New bacterium with low carbohydrate secretion

Abstract

The present disclosure is in the field of dairy technology. It relates to a method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates comprising the steps: (a) providing a mother strain which is able to ferment lactose and glucose; (b) selecting a mutant strain derived from the mother strain which mutant strain has a change in: (i) one or more of the genes encoding a protein involved in the metabolism of galactose wherein said strain is able to ferment galactose; and (ii) the lacS gene encoding a lactose permease which change results in at least one mutation in the amino acid sequence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID NO:2; and wherein the mutant strain has a Lac(+) Glu(+) Gal(+) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain. It furthermore relates to Streptococcus thermophilus strains exhibiting a low excretion of carbohydrates, Composition comprising said strains, methods for manufacturing a fermented product, fermented products, and use of said strains.

Description

[0001] NEW BACTERIUM WITH LOW CARBOHYDRATE SECRETION

[0002] FIELD OF THE INVENTION

[0003] The present disclosure lies within the field of dairy and fermented products. More specifically it relates to strains exhibiting low excretion of carbohydrates while being able to metabolize several carbohydrates.

[0004] BACKGROUND OF THE INVENTION

[0005] Lactic acid bacteria (LAB) have been used over decades for preparing fermented food products. During fermentation, lactic acid and other organic compounds are produced by the lactic acid bacteria, thereby reducing the pH of the food product.

[0006] Lactose is the main carbon and energy source for Streptococcus thermophilus and it is metabolized by fermentation into lactic acid. The disaccharide lactose is imported into the cell by a lactose permease (LacS) and is hydrolyzed to the monosaccharides glucose and galactose by a p-galactosidase (LacZ). Glucose is metabolized via the glycolytic pathway, whereas galactose is not used in most S. thermophilus strains and is secreted into the medium by the LacS antiporter which allows uptake of lactose from the medium in exchange for galactose. Galactose accumulation in dairy products can lead to unfavorable events, such as e.g., browning of heat-treated Mozzarella on pizza where browning is believed to be due to the Maillard reaction where galactose as reducing sugar is reacting with amino acids / peptides, cheese fractures due to CO2 overproduction by heterofermentative bacteria and toxic effects on people unable to metabolize galactose i.e. suffering from galactosemia. Thus, strains capable of utilizing galactose could be beneficial and desirable in the manufacture of dairy products. In galactose fermenting or galactose positive (Gal+) strains, this sugar is metabolized via the Leloir pathway which includes the four enzymes: galactose mutarotase (GalM), galactokinase (GalK), galactose-l-phosphate uridylyltransferase (GalT) and UDP- glucose 4-epimerase (GalE). In S. thermophilus there are five genes related to galactose metabolism (gaIR, galK, ga!T, galE, ga!M) which are located upstream of the lac operon {JacSZ). Genes galK, gall, and galE are under the control of the same promoter and constitute the gal operon. The single nucleotide polymorphisms (SNPs) localized in the galKTE promoter was proposed as the cause of the low efficiency in initiating the galKTE operon transcription leading to the inability to metabolize galactose for most S. thermophilus strains (galactosenegative strains; Gal( )). WO2019 / 042881 (Chr. Hansen A / S) describes how to generate S. thermophilus strains with up to 50% reduced amount of galactose excretion as compared to a reference strain.

[0007] F. de Vin, et al. (Applied and Environmental Microbiology, July 2005 Vol.71(7) p.3659-67) described a strain IMDOST40 capable of fermenting lactose, glucose and galactose. It is stated that "AH the strains, except for strain IMDOST40, excreted galactose into the medium during growth on lactose, indicating the activity of the lactose-galactose antiport uptake system". However, nothing is described regarding the secretion of glucose or allolactose, nor is lactose permease mentioned.

[0008] Accordingly, there is still a need for development of strains excreting no or low amounts of carbohydrates.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure provides in a first aspect a method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates comprising the steps:

[0011] (a) providing a mother strain which is able to ferment lactose and glucose;

[0012] (b) selecting a mutant strain derived from the mother strain which mutant strain has a change in: (i) one or more of the genes encoding a protein involved in the metabolism of galactose wherein said strain is able to ferment galactose; and (ii) the LacS gene encoding a lactose permease which change results in at least one mutation in the amino acid seguence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID No:2; and wherein the mutant strain has a Lac( + ) Glu( + ) Gal( + ) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain.

[0013] In a second aspect the present disclosure provides a Streptococcus thermophilus strain with a Lac(+) Glu(+) Gal(+) phenotype and at least one mutation in the lacS gene encoding a lactose permease wherein the mutation leads to an amino acid substitution at a position corresponding to position 148, 377 and / or 476 in SEQ ID No:2.

[0014] In a third aspect the present disclosure provides a method for manufacturing a fermented product comprising the steps: (a) adding the strain according to any one of claims 9-16 or the composition according to 16 to a milk base; and (b) fermenting the milk base to obtain a fermented product, preferably a fermented dairy product.

[0015] In a fourth aspect the present disclosure provides a fermented product comprising the strain or the composition according to the present disclosure. In a fifth aspect the present disclosure provides use of the strain or the composition according to the present disclosure for reducing the content of carbohydrates in a fermented product and / or for reducing the fermentation time.

[0016] BRIEF DESCRIPTION OF THE FIGURE AND SEQUENCES FIGURE 1

[0017] Figure 1 shows the acidification profiles of DSM 35264 and DSM 33719.

[0018] Table 1. List of Lactose permease (lacS) gene and amino acid sequences (1-16). Mother strains are: DSM 33677, DSM 33719, and DSM 35263. Table 2. List of galactokinase (galK), glucokinase (glcK) and phosphoglucomutase (pgm) gene and amino acid sequences (17-22). Mutants of the mother strain DSM 33677 are: DSM 33719, DSM 35264, DSM 35263 and DSM 35265. DETAILED DESCRIPTION OF THE INVENTION

[0019] Streptococcus thermophilus strains are generally unable to use galactose resulting from hydrolyzis of lactose into its monosaccharides glucose and galactose, and secretes it out into the milk during acidification. The glucose part of lactose is fully sufficient to support milk acidification. There are at least two applications where a solution with less carbohydrate secretion would be highly desirable. First, in mixed cultures where secreted sugar of a first strain can be utilized by a second strain and thus result in undesired fermentation. Secondly, it is known that during heating of mozzarella cheese presence of galactose may lead to browning due to the Maillard reaction.

