistent with those for scrA 829 FS and pscR 829 We also show that neither the addition of the mutation nor the addition of the pglcU mutation had a negative effect on the acidification kinetics (gradient from pH 6 to 5.5) and remained acceptable at industrial levels for producing fermented dairy products.

[0243] In conclusion, scrA 829 FS and pscR 829 and pglcU mutations, glcK 378 -ManM 12997 and ccpA 855 -ManM 12997 The double mutant does not affect the ability to control the final pH in normal fermented milk. 829 FS and pscR 829 The mutation makes it possible to extend this property in milk supplemented with sucrose.

[0244] [Table 4]

[0245] Example 6. glcK E275K and manM 12997Combination with mutations or ccpA 855 and manM 12997 In addition to any of the combinations with the mutations, scrA 829 FS and pscR 829 Improved glucose release and lactose consumption using S. thermophilus with mutations (with or without the pglcU mutation). In addition to controlling the final pH of the fermented milk (see Example 5), lactose-positive, galactose-negative S. thermophilus strains carrying a mutation in the glcK gene and / or the ccpA gene in combination with a mutation in one of the genes encoding the proteins of the mannose-glucose specific PTS have been shown to be interesting for over-consuming lactose and releasing glucose (WO2019197051).

[0246] scrA for lactose overconsumption and glucose release 829 FS and pscR 829To evaluate the effect of the mutations as well as the pglcU mutation, the engineered strains ST1-GM-AB, ST1-GM-ABU1 and ST1-CM-ABU1 described in Example 5 were further investigated. These strains and the appropriate control strains (ST1-GM and ST1) were used to ferment sweet milk as described in Example 5. Samples were taken before incubation and at 24 hours of incubation. Biochemical analysis was performed as follows: 5 g of each sample was diluted with 25 g of 0.025N H2SO4 and then centrifuged at 4600 rpm for 10 minutes at 4°C. The supernatant was filtered through a 0.2 μm nylon filter (Phenomenex, Germany, Aschaffenburg) directly into a 2 ml HPLC vial. Glucose and lactose were separated by high performance liquid chromatography (Agilent 1200 HPLC) equipped with a refractive index detector using an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories Inc.) at 35 °C with 12.5 mM H2SO4 and 0.6 ml min as eluents. -1 The results were analyzed using the Chemstation reprocessing software (Agilent).

[0247] The results are shown in Table 5. The parent strain (ST1) consumed limited amounts of lactose (less than 50% considering the initial concentration in sweet milk of 145 mM) and no measurable amount of glucose was detected from carbohydrate hydrolysis. As expected and as shown previously (WO2019197051), glcK 378 and ManM 12997 The double mutant (ST1-GM) carrying both mutations was able to consume more lactose (only 44 mM remained) and release up to 44 mM glucose. 829 FS and pscR 829Introducing the mutations (producing ST1-GM-AB) resulted in a strain that consumed more lactose (16 mM residual lactose) with the consequence of releasing more glucose (71 mM). Finally, the introduction of a mutation in the glcU promoter region of the ST1-GM-AB genome allowed it to hydrolyze the entire lactose present in milk within 24 hours of fermentation and, more surprisingly (the pglcU mutation increased the scrA 829 FS -pscrB 829 Taking into account the improved growth of the mutant on glucose, a strain (ST1-GM-ABU1) is obtained that releases up to 91 mM more glucose. 378 Instead of mutations in ccpA 855 Similar behavior was observed in the strain carrying the mutation (strain ST1-CM-ABU1).

[0248] In conclusion, scrA 829 FS and pscR 829 The glcK and pglcU mutations were found to be involved in the regulation of lactate fermentation. 378 and ManM 12997 or ccpA 855 and ManM 12997 The phenotype conferred by the mutations (excessive consumption of lactose and release of glucose) is not adversely affected: on the contrary, these mutations make it possible to obtain strains which hydrolyze more lactose and release more glucose.