[0020] The amount of galactose secreted resulting from milk acidification may vary from 5-10g / L. This secretion is due to the function of lactose permease (LacS) that acts as an antiporter and secrete one molecule of galactose per one molecule of lactose taken up. In galactose positive mutants, the secretion of galactose is typically less but still significant and lead often also to a higher final pH. In "Sweety mutants" as described in W02022 / 180071, the glucose is not consumed but secreted to sweeten the milk. Thus only the galactose part is consumed for growth. Yet, still up to 15-18 g / l galactose can be secreted in Sweety strains. This is a highly inefficient way of using the lactose and result also in a slower acidification performance of milk.

[0021] To overcome this performance issue of Sweety strains, we aimed at optimizing growth on the galactose part. A manual adaptive laboratory evolution was initialized using a Sweety strain, DSM 33719, as starting point and then grow for 3 weeks in broth with increasing levels of galactose. Out of several adapted mutants DSM 35264 was also able to acidify milk significantly faster than the mother strain. From another mother strain DSM 35263 exposed to ALE on galactose the mutant DSM 35265 was obtained.

[0022] METHOD FOR PRODUCING THE STRAIN

[0023] In S. thermophilus, lactose is generally transported by the permease (lacS) and hydrolyzed by p-galactosidase (lacZ) to yield glucose and galactose. The genes encoding lacS and lacZ constitute the lactose (Lac) operon. The lactose permease (lacS) of S. thermophilus is a 634 amino acid chimeric protein consisting of an amino-terminal carrier domain (AA 1-490) and a carboxyl-terminal phosphoenolpyruvate:sugar phosphotransferase system (PTS) IIA protein domain (AA 500-604). Although it is expected that the transport of lactose will be affected by mutations in the lactose permease it is surprising that the excretion of other carbohydrates will be reduced. In one aspect the present disclosure relates to a method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates comprising the steps:

[0024] (a) providing a mother strain which is able to ferment lactose and glucose;

[0025] (b) selecting a mutant strain derived from the mother strain which mutant strain has a change in:

[0026] (I) one or more of the genes encoding a protein involved in the metabolism of galactose wherein said strain is able to ferment galactose; and

[0027] (II) the LacS gene encoding a lactose permease which change results in at least one mutation in the amino acid sequence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID No:2; and wherein the mutant strain has a Lac( + ) Glu( + ) Gal( + ) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain.

[0028] The mutations identified in the present disclosure are all located in the carrier domain. In one embodiment the present disclosure relates to the method, wherein the change in the LacS gene results in at least one mutation in the amino acid sequence of lactose permease carrier domain.

[0029] In one embodiment the present disclosure relates to the method, wherein the amino acid sequence of lactose permease at the position corresponding to position 148 is Histidine (H), position 377 is Cysteine (C), and position 476 is Lysine (K).

[0030] Further embodiments relating to the carbohydrates and their concentrations are described in detail in the section "STRAIN" infra and is equally relevant for this section. In fact description and definitions disclosed in one section are not bound to that sections only but are equally relevant for all sections of the present disclosure. In one embodiment the present disclosure relates to the method according to the present disclosure, wherein the carbohydrate is one or more selected from: lactose, allolactose, glucose, and galactose. In one embodiment the present disclosure relates to the method according to the present disclosure, wherein the reduced secretion of carbohydrates results in a concentration of glucose and / or galactose in a milk base fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL.

[0031] Lactose utilization by S. thermophilus starts with the import by lactose permease. Once inside the bacteria glucose and galactose resulting from the hydrolysis of lactose are metabolized though the glycolytic pathway and the Leloir pathway respectively. These pathways are interlinked through the phosphoglucomutase. Strains of the present disclosure are in contrast to most S. thermophilus strains able to metabolize galactose. Galactokinase (galK, EC 2.7.1.6) is the first enzyme in the Leloir pathway through which galactose is metabolized. It converts a-galactose to galactose-1- phosphate (GallP). GalK is part of the galactose operon which further consist galT and galE. Strains of the present disclosure have shown to be mutated in a position corresponding to position 47 of SEQ ID No: 18 which is in the galactose binding domain.

[0032] Strains of the present disclosure are like most S. thermophilus strains able to metabolize glucose. Glucokinase (glcK, EC 2.7.1.2) is a hexokinase enzyme, catalyzing the first step in glycolysis, namely the phosphorylation of glucose to glucose-6-phosphate (Glu6P). Strains of the present disclosure have shown to be mutated in a position corresponding to position 16 of SEQ ID No:20.

[0033] Phosphoglucomutase (pgm, EC 5.4.2.2) is an enzyme that convert the reversible reaction between p-glucose-l-phosphate (GlulP) to glucose-6-phosphate (Glu6P), thereby linking the glycolysis and the Leloir pathways. Strains of the present disclosure have shown to be mutated in a position corresponding to position 242 of SEQ ID No:22.

[0034] In one embodiment the present disclosure relates to the method according to the present disclosure, wherein the genes involved in the metabolism of galactose is selected from galactokinase (galK), glucosekinase (glcK) and phosphogiucomutase (pgm).

[0035] In one embodiment at least one of genes encoding galactokinase (galK), glucosekinase (glcK) and phosphogiucomutase (pgm) is mutated, preferably galK. If two of the genes are mutated the preferred combination is galK+glcK or galK+pgm. Preferably, all three genes are mutated. If the mutated gene is galK the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No: 18. If the mutated gene is glcK the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No:20. If the mutated gene is pgm the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22.

[0036] In one embodiment the present disclosure relates to the method, wherein the mutated gene is: galK and the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No: 18; glcK and the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No: 20; and / or pgm and the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22.

[0037] In the method for producing a S. thermophilus strain exhibiting low excretion of carbohydrates the steps 1 (b) (i) providing the galactose fermenting ability and 1 (b) (ii) providing the mutated lactose permease, may be conducted in any order. In one embodiment the present disclosure relates to the method, wherein step l(b)(i) is conducted before step l(b)(ii), or step l(b)(ii) is conducted before step l(b)(i).

[0038] The method may be used with any mother strain of the species S. thermophilus. The mother strain may be an unmutated strain into which the gene changes are introduced such as e.g. DSM 33677. Alternatively, the mother strain may be selected from a strain already comprising one or more of the mutations such as e.g. DSM 33719 or DSM 35263. Thus in some embodiments of the methods of the present invention, the mother strain is a Gal(+) strain.

[0039] In one embodiment the present disclosure relates to the method according to the present disclosure, wherein the mother strain is selected from: DSM 33677; DSM 33719; DSM 35263; or any mutants or variants thereof.

[0040] The method may be performed as described in the Examples herein, such as, but not limited to, as described in Example 1.

[0041] Thus, in some embodiments of the method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates of the present invention, the mutant may be obtained, for example, but not limited to, by the approaches for obtaining mutant strains from a mother strain described herein, such as approaches selected from the group consisting of: adaptive laboratory evolution (ALE), chemical mutagenesis, radiation mutagenesis, selection of naturally occurring mutants, and genetic engineering.

[0042] In preferred embodiments of the method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates of the present invention, the mother strain is subjected to adaptive laboratory evolution (ALE), preferably under aerobic conditions, before the step of selecting the mutant strain.

[0043] Adaptive laboratory evolution (ALE) for obtaining improved Streptococcus thermophilus strains capable of fermenting galactose (Gal+) may comprise the following steps:

[0044] Cultivating the mother strain under selective conditions in a medium where galactose is the primary or sole carbon source, thereby applying selective pressure for the emergence or enhancement of galactose fermentation capability.

[0045] Seriallytransferring or sub-culturing the culture of the previous stepover multiple generations, maintaining the selective environment to favor the growth of mutants with improved galactose utilization.

[0046] Monitoring growth and adaptation by measuring relevant parameters such as optical density or substrate consumption.

[0047] After a defined number of passages or upon observation of improved growth compared to the mother strain, individual clones or populations are isolated. These isolates are then screened and characterized for the desired Gal+ phenotype, for example by assessing their ability to grow in galactose-containing media. Further genetic and / or phenotypic analyses may be performed to confirm the presence of beneficial mutations of interest and validate the improved properties of the evolved strains, as for example performed in step b) of the methods of the present invention.

[0048] In some embodiments, the method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates of the present invention comprsises the steps of :

[0049] • (a) Providing a mother strain which is able to ferment lactose and glucose;

[0050] • (b) Selecting a mutant strain derived from the mother strain, wherein the selection comprises:

[0051] (i) Cultivating the mother strain under selective conditions in a medium comprising galactose, preferably where galactose is the primary or sole carbon source, thereby applying selective pressure for the emergence or enhancement of galactose fermentation capability;

[0052] (ii) Serially transferring or sub-culturing the culture over multiple generations, maintaining the selective environment to favor the growth of mutants with improved galactose utilization;

[0053] (iii) Optionally monitoring growth and adaptation by measuring relevant parameters such as optical density or substrate consumption;

[0054] (iv) After a defined number of passages or upon observation of improved growth, isolating individual clones or populations;

[0055] (v) Screening the isolates for the desired Gal(+) phenotype, for example by assessing their ability to grow in galactose-containing media;

[0056] (vi) Optionally determining that the mutant has a change in one or more of the genes encoding a protein involved in the metabolism of galactose;

[0057] (vii) Determining that the mutant has a change in the LacS gene encoding a lactose permease which change results in at least one mutation in the amino acid sequence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID No:2, preferably by sequencing;

[0058] (viii) Confirming that the selected mutant strain exhibits a Lac(+), Glu(+), Gal(+) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain, for example as determined by carbohydrate analysis of fermented milk (e.g., using HPLC).

[0059] The above steps may be performed for example as recited in the Examples herein.

[0060] In certain embodiments, the step of selecting a mutant strain as recited in step (b) (i) may be performed by isolating and screening mutants for the ability to ferment galactose, wherein the mother strain may already possess the Gal(+) phenotype, and the ALE process is used to further enhance galactose utilization or other desired traits.

[0061] In further embodiments, the step of selecting a mutant strain as recited in step (b)(ii) may comprise identifying a mutant with at least one mutation in the LacS gene encoding a lactose permease, wherein the mutation results in an amino acid substitution at a position corresponding to position 148, 377, or 476 in SEQ ID No:2.

[0062] STRAINS

[0063] In the present context, the term "lactic acid bacteria" or "LAB" is used to refer to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram-positive, low-GC, acid tolerant, non-sporulating, nonrespiring, rod-shaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of dairy product. As used herein, the term "lactic acid bacteria" encompasses, but is not limited to, bacteria belonging to the genus of Streptococcus spp., Lactobacillus spp., Lactococcus spp., Leuconostoc spp., such as e.g. Streptococcus thermophilus, Lactobacillus delbrueckii subsp. buigaricus, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Lactococcus lactis, and Lacticaseibacillus paracasei subsp. paracasei.

[0064] In the present context, the term "mutant" should be understood as a strain derived from a strain of the present disclosure, for example by means of e.g. genetic engineering, radiation and / or chemical treatment. As shown in the examples herein it is also possible to obtain mutants by Adaptive Laboratory Evolution (ALE). It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties in particular in relation to the effects on secretion of carbohydrates, acidification time, and texturizing properties, as the deposited strain. Respective mutants represent embodiments of the present disclosure. The term "mutant" refers in particular to a strain obtained by subjecting a strain of the present disclosure to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening / selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening / selection steps) are carried out. In a presently preferred mutant, less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain.

[0065] In the present context, the term "variant" or "variant strain" should be understood as a strain which is functionally equivalent to a strain of the present disclosure, e.g. having substantially the same, or improved, properties or characteristics e.g. texture, acidification speed, viscosity, gel firmness, mouth coating, flavor, post acidification and / or phage robustness). Such variants, which may be identified using appropriate screening techniques, are a part of the present invention.