[0249] [Table 5]

[0250] Example 7. scrA 829 FS -pscrB 829 The double mutation impairs the transport of sucrose and glucose into cells, and the pglcU mutation restores the transport of glucose into cells of the double mutant. To elucidate the mechanism underlying the sucrose-negative and glucose-slow growth phenotype, we analyzed the quiescent (non-growing) cells of ST1 and their scrA 829 FS -pscrB 829 (ST1-AB) and scrA 829 FS -pscrB 829 -pglcU IN_T (ST1-ABU1) mutant was used to carry out experimental investigations into carbohydrate transport.

[0251] The strain was pre-cultured overnight at 37° C. in M17 containing 30 g / L lactose. The pre-culture was used to grow M17 containing 30 g / L lactose to a final OD of 0.05. 600 The cells were seeded at 1000 x g for 30 min and incubated at 42° C. for 4 h. The cells were harvested by centrifugation at 2450 g for 7 min, washed twice with 4% (w / v) β-glycerophosphate buffer, and then resuspended in 10 mL of β-glycerophosphate buffer. The OD of the final cell suspension was 600 was recorded. The cell suspension was pre-warmed at 42°C and glucose, lactose or sucrose was added to a final concentration of 3.5g / L, 7.0g / L or 7.0g / L, respectively. After 3 min of incubation at 42°C, the reaction medium was filtered through a 0.2 μm nylon membrane (PHENEX GF / NY) and carbohydrate loading was performed by high performance liquid chromatography (HPLC Agilent 1100 or 1200) measurement. A guard column (Security Guard Cartridges, Carbo-Pb 4x3.0mm ID, Phenomenex) was injected followed by injection of the sample into the ion exchange column. The ion exchange column was Pb2+ (Rezex RPM-monosaccharide Pb+2, 300×7.8mm, 8μm, Phenomenex). Elution was performed in isocratic mode with ultrapure water and carbohydrates were detected by refractometer. Quantification was performed by external calibration. Standard solutions were prepared in β-glycerophosphate 4% (w / v) ranging from 0 to 7 g / L for each carbohydrate. All experiments were performed in duplicate. The rate of sugar consumption was calculated in μmol / (u.OD*min) as follows: Carbohydrate uptake activity = [([sugar] T0x2 / 1.8x1000 / MW 糖 )-([sugar] T+3’ x1000 / MW 糖 )] / OD 600 / 3 [sugar] T0 : Initial carbohydrate concentration (g / L) [sugar] T+3’ : Final carbohydrate concentration measured after 3 min of contact (g / L) Volume of cell suspension: 1.8mL Volume of cell suspension and sugar solution: 2 mL MW 糖 : Molecular weight of carbohydrate (g / mol) O.D. 600 : Cell concentration at the start of the test

[0252] The results are shown in Table 6. The parent strain ST4 (DSM28255) was able to efficiently transport lactose, sucrose and glucose with consumption rates of 0.236, 0.180 and 0.108 μmole / u.OD*min, respectively. These values ​​were consistent with the scrA 829 FS -pscrB 829 The double mutant (ST4-AB) showed a dramatic decrease in sucrose and glucose (0.032 μmole / u.OD*min and 0.011 μmole / u.OD*min, respectively), but no significant difference in lactose (0.201 μmole / u.OD*min). IN_T The introduction of the mutation did not significantly affect the transport of lactose into the cells (0.227 μmole / u.OD*min) and did not improve the transport of sucrose (0.004 μmole / u.OD*min). IN_T The mutation completely restored glucose transport into the cell (0.102 μmole / u.OD*min). 829 FS -pscrB 829 The double mutation impairs the transport of sucrose and glucose into cells, and pglcU IN_T The mutation restores glucose transport into cells of the double mutant.