[0066] For purposes of the present disclosure, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. (2000) Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -no brief option) is used as the percent identity and is calculated as follows:

[0067] (Identical Residues x 100) I (Length of Alignment - Total Number of Gaps in Alignment)

[0068] In the present description and claims the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, amino acid changes in mutants and variants of the invention are described by use of the following nomenclature: amino acid residue in the parent enzyme; position; substituted amino acid residue(s). According to this nomenclature, the substitution of, for instance, an alanine residue for a glycine residue at position 20 is indicated as Ala20Gly or A20G. The deletion of alanine in the same position is shown as Ala20* or A20 *. The insertion of an additional amino acid residue (e.g. a glycine) is indicated as Ala20AlaGly or A20AG. The deletion of a consecutive stretch of amino acid residues (e.g. between alanine at position 20 and glycine at position 21) is indicated as DELTA(Ala20-Gly21) or DELTA(A20-G21). When a parent enzyme sequence contains a deletion in comparison to the enzyme sequence used for numbering an insertion in such a position (e.g. an alanine in the deleted position 20) is indicated as *20Ala or *20A. Multiple mutations are separated by a plus sign or a slash. For example, two mutations in positions 20 and 21 substituting alanine and glutamic acid for glycine and serine, respectively, are indicated as A20G+E21S or A20G / E21S. When an amino acid residue at a given position is substituted with two or more alternative amino acid residues these residues are separated by a comma or a slash. For example, substitution of alanine at position 30 with either glycine or glutamic acid is indicated as A20G,E or A20G / E, or A20G, A20E. When a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 20 is mentioned but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid residue (i.e. any one of R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

[0069] In the present context, a mutation in the gene (gene mutation) is to be understood as an alteration in the nucleotide sequence of the genome of an organism resulting in changes in the phenotype of said organism, wherein the alteration may be a deletion of a nucleotide, a substitution of a nucleotide by another nucleotide, an insertion of a nucleotide, or a frameshift. In the context of the present invention, a deletion is to be understood as a genetic mutation resulting in the removal of one or more nucleotides of a nucleotide sequence of the genome of an organism; a insertion is to be understood as the addition of one or more nucleotides to the nucleotide sequence; a substitution (or point mutation) is to be understood as a genetic mutation where a nucleotide of a nucleotide sequence is substituted by another nucleotide; a frameshift is to be understood as a genetic mutation caused by a insertion or deletion of a number of nucleotides in a nucleotide sequence that is not divisible by three, therefore changing the reading frame and resulting in a completely different translation from the original reading frame; an introduction of a stop codon is to be understood as a point mutation in the DNA sequence resulting in a premature stop codon; a inhibition of substrate binding of the encoded protein is to be understood as any mutation in the nucleotide sequence that leads to a change in the protein sequence responsible for preventing binding of a substrate to its catalytic site of the protein. Furthermore, a knockout mutant is to be understood as genetic mutation resulting in the removal or deletion of a gene, such as an entire gene or an entire open reading frame from the genome of an organism. In the present description and claims the conventional one-letter code for nucleotides is used following the analogous principles as described for amino acids nomenclature supra.

[0070] Algorithms for aligning sequences and determining the degree of sequence identity between them are well known in the art. For the purpose of the present invention a process may be carried out for aligning nucleotide sequences using blastn as provided by the National Center for Biotechnology Information (NCBI) on https: / / blast.ncbi.nlm.nih.gov applying standard parameter.

[0071] In one aspect the present disclosure relates to a Streptococcus thermophilus strain obtained by the method according to the present disclosure.

[0072] In the present context, the terms "Galactose fermenting", "Galactose-positive" or "Gal(+)" is used to refer to the ability of a lactic acid bacteria to metabolize galactose and utilize galactose as energy source when galactose is present as sole carbon source. Galactose fermentation can be defined as reduction of pH of at least 1.0 after 16 hours incubation at 37°C in M17 with 2% galactose (galactose added as sole carbohydrate), inoculated in an amount of at least 104cells / mL

[0073] The terms "Lactose fermenting", "Lactose-positive" or "Lac(+)" is used to refer to the ability of a lactic acid bacteria to metabolize lactose and utilize lactose as energy source when lactose is present as sole carbon source. The terms "Glucose fermenting", "Glucose-positive" or "Glu(+)" is used to refer to the ability of a lactic acid bacteria to metabolize glucose and utilize glucose as energy source when glucose is present as sole carbon source.

[0074] In one embodiment the present disclosure relates to a Streptococcus thermophilus strain with a Lac(+) Glu(+) Gal(+) phenotype and at least one mutation in the lacS gene encoding a lactose permease wherein the mutation leads to an amino acid substitution at a position corresponding to position 148, 377 and / or 476 in SEQ ID No:2.

[0075] In one embodiment the present disclosure relates to the strain, wherein the amino acid substitution is 148H, 377C and / or 476K.

[0076] In one embodiment the present disclosure relates to the strain, wherein the strain comprises a lactose permease with a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID No:4; SEQ ID No:6; SEQ ID No:8; SEQ ID No: 10; SEQ ID No: 12; SEQ ID No: 14; or SEQ ID No: 16.

[0077] In one embodiment the present disclosure relates to the strain, wherein the strain comprises a LacS gene with a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No: 11; SEQ ID No: 13; or SEQ ID No: 15.

[0078] The Lac(+) Glu(+) Gal(+) phenotype of the strain is a result of the genes involved in the carbohydrate metabolism. The presence of mutations in the genes encoding galactokinase (galK), glucosekinase (glcK) and phosphoglucomutase (pgm) have been observed in the strains of the present disclosure. See the description of these enzymes supra. In one embodiment the present disclosure relates to the strain, wherein the strain has a mutation in one or more of the genes selected from galactokinase (galK), glucosekinase (glcK) and phosphoglucomutase (pgm).

[0079] As described supra, at least one of genes encoding galactokinase (galK), glucosekinase (glcK) and phosphoglucomutase (pgm) is mutated in the strain, preferably galK. If two of the genes are mutated the preferred combination is galK+glcK or galK+pgm. Preferably, all three genes are mutated. If the mutated gene is galK the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No: 18. If the mutated gene is glcK the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No:20. If the mutated gene is pgm the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22. In one embodiment the present disclosure relates to the strain, wherein the mutated gene is: galK and the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No: 18; glcK and the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No:20; and / or pgm and the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22.

[0080] It is well known that a change in an amino acid may be caused by several changes in the gene sequence. The various nucleotide triplets i.e. codons encoding selected amino acids are listed in the table below. It is thus to be understood that any change in the gene sequence providing a codon that lead to an amino acid change identical to those described herein are also part of the present disclosure. An example is the change at position 148 to histidine in the LacS amino acid sequence shown as SEQ ID No:4 caused by a nucleotide change providing the codon "cat". However a change to histidine can also be caused by the codon "cac" which is thus included.