[0253] [Table 6]

[0254] array SEQ ID NO:2 [ka] SEQ ID NO:22 [ka] SEQ ID NO:25 (The bold underlined X corresponds to the aa at position 275 and can be any naturally occurring amino acid but is not Glu.) [ka] SEQ ID NO:45 [ka] SEQ ID NO:23 [ka] SEQ ID NO:65 [ka] SEQ ID NO:71 [ka] SEQ ID NO:78 [ka] SEQ ID NO:111 [ka] SEQ ID NO:112 [ka] SEQ ID NO:130 [ka] SEQ ID NO:157 [ka] SEQ ID NO:158 [ka] SEQ ID NO:166 [ka] SEQ ID NO:206 [ka] SEQ ID NO:207 MAEKIQLSQADRKKGLVALTILARCMEL SEQ ID NO:208 MAEKIQLSQADRKKGLVALTILARFMEL SEQ ID NO:209 MSEKIQLSQADRKKGLVALTILARFMEL SEQ ID NO:210 MAEKIQLSQADRRKGLVALTILARFMEL SEQ ID NO:211 MSEKIQLSQADHKKGLVALTILARFMEL SEQ ID NO:212_partial scrA829 [ka] SEQ ID NO:213_pscrB829 [ka] SEQ ID NO:214 [ka] SEQ ID NO:215 [ka] SEQ ID NO:216 [ka] SEQ ID NO:217 [ka] SEQ ID NO:218 [ka] SEQ ID NO:219 [ka] SEQ ID NO:220 [ka] SEQ ID NO:221 [ka] SEQ ID NO:222>ST12997_manM_DNA_mutation [ka] SEQ ID NO:223>ST12997_manM_protein_mutation [ka]

[0255] KK DGCC numbers are internal references to the DuPont Danisco collection. DSM numbers are numbers assigned by the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, following deposits made in accordance with the Budapest Treaty.

[0256] As far as the Streptococcus thermophilus strain DGCC7710, deposited in accordance with the Budapest Treaty at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH on 14 January 2014 under number DSM 28255, is concerned, the Applicant confirms that the depositor, Danisco Deutschland GmbH, Busch-Johannsen-Strasse 1, D-25899 Niebuell, Germany, has authorized the Applicant (DuPont Nutrition Biosciences ApS) to refer to the deposited biological material in the present application. The expressions "strain DGCC7710" and "DGCC7710 derivatives" are used interchangeably with the expressions "strain DSM 28255" and "DSM 28255 derivatives".

[0257] We designed a derivative of strain DGCC7710 in which the glcK gene encodes glucokinase and in which the glutamic acid (E) at position 275 was replaced by the amino acid lysine (K). This derivative (DGCC12534) was deposited at the DSMZ on August 15, 2017 under the accession number DSM32587. - the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM33651 (herein referred to as ST3 or DSM33651).

[0258] The following strains have been deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH in the name of DuPont Nutrition Biosciences ApS: Streptococcus thermophilus strain ST1-ABU1 (deposited as DSM34134), Streptococcus thermophilus strain ST1-ABU4 (deposited as DSM34140), Streptococcus thermophilus ST1-GM-ABU1 strain (deposited as DSM34138), Streptococcus thermophilus ST1-CM-ABU1 strain (deposited as DSM34139), Streptococcus thermophilus ST2 strain (deposited as DSM34132), Streptococcus thermophilus strain ST2-ABU2 (deposited as DSM34147), Streptococcus thermophilus strain ST2-ABU3 (deposited as DSM34146). - the Streptococcus thermophilus strain deposited on February 16, 2022 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM34172 (herein referred to as ST1 or DSM34172).

[0259] Applicants request that until the date a patent is granted, only samples of the deposited microorganisms described herein may be made available to practitioners.

[0260] In respect of the indication that a European patent is sought, samples of the deposited microorganisms shall only be made available by furnishing such samples to experts designated by the claimant requesting samples and recognized by i) the applicant and / or ii) the European Patent Office, whichever is applicable, until the publication of the reference to the grant of the European patent or the date on which the application is refused or withdrawn or considered withdrawn.