[0081] Table 3. List of selected codons and their amino acids. The strain exhibit a low excretion of at least one carbohydrate selected from Lactose, glucose, galactose and allolactose. The at least one carbohydrate may be one, two, three or four carbohydrates in any combination. In one embodiment the present disclosure relates to the strain exhibiting low excretion of one carbohydrate selected from lactose, glucose, galactose, or allolactose. In one embodiment the present disclosure relates to the strain exhibiting low excretion of two carbohydrates selected from lactose+glucose, lactose+galactose, lactose+allolactose, glucose+galactose, glucose+allolactose, or galactose+allolactose. In one embodiment the present disclosure relates to the strain exhibiting low excretion of three carbohydrates selected from lactose+glucose+galactose, lactose+glucose+allolactose, or glucose+galactose+allolactose. In one embodiment the present disclosure relates to the strain exhibiting low excretion of four carbohydrates selected from lactose+glucose+galactose+allolactose.

[0082] The low excretion of carbohydrates in the milk fermented with the strain of the present disclosure is below 1.1 mg / mL, preferably in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL. In one embodiment the present disclosure relates to the strain, wherein the amount of glucose in milk fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL. In one embodiment the present disclosure relates to the strain, wherein the amount of galactose in milk fermented with the strain is in the range of 0-1.0 mg / mL; 0- 0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL. In one embodiment the present disclosure relates to the strain, wherein the amount of allolactose in milk fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL. In one embodiment the present disclosure relates to the strain, wherein the amount of glucose and / or galactose in milk fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL.

[0083] In one embodiment the present disclosure relates to the strain, wherein the strain is (a) derived from a mother strain selected from: DSM 33677; DSM 33719 or DSM 35263; or (b) selected from: DSM 35264; DSM 35265; and mutants or variants thereof.

[0084] COMPOSITIONS

[0085] In one aspect the present disclosure relates to a composition, as a mixture or as a kit-of-part, comprising the strain according to the present disclosure.

[0086] LAB are most commonly added to milk in the form of a starter culture. In one embodiment the present disclosure relates to a composition, wherein said composition is a starter culture. The terms "starter culture" or "starter" as used in the present context refer to a culture of one or more food-grade microorganisms, in particular to lactic acid bacteria, which are responsible for the acidification of the milk base. Starter cultures may be fresh, but are most frequently frozen or freeze-dried. These products are also known as "Direct Vat Set" (DVS) cultures and are produced for direct inoculation of a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product such as a fresh dairy product or a cheese.

[0087] The composition may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof. The composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and / or nutrients, more preferably cryoprotectants, lyoprotectants and / or antioxidants and most preferably cryoprotectants or lyoprotectants, or both. Use of protectants such as cryoprotectants and lyoprotectants are known to a person skilled in the art. Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate). Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C). The composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and / or flavorants.

[0088] The composition of the present disclosure may be provided in several forms. It may be a powder, pellets or tablets. It may be a frozen form, dried form, freeze dried form, or liquid form. Thus, in one embodiment the composition is in frozen, dried, freeze-dried or liquid form. In one embodiment the composition or starter culture comprises lactic acid bacteria in a concentration of at least 107colony forming units per g (CFU / g) material, in a concentration of at least 108, at least 109, at least 1010, or in a concentration of at least 1011CFU / g material. In one embodiment of the present disclosure, the compositions or starter cultures comprise strains of the species Streptococcus thermophilus, wherein the strain is (a) derived from a mother strain selected from : DSM 33677; DSM 33719 or DSM 35263; or (b) selected from: DSM 35264; DSM 35265; and mutants or variants thereof. FERMENTED PRODUCTS AND METHODS FOR THEIR MANUFACTURE

[0089] Fermented products can be manufactured by adding the strains or the compositions of the present disclosure to a milk base.

[0090] In the present context, the term "milk" is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals or by plants. In accordance with the present disclosure the milk may have been processed and the term "milk" includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-reduced milk, or concentrated milk. Fat-free milk is non-fat or skim, milk product. Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat. Full fat milk often contains 2% fat or more. The term "milk" is intended to encompass milks from different mammal and plant sources. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, lama, mare and deer. Plant sources of milk include, but are not limited to, milk extracted from soy bean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed. In the methods and products of the present disclosure, milk derived from cows is most preferably used as a starting material for the fermentation.

[0091] The term "milk" also includes fat-reduced and / or lactose-reduced milk products. Respective products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatograph and centrifugation.

[0092] The term "milk base" is broadly used in the present disclosure to refer to a substrate based on milk or milk components which can be used as a medium for growth and fermentation of LAB. The milk base comprises components derived from milk and any other component that can be used for the purpose of growing or fermenting LAB incl. plant based components.

[0093] Prior to fermentation, the milk base may be homogenized and pasteurized according to methods known in the art. "Homogenizing" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices. "Pasteurizing" as used herein means treatment of the milk base to reduce or eliminate the presence of live organisms, such as microorganisms. Preferably, pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow. Fermentation is carried out to produce fermented products, preferably dairy products. The terms "fermented product", "milk product" or "dairy product" refer to products obtainable by the fermentation methods of the present disclosure and include cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais and cream cheese.

[0094] The term "cheese" is understood to encompass any cheese, including hard, semi-hard and soft cheeses, such as cheeses of the following types: Cottage, Feta, Cheddar, Parmesan, Mozzarella, Emmentaler, Danbo, Gouda, Edam, Feta-type, blue cheeses, brine cheeses, Camembert and Brie. Cheese that is heated before consumption is of particular interest such as e.g. cheese used for pizza, lasagna and the like. The person skilled in the art knows how to convert the coagulum into cheese, methods can be found in the literature, see e.g. Kosikowski, F. V., and V. V. Mistry, "Cheese and Fermented Milk Foods", 1997, 3rd Ed. F. V. Kosikowski, L. L. C. Westport, CT. As used herein, a cheese which has a NaCI concentration below 1.7% (w / w) is referred to as a "low-salt cheese".

[0095] In the context of the present disclosure, the term "yoghurt" refers to products comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lacticaseibacillus paracasei subsp. paracasei. The lactic acid strains other than Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, are included to give the finished product various properties, such as the property of promoting the equilibrium of the flora. As used herein, the term "yoghurt" encompasses set yoghurt, stirred yoghurt, drinking yoghurt, Petit Suisse, heat treated yoghurt, strained or Greek style yoghurt characterized by a high protein level and yoghurt-like products.

[0096] In particular, term "yoghurt" encompasses, but is not limited to, yoghurt as defined according to French and European regulations, e.g. coagulated dairy products obtained by lactic acid fermentation by means of specific thermophilic lactic acid bacteria only (i.e. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus') which are cultured simultaneously and are found to be live in the final product in an amount of at least 10 million CFU (colony-forming unit) I g. Yoghurt may optionally contain added dairy raw materials (e.g. cream) or other ingredients such as sugar or sweetening agents, one or more flavoring(s), fruit, cereals, or nutritional substances, especially vitamins, minerals and fibers, as well as stabilizers and thickeners. Optionally the yoghurt meets the specifications for fermented milks and yoghurts of the AFNOR NF 04-600 standard and / or the codex StanA-IIa-1975 standard. In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m / m) of the finished product.

[0097] In one aspect the present disclosure relates to a method for manufacturing a fermented product comprising the steps: (a) adding the strain or the composition according to the present disclosure to a milk base; and (b) fermenting the milk base until a target pH is reached to obtain a fermented product, preferably a fermented dairy product. In one embodiment the present disclosure relates to the method wherein the target pH is no more than pH 4.70: 4.65; 4.60; 4.55; 4.50; 4.45; 4.40; 4.35; 4.30; 4.25; 4.20; 4.15; 4.10; 4.05; 4.00; 3.95; 3.90; 3.85; 3.80; 3.75; 3.70; 3.65; 3.60; 3.55; 3.50, or is in the range of pH 4.70- 4.00; 4.70-4.10; 4.70-4.20; 4.70-4.30; 4.70-4.40; 4.70-4.45; 4.65-4.50; 4.60-4.55; 4.50-3.50; 4.50-4.05; 4.45-4.10; 4.45-4.15; 4.40-4.20; 4.40-4.25; 4.35-4.30; 4.00-3.50; 3.95-3.50; 3.90-3.55; 3.85-3.60; 3.80-3.65; 3.75-3.60, or is about pH 4.70: 4.65; 4.60; 4.55; 4.50; 4.45; 4.40; 4.35; 4.30; 4.25; 4.20; 4.15; 4.10; 4.05; 4.00; 3.90; 3.80; 3.70; 3.60; 3.50.

[0098] In one embodiment the present disclosure relates to the method wherein the fermented dairy product is selected from cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais and cream cheese.

[0099] In another aspect the present disclosure relates to a fermented product comprising the strain or the composition according to the present disclosure.

[0100] In one embodiment the present disclosure relates to a fermented product according to the present disclosure, wherein the fermented product is obtained by the method according to the present disclosure.

[0101] In connection with S. thermophilus, the term "CFU" means colony forming units as determined by growth (forming a colony) on an M17 agar plate incubated at aerobic conditions at 37°C for 3 days.

[0102] The M17 agar has the following composition (g / l) :

[0103] Tryptone: 2.5 g

[0104] Peptic digest of meat: 2.5 g

[0105] Papaic digest of soybean meal: 5.0 g

[0106] Yeast extract: 2.5 g

[0107] Meat extract: 5.0 g

[0108] Lactose: 5.0 g

[0109] Sodium-glycero-phosphate: 19.0 g Magnesium sulphate, 7 H2O: 0.25 g Ascorbic acid : 0.5 g Agar: 15.0 g

[0110] Milli-Q water: 1000 ml. final pH 7.1 ±0.2 (25°C)

[0111] In one embodiment the present disclosure relates to a fermented product, wherein the strain is present in a concentration of at least 107, at least 108, at least 109, at least 1010, at least 1011, or at least 1012CFU / g product. In one embodiment the present disclosure relates to a fermented product, wherein the strain is present in a concentration of at least 107CFU / g product.

[0112] USE

[0113] It has surprisingly been observed that the Streptococcus thermophilus strains of the present disclosure exhibit a low excretion of carbohydrates. Use of the strains for fermentation lead to a reduced concentration of carbohydrates as compared to the use of the mother strain. In one embodiment the present disclosure relates to use of the strain, wherein the content of carbohydrates in a fermented product is reduced as compared to a fermented product manufactured by use of the mother strain. The strains of the present disclosure such as DSM 35264 and DSM 35265 excrete low amounts of galactose which reduce accumulation of galactose in the fermented product and accordingly reduce browning during exposure to heattreatment. In one embodiment the present disclosure relates to use of the strain to reduce browning of the fermented product during exposure to heat-treatment.

[0114] One of the strains of the present disclosure has shown a reduced fermentation time, i.e. it acidifies faster to a target pH as compared to the mother stain. In one embodiment the disclosure relates to use of the strain, wherein the fermentation time is reduced in the manufacturing of a fermented product as compared to use of the mother strain.

[0115] Use of the strain by inclusion in a starter culture together with other strains that metabolizes the carbohydrates which are excreted in low amount by the strains of the present disclosure may be a tool for controlling the fermentation. In particular, when the carbohydrate in question is limited. In one embodiment the present disclosure relates to use of the strain in a starter culture. In one embodiment the present disclosure relates to use of the strain in a starter culture for controlling the fermentation.

[0116] It has also been observed that the strains of the present disclosure provide increased texture in fermented milk. In one embodiment the present disclosure relates to use of the strain for increasing texture in fermented milk as compared to use of the mother strain. In one aspect the present disclosure relates to use of the strain or the composition according to the present disclosure for: reducing the content of carbohydrates in a fermented product; reducing the fermentation time; and / or increasing the texture in a fermented product.

[0117] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be constructed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0118] SEQUENCES

[0119] Gene sequences are provided in lowercase, and upstream nucleotides are underlined. Protein sequnces are provided in UPPERCASE.

[0120] SEQ ID No:l lacS gene incl. 100 bp upstream. atatattaataaaaatttaataaaaaacactaaaattattaactacataaaccaattttcatataatataaacatattcaaataatag aaaatttccaaaatqaaaaaatctaaaqatcaaataaaatctcatttatcctacacaactqatacttttqgtaacaacatcttctata caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcctgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtaggt gttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatcg ttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaact agccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaat acacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttgg tttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggggt atcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttccttgt tgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacaccgtgatgaatcacttactttgtcagttcgtccac ttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcatca acaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattgctc gtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaatgaa gttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcaggt agcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctactcg ccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaaggattc attagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtcttg atgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaagctt tgtctgaagttattaccttcaaaaaaggagaataa

[0121] SEQ ID No:2 LacS amino acid sequence.