Claims

1. 1. A lactose-positive, sucrose-negative Streptococcus thermophilus strain carrying one or more mutations, said one or more mutations being 1) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or transport sucrose; and i) one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene; ii) optionally, further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype; and one or more mutations including at least 2) Optionally, one or more further mutations affecting a glucose transporter, e.g., glcU and / or its expression, which restore or improve glucose consumption of the strain upon growth on glucose as the sole source of carbohydrate. A lactose positive, sucrose negative Streptococcus thermophilus strain selected from the group consisting of:

2. 2. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 1, which is glucose-positive or glucose-slow growing.

3. 3. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 1 or 2, having one or more mutations in the sucrose regulon in one or more of the scrA and scrB genes.

4. The mutation in one or more genes of the sucrose regulon is in the scrA gene and effectively disrupts gene function, e.g., EIIABC スクロース A frameshift in the open reading frame of a gene encoding a protein is caused, and the truncated EIIABC encoded by the scrA gene is スクロース 4. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 3, wherein the mutation is due to an insertion or deletion that results in the translation of a protein, for example an insertion or deletion of a nucleotide in the open reading frame (ORF) of the scrA gene, such as the nucleotide corresponding to or surrounding position 133 of SEQ ID NO:

212.

5. 5. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 4, having a mutation in the promoter region regulating expression of any one of the scrA, scrB and / or scrR genes.

6. The mutation may be a SNP in the promoter region of the scrA and / or scrB gene, such as a mutation near the -10 and -35 sequences of the promoter, such as a T to G mutation corresponding to position 38 of SEQ ID NO:213 upstream of the scrB promoter, such as a mutation comprising the entire sequence identified by SEQ ID NO:213 or a variant sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB ８２９ 6. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 5, wherein the lactose-positive, sucrose-negative Streptococcus thermophilus strain is

7. 7. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 6, comprising a mutation affecting said glucose transporter, said mutation restoring or improving glucose consumption of said strain when grown on glucose as the sole source of carbohydrate, e.g. completely restoring growth on glucose compared to that of the lactose-positive and sucrose-positive Streptococcus thermophilus parent strain.

8. The mutation affecting the glucose transporter may affect glcU and / or its expression, for example located within the promoter region of glcU, e.g. a) an insertion of a T nucleotide in the polyT region at a position corresponding to position −75 of the glcU promoter identified as SEQ ID NO:216 (pglcU ＩＮ＿Ｔ ), e.g., a mutation containing a T nucleotide at position 102 of SEQ ID NO:217, e.g., a mutation comprising the entire sequence identified by SEQ ID NO:217 or a variant sequence which is at least about 90%, e.g., at least about 95%, identical thereto; b) an insertion of a C nucleotide in the polyT region at a position corresponding to position −75 of the glcU promoter identified as SEQ ID NO: 216 (pglcU ＩＮ＿Ｃ ), e.g., a mutation containing a C nucleotide at position 102 of SEQ ID NO:218, e.g., a mutation comprising the entire sequence identified by SEQ ID NO:218 or a variant sequence which is at least about 90%, e.g., at least about 95%, identical thereto; c) a T to C nucleotide mutation in the polyT region at a position corresponding to position −81 of the glcU promoter identified as SEQ ID NO: 216 (pglcU ＳＮＰ＿Ｃ ), e.g., a mutation containing a C nucleotide at position 96 of SEQ ID NO:219, e.g., a mutation comprising the entire sequence identified by SEQ ID NO:219 or a variant sequence which is at least about 90%, e.g., at least about 95%, identical thereto; d) a mutation comprising an insertion of a sequence at a position corresponding to nucleotide positions 110-111 of the glcU promoter identified as SEQ ID NO:216, e.g., an insertion of a sequence identified by nucleotides 1-1321 of SEQ ID NO:221, e.g., the entire sequence identified by SEQ ID NO:220 or a variant sequence which is at least about 90%, e.g., at least about 95% identical thereto, or SEQ ID NO:221 or a variant sequence which is at least about 90%, e.g., at least about 95% identical thereto (pglcU ＩＳ ) 8. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 7, wherein the mutation is selected from the group consisting of:

9. a) a mutation in the glcK gene encoding glucokinase, which mutation significantly reduces, but does not reduce, the glucokinase activity of said glucokinase in said strain, and / or b) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene results in a stop codon at position 180 of SEQ ID NO:222, EIIC Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO: 167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ (resulting in a mutant allele), a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41 9. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 1, further comprising one or more mutations selected from:

10. 10. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 9, which is galactose-negative, for example due to a mutation in the glcK and / or ccpA genes, optionally combined with a mutation in one of the genes encoding a protein of the mannose-glucose specific PTS.

11. 11. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 9 or 10, having a mutation in the glcK gene encoding glucokinase, said mutation providing for a significantly reduced, but not zero, glucokinase activity in said strain.

12. The mutant glcK gene is a) a glucokinase having at position 275 an amino acid which is not glutamic acid, in particular which is not an acidic amino acid, in particular which is lysine; b) at position 275, an amino acid which is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine (glcK ３７８ and having an arginine at position 278 and / or a serine at position 279; and c) a glucokinase having at position 144 an amino acid which is not glycine, in particular which is not an aliphatic amino acid, in particular which is serine; 12. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of claims 9 to 11, which encodes a glucokinase selected from the group consisting of:

13. The mutant glcK gene encodes glucokinase and has the sequence a) the sequence defined in SEQ ID NO: 25, in which the amino acid at position 275 is not glutamic acid, in particular is not an acidic amino acid, in particular is lysine, and b) a GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 25, wherein the amino acid of said glucokinase corresponding to position 275 of SEQ ID NO: 25 (or the amino acid at position 275 of said glucokinase) is not glutamic acid, in particular is not an acidic amino acid, in particular is a lysine; c) the sequence defined in SEQ ID NO: 46, in which the amino acid at position 144 is not a glycine, in particular is not an aliphatic amino acid, in particular is a serine; and d) A GlcK variant sequence having at least 90% similarity or identity to SEQ ID NO: 46, wherein the amino acid of said glucokinase corresponding to position 144 of SEQ ID NO: 46 (or the amino acid at position 144 of said glucokinase) is not glycine, in particular is not an aliphatic amino acid, in particular is serine.

13. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 9 to 12, selected from the group consisting of:

14. 14. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 13, further carrying a mutation in the ccpA gene.

15. 15. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 14, further carrying a mutation in the gene encoding a protein of the mannose-glucose specific PTS, such as a gene selected from the group consisting of the manL gene, the manM gene and the manN gene.

16. 16. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 14 or 15, wherein the ccpA gene harbors a mutation selected from the group consisting of a nonsense mutation located between nucleotides 1 and 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, resulting in a frameshift of the open reading frame of the ccpA gene.

17. The sequence of the mutant ccpA gene is a) a sequence as defined in SEQ ID NO: 71, and b) a ccpA variant sequence having at least 90%, such as at least 95%, identity to SEQ ID NO: 71; 17. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of claim 16, selected from the group consisting of:

18. During the 24 hour fermentation, the milk can be fermented as described in Example 5, a) the concentration of lactose remaining in the fermented milk is less than 60 mM; and / or b) The pH of the milk is adjusted to a pH that ensures a rate of acidification of less than 0.1 mU pH / min (pH ＳＴＯＰ ) and the pH ＳＴＯＰ 18. The lactose-positive, sucrose-negative Streptococcus thermophilus strain of any one of claims 1 to 17, wherein the pH is 4.4 to 5.3, and optionally the gradient from pH 6 to pH 5.5 is at least -0.008 Up / min.

19. 19. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 18, having the ability to over-consume lactose compared to a parent strain not carrying said one or more mutations, e.g. when fermenting sweet milk as described and assayed according to Example 6, the amount of lactose remaining is less than 100 mM, such as 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, 8 mM, 6 mM, 4 mM, 2 mM or 1 mM.