[0122] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0123] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW SMIPALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT

[0124] SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV VFMIISDSVEYGQWKTGHRDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENEVKANVVSLVTPTTGYLVDLSSVN DEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYVE QGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0125] SEQ ID No:3 lacS C543A gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcatgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtagg tgttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatc gttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaa ctagccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaa tacacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttg gtttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggg gtatcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttcctt gttgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacaccgtgatgaatcacttactttgtcagttcgtcc acttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcat caacaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattg ctcgtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaatg. aagttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcag gtagcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctact cgccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaagga ttcattagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtc ttgatgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaag ctttgtctgaagttattaccttcaaaaaaggagaataa

[0126] SEQ ID No:4 LacS P148H amino acid sequence.

[0127] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0128] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW SMIHALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV VFMIISDSVEYGQWKTGHRDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENEVKANVVSLVTPTTGYLVDLSSVN

[0129] DEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYVE QGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0130] SEQ ID No:5 lacS C1229T gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcctgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtaggt gttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatcg ttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaact agccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaat acacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttgg tttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggggt atcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttccttgt tgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacactgtgatgaatcacttactttgtcagttcgtccact tattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcatca acaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattgctc gtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaatgaa gttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcaggt agcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctactcg ccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaaggattc attagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtcttg atgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaagctt tgtctgaagttattaccttcaaaaaaggagaataa

[0131] SEQ ID No:6 LacS R377C amino acid sequence.

[0132] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0133] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW SMIPALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT

[0134] SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV VFMIISDSVEYGQWKTGHCDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENEVKANVVSLVTPTTGYLVDLSSVN DEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYVE QGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0135] SEQ ID No:7 lacS G1526A gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcctgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtaggt gttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatcg ttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaact agccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaat acacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttgg tttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggggt atcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttccttgt tgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacaccgtgatgaatcacttactttgtcagttcgtccac ttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcatca acaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattgctc gtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaataaag ttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcaggta gcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctactcgc catgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaaggattca ttagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtcttga tgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaagcttt gtctgaagttattaccttcaaaaaaggagaataa

[0136] SEQ ID No:8 LacS E476K amino acid sequence.

[0137] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0138] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW

[0139] SMIPALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT

[0140] SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG

[0141] KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV

[0142] VFMIISDSVEYGQWKTGHRDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENKVKANVVSLVTPTTGYLVDLSSV

[0143] NDEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYV EQGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0144] SEQ ID No:9 lacS C543A+C1229T gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcatgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtagg tgttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatc gttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaa ctagccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaa tacacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttg gtttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggg gtatcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttcctt gttgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacactgtgatgaatcacttactttgtcagttcgtcc acttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcat caacaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattg ctcgtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaatg. aagttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcag gtagcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctact cgccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaagga ttcattagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtc ttgatgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaag ctttgtctgaagttattaccttcaaaaaaggagaataa

[0145] SEQ ID No: 10 LacS P148H + R.377C amino acid sequence.

[0146] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0147] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW SMIHALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV VFMIISDSVEYGQWKTGHCDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENEVKANVVSLVTPTTGYLVDLSSVN

[0148] DEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYVE QGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0149] SEQ ID No: ll lacS C543A+G1526A gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcatgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtagg tgttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatc gttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaa ctagccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaa tacacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttg gtttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggg gtatcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttcctt gttgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacaccgtgatgaatcacttactttgtcagttcgtcc acttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcat caacaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattg ctcgtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaata aagttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcag gtagcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctact cgccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaagga ttcattagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtc ttgatgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaag ctttgtctgaagttattaccttcaaaaaaggagaataa

[0150] SEQ ID No: 12 LacS P148H+E476K amino acid sequence.

[0151] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0152] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW

[0153] SMIHALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT

[0154] SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG

[0155] KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV

[0156] VFMIISDSVEYGQWKTGHRDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENKVKANVVSLVTPTTGYLVDLSSV

[0157] NDEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYV EQGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0158] SEQ ID No: 13 lacS C1229T+G1526A gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcctgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtaggt gttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatcg ttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaact agccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaat acacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttgg tttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggggt atcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttccttgt tgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacactgtgatgaatcacttactttgtcagttcgtccact tattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcatca acaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattgctc gtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaataaag ttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcaggta gcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctactcgc catgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaaggattca ttagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtcttga tgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaagcttt gtctgaagttattaccttcaaaaaaggagaataa

[0159] SEQ ID No: 14 LacS R.377C+E476K amino acid sequence.

[0160] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP

[0161] LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW

[0162] SMIPALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT

[0163] SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG

[0164] KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV

[0165] VFMIISDSVEYGQWKTGHCDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENKVKANVVSLVTPTTGYLVDLSSV

[0166] NDEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYV EQGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0167] SEQ ID No: 15 lacS C543A+C1229T+G1526A gene incl. 100 bp upstream. caaccttgtcaacatactttatcatgtttgtgacaactcacttgtttaacacaggtgatccaaagcaaaatagtcactacgtactatta atcactaacattatctctattttgcgtatcttggaagtatttatcgatccattgatcggtaatatgattgataacactaatactaagtatg gtaaattcaaaccatgggtagttggtggtggtatcatcagttctatcaccttgttgcttctcttcaccgatttaggtggtttgaataaaa caaatcctttcttgtaccttgtactttttggaattatctaccttgtaatggatgtcttctactcgattaaagatatcggtttctggtcaatg attcatgccttgtctcttgatagtcacgaacgtgaaaaaatggcaacttttgcccgtattggttctacgattggtgccaatattgtagg tgttgccatcatgccaatcgttttgttcttctctatgacgaacaatagtggctctggagataaatctggatggttctggtttgcatttatc gttgctctcattggtgtgattacatcaattgctgttggtattggtacacgtgaagttgagtcaaaaattcgtgataataacgaaaaaa ctagccttaaacaagtctttaaggttcttggtcaaaacgaccaattgatgtggttatctcttggatattggttctatggtcttggtattaa tacacttaatgctcttcaactttattatttcacatttatccttggtgattcaggtaaatactcaattctttacggattgaatacagttgttg gtttggtttcagtttcactcttccctaccctagctgataaattcaaccgtaaacgtttgttctacggatgtattgcagtaatgctcgggg gtatcggaatatttagtattgcaggtacatcacttccaataatcttgactgcagctgaactcttcttcattccacaacctcttgtgttcctt gttgtctttatgattatctctgactcagtagaatatggtcaatggaaaacgggacactgtgatgaatcacttactttgtcagttcgtcc acttattgataaacttggtggtgcgatgtcaaactggcttgtttctacatttgccgtagctgccggtatgacaacaggtgcctcagcat caacaattacaacacatcaacagtttatctttaagcttggcatgtttgctttcccagcagcaacaatgcttatcggtgccttcattattg ctcgtaaaatcactttgactgaagcacgtcacgctaaaattgttgaagaattggaacatcgctttagcgtagcaacttctgaaaata aagttaaagctaacgtcgtatctcttgtaacccctacaactggttatttggttgatctctcaagtgttaatgatgaacactttgcttcag gtagcatgggtaaaggtttcgccattaaacctactgatggagctgtctttgcaccaattagtggtaccattcgtcaaattcttcctact cgccatgcagttggtattgaaagtgaagatggtgtcattgttcttatccacgttggcatcggaacagttaaacttaatggtgaagga ttcattagttacgtagaacaaggtgatcgtgttgaagttggacaaaaacttcttgagttctggtcaccaattattgagaaaaatggtc ttgatgacacagtacttgtcactgtaactaattcagaaaaattcagtgctttccatcttgaacaagaagttggagaaaaggtagaag ctttgtctgaagttattaccttcaaaaaaggagaataa