20. 20. The lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 19, having the ability to release increased amounts of glucose compared to a parent strain not carrying said one or more mutations, e.g. glucose release as described and assayed according to Example 6 is higher than 10 mM, such as higher than 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM or 90 mM.

21. 21. A lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 20, which releases a concentration of glucose assayed as described in Example 6 that is at least 50 mM, such as at least 60, 70, 80 or 90 mM, or which releases a concentration of glucose that is at least 150% or at least 200%, such as 300%, 400%, 500% increased compared to the glucose concentration released by the parent strain, both when assayed as described in Example 6.

22. 22. A composition comprising at least one, in particular one lactose positive, sucrose negative Streptococcus thermophilus strain according to any one of claims 1 to 21, in particular in combination with another lactic acid bacterium, in particular in combination with one or more strains selected from the group consisting of a strain of the genus Lactobacillus, such as a Lactobacillus delbrueckii subsp. bulgaricus strain, a strain of the genus Lactococcus, such as a Lactococcus lactis strain or a strain of the genus Bifidobacterium.

23. 22. A method for producing a fermented milk product, in particular fermented milk, comprising inoculating a milk substrate with a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or with the composition according to claim 19, and fermenting the inoculated milk to obtain a fermented milk product.

24. Use of a lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or a composition according to claim 22 for the production of a fermented milk product.

25. Fermented milk product comprising at least one, in particular one, lactose-positive, sucrose-negative Streptococcus thermophilus strain according to any one of claims 1 to 21 or obtainable by the method according to claim 23.

26. Streptococcus thermophilus strains, such as lactose-positive, sucrose-negative Streptococcus thermophilus strains, - strains deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34132 in the name of DuPont Nutrition Biosciences ApS; - strains deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34134 in the name of DuPont Nutrition Biosciences ApS; - strains deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34138 in the name of DuPont Nutrition Biosciences ApS; - strains deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34139 in the name of DuPont Nutrition Biosciences ApS; - strains deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34140 in the name of DuPont Nutrition Biosciences ApS; - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34146 in the name of DuPont Nutrition Biosciences ApS, - the strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH under the number DSM 34146 in the name of DuPont Nutrition Biosciences ApS The strain deposited under the number DSM 34147 in the name of ApS, and - Strain deposited under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH according to the Budapest Treaty on February 16, 2022 A Streptococcus thermophilus strain selected from the group consisting of:

27. - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence of scrA corresponds to the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutated sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or said promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 34172 (herein referred to as ST1 or DSM 34172 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK ３７８ 222, e.g., by an insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene, resulting in a stop codon at position 180 of SEQ ID NO:

222. Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity at least 95% to SEQ ID NO:71; - a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under number DSM 34172 according to the Budapest Treaty (herein referred to as strain ST1 or DSM 34172), which has the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK ３７８ ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene results in a stop codon at position 180 of SEQ ID NO:222, EIIC Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO: 167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ having a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity with SEQ ID NO: 71, at least 95% A strain comprising one or more of: - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of scrA corresponds to the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutated sequence which is at least about 90%, such as at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ under the name of Danisco Deutschland GmbH under number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or said promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 (herein referred to as ST4 or DSM 28255 strain), in which the sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK ３７８ 222, e.g., by an insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene, resulting in a stop codon at position 180 of SEQ ID NO:

222. Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity at least 95% to SEQ ID NO:71; - a strain of Streptococcus thermophilus, such as the strain deposited on 14 January 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255 according to the Budapest Treaty (herein referred to as the ST4 or DSM 28255 strain), which has the following characteristics: 1) The sequence of the open reading frame (ORF) of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK ３７８ ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene results in a stop codon at position 180 of SEQ ID NO:222, EIIC Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ having a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity with SEQ ID NO: 71, at least 95% A strain comprising one or more of: - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), wherein the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited under the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on January 18, 2022 under the number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutated sequence that is at least about 90%, such as at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited under the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on January 18, 2022 under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence as set forth in any one of SEQ ID NOs: 217 to 220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), wherein the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), wherein the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 18, 2022 at the Leibniz-Institut DSMZ under the name of DuPont Nutrition Biosciences ApS under number DSM 34132 (herein referred to as ST2 or DSM 34132 strain), wherein the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK ３７８ 222, e.g., by an insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene, resulting in a stop codon at position 180 of SEQ ID NO:

222. Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71; - a strain of Streptococcus thermophilus, such as the strain deposited on January 18, 2022 at the Leibniz-Institut DSMZ in the name of DuPont Nutrition Biosciences ApS under number DSM 34132 according to the Budapest Treaty (herein referred to as the ST2 or DSM 34132 strain), which has the following characteristics: 1) The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK ３７８ ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene results in a stop codon at position 180 of SEQ ID NO:222, EIIC Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ having a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 A strain comprising one or more of: - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutated sequence that is at least about 90%, such as at least about 95%, identical thereto (pscrB ８２９ ),KK, - Streptococcus thermophilus deposited on September 29, 2020 at DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 In one embodiment, the strain corresponds to a S. thermophilus strain (referred to herein as ST3 or DSM33651), in which the locus that was the promoter region upstream of the glcU gene (pglcU) has been replaced by a corresponding sequence set forth in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ),KK, - a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under accession number DSM 33651 (herein referred to as ST3 or DSM 33651), in which the sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), and the sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), and the locus that was the promoter region upstream of the glcU gene (pglcU) is replaced by the corresponding sequence shown in any one of SEQ ID NOs: 217-220 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a variant sequence that is at least about 90%, such as at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), optionally the glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence that is at least about 90%, e.g., at least about 95%, identical thereto, and the amino acid at position 275 is a lysine (glcK ３７８ 222, e.g., by an insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene, resulting in a stop codon at position 180 of SEQ ID NO:

222. Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ a mutation comprising an insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele, and optionally further comprising a ccpA sequence of SEQ ID NO:71 or a ccpA variant sequence having at least about 90%, such as at least 95%, identity to SEQ ID NO:71; - a strain of Streptococcus thermophilus deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651 (herein referred to as ST3 or DSM 33651), which has the following characteristics: 1) The sequence of scrA is the truncated EIIABC スクロース 212 or a mutant sequence that is at least about 90%, such as at least about 95%, identical thereto, resulting in a frameshift in translation in ８２９ ＦＳ ), 2) The sequence upstream of the start codon of the scrB gene is replaced by the corresponding sequence shown in SEQ ID NO: 213 or a mutant sequence that is at least about 90%, for example at least about 95%, identical thereto (pscrB ８２９ ), 3) The locus that was the promoter region (pglcU) upstream of the glcU gene is replaced by a corresponding sequence shown in any one of SEQ ID NOs: 217 to 220 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto, or the promoter comprises the sequence of SEQ ID NO: 221 or a mutant sequence that is at least about 90%, for example at least about 95% identical thereto (pglcU ＳＮＰ＿Ｃ , pglcU ＩＮ＿Ｔ , pglcU ＩＮ＿Ｃ , pglcU ＩＳ ), 4) The glcK gene encoding glucokinase contains a sequence defined by SEQ ID NO:25 or a variant sequence which is at least about 90%, for example at least about 95%, identical thereto, and the amino acid at position 275 is lysine (glcK ３７８ ), 5) For example, the insertion of one nucleotide C at any one of positions 438, 439 or 440 of SEQ ID NO:223 in the manM gene results in a stop codon at position 180 of SEQ ID NO:222, EIIC Ｍａｎ A frameshift is generated in the open reading frame of a gene encoding a protein (manM １２９９７ ), and / or SEQ ID NO: 80 (IIAB Ｍａｎ A substitution of nucleotide G to T at position 916 of SEQ ID NO:79 of the manL gene resulting in a stop codon at position 306 of the protein (manL １３７４０ ), and / or a substitution of the nucleotide G by the nucleotide T at position 625 of manM resulting in a stop codon at position 209 (manM １６４０４ ) and / or the IID at position 28 of SEQ ID NO:167 Ｍａｎ Generates a frameshift in the manN gene, resulting in a truncation of the protein (manN １３７４１ having a mutation involving the insertion of one nucleotide A in a stretch of five nucleotide A at positions 37-41, resulting in a mutant allele; 6) having a ccpA sequence of SEQ ID NO: 71 or a ccpA variant sequence having at least about 90%, for example at least 95%, identity to SEQ ID NO: 71 Strains containing one or more of the following: A lactose positive, sucrose negative Streptococcus thermophilus strain selected from the group consisting of:

28. A polynucleotide comprising one or more mutations in one or more genes of a sucrose regulon, said one or more mutations being a) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255, wherein said polynucleotide replaces the corresponding sequence in DSM 28255, b) a strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34132 at the Leibniz-Institut DSMZ in accordance with the Budapest Treaty on January 18, 2022 under the name of DuPont Nutrition Biosciences ApS, wherein said polynucleotide replaces the corresponding sequence in DSM 34132; c) a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651, wherein the polynucleotide replaces the corresponding sequence in DSM 33651; d) A strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on February 16, 2022, wherein the polynucleotide replaces the corresponding sequence in DSM 34172. A polynucleotide which provides a significant reduction in the ability to take up and consume any sucrose in a strain selected from the group consisting of:

29. 1. A polynucleotide comprising one or more mutations affecting a glucose transporter, said one or more mutations being a) a strain corresponding to the Streptococcus thermophilus strain deposited in accordance with the Budapest Treaty on January 14, 2014 at the Leibniz-Institut DSMZ in the name of Danisco Deutschland GmbH under the number DSM 28255, wherein said polynucleotide replaces the corresponding sequence in DSM 28255, b) a strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34132 at the Leibniz-Institut DSMZ in accordance with the Budapest Treaty on January 18, 2022 under the name of DuPont Nutrition Biosciences ApS, wherein said polynucleotide replaces the corresponding sequence in DSM 34132; c) a strain corresponding to the Streptococcus thermophilus strain deposited on September 29, 2020 at the DSMZ [Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig-Germany] under the accession number DSM 33651, wherein the polynucleotide replaces the corresponding sequence in DSM 33651; d) A strain corresponding to the Streptococcus thermophilus strain deposited under the number DSM 34172 in the name of DuPont Nutrition Biosciences ApS at the Leibniz-Institut DSMZ according to the Budapest Treaty on February 16, 2022, wherein the polynucleotide replaces the corresponding sequence in DSM 34172. A polynucleotide which restores or improves glucose consumption in a strain selected from the group consisting of:

30. 30. The polynucleotide according to claim 28 or 29, wherein the one or more mutations are as defined in any one of claims 3 to 8.

31. 1. A method for selecting a lactose-positive, sucrose-negative Streptococcus thermophilus strain, comprising: 1) A strain of Streptococcus thermophilus, a) one or more mutations in one or more genes of the sucrose regulon that provide a significant reduction in the ability of the strain to grow on sucrose and / or to transport sucrose, and i) one or more of the scrA gene, and / or the scrB gene, and / or the scrR gene; ii) optionally, further mutations in the promoter regions regulating the expression of the scrA, scrB and / or scrR genes, e.g. mutations that protect the strain from reversion to a sucrose-negative phenotype; One or more mutations including at least Providing a strain of Streptococcus thermophilus comprising: 2) screening for strains of Streptococcus thermophilus from step 1) that have a significant reduction in the ability to grow on sucrose and transport sucrose; 3) Optionally, screening, optionally before, simultaneously with or after any one of steps 1 and 2, for Streptococcus thermophilus strains with restored or improved glucose consumption resulting from one or more additional mutations affecting a glucose transporter, e.g. affecting glcU and / or its expression; The method includes:

32. The method of claim 31, wherein the strain is according to any one of claims 1 to 21.

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