[0168] SEQ ID No: 16 LacS P148H + R.377C+E476K amino acid sequence.

[0169] MEKSKGQMKSRLSYAAGAFGNDVFYATLSTYFIMFVTTHLFNTGDPKQNSHYVLLITNIISILRILEVFIDP LIGNMIDNTNTKYGKFKPWVVGGGIISSITLLLLFTDLGGLNKTNPFLYLVLFGIIYLVMDVFYSIKDIGFW SMIHALSLDSHEREKMATFARIGSTIGANIVGVAIMPIVLFFSMTNNSGSGDKSGWFWFAFIVALIGVIT SIAVGIGTREVESKIRDNNEKTSLKQVFKVLGQNDQLMWLSLGYWFYGLGINTLNALQLYYFTFILGDSG KYSILYGLNTVVGLVSVSLFPTLADKFNRKRLFYGCIAVMLGGIGIFSIAGTSLPIILTAAELFFIPQPLVFLV VFMIISDSVEYGQWKTGHCDESLTLSVRPLIDKLGGAMSNWLVSTFAVAAGMTTGASASTITTHQQFIF KLGMFAFPAATMLIGAFIIARKITLTEARHAKIVEELEHRFSVATSENKVKANVVSLVTPTTGYLVDLSSV NDEHFASGSMGKGFAIKPTDGAVFAPISGTIRQILPTRHAVGIESEDGVIVLIHVGIGTVKLNGEGFISYV EQGDRVEVGQKLLEFWSPIIEKNGLDDTVLVTVTNSEKFSAFHLEQEVGEKVEALSEVITFKKGE*

[0170] SEQ ID No: 17 ga / K gene sequence. atgaatacatcacagttaagagaaaagtttaaagaagtttttggtgtagaagcagatcatactttcttttcaccaggtcgtattaattt gattggtgagcatacggactacaatggaggtaacgtccttccggtagctattaccctaggtacttacggagcggcccgcaaacgtg atgacaaagttttgcgtttcttctcagctaactttgaagagaagggaatcatcgaagtgccacttgaaaatcttcgttttgaaaaaga acacaactggacaaactatccaaaaggtgttcttcatttcttgcaagaagctgggcatacgattgattcaggtatggatatttacat ctatggtaacattccaaacggatcaggcttgtcatcatcatcatctttggaattattgattggtgttattgttgaaaaactttatgacct taaattggaacgcctggacttggttaaaatcggaaaacaaacggaaaatgactttattggcgttaactctggtatcatggaccaatt cgctattggtatgggagctgatcaatgtgcgatttacttggacacaaatactctaaagtatgacttggtaccccttgacctcaaggat aatgtcgtagtcatcatgaacactaacaaacgtcgtgaattggctgattctaaatacaatgaacgtcgtgctgaatgtgaaacagc agtatctgaactacaagaaaaattggatatccaaactctcggtgaattagacttcttgacatttgacgcatacagctatttgattaaa gatgaaaaccgtatcaaacgtgcacgccatgtagttcttgaaaatcaacgtacacttcaagctcgtaaagctcttgaaacaggaga tttggaaggctttggacgccttatgaatgcttctcatgtgtcattggaatatgattacgaagttacaggtcttgaacttgatactttggc acacacagcttgggaacaagaaggagtattaggagcccgcatgacaggagctggtttcggtggatgtgccattgcacttgtaaac aaagacaaagttgaagacttcaaaaaagcagttggtcaacgctatgaagaagtcgttggttatgcaccaagcttctatattgccga agtaactggtggttcacgagtacttgattaa

[0171] SEQ ID No: 18 GalK amino acid sequence.

[0172] MNTSQLREKFKEVFGVEADHTFFSPGRINLIGEHTDYNGGNVLPVAITLGTYGAARKRDDKVLRFFSANF EEKGIIEVPLENLRFEKEHNWTNYPKGVLHFLQEAGHTIDSGMDIYIYGNIPNGSGLSSSSSLELLIGVIV EKLYDLKLERLDLVKIGKQTENDFIGVNSGIMDQFAIGMGADQCAIYLDTNTLKYDLVPLDLKDNVVVIM NTNKRRELADSKYNERRAECETAVSELQEKLDIQTLGELDFLTFDAYSYLIKDENRIKRARHVVLENQRTL QARKALETGDLEGFGRLMNASHVSLEYDYEVTGLELDTLAHTAWEQEGVLGARMTGAGFGGCAIALVN KDKVEDFKKAVGQRYEEVVGYAPSFYIAEVTGGSRVLD SEQ ID No: 19 glcK gene sequence. atgagtaagaaactcttaggtattgaccttggtggaacaactgttaagtttggtattttgactgcagatggtgaagttcaagaaaaa tgggctattgaaacaaatacgtttgaaaatggtagccacattgttcctgacattgtagaatctttgaaacaccgtttggaattgtatg gacttactgctgaagattttattggaattggtatgggatctccaggtgcagttgaccgagaaaataaaacagtaacgggtgccttta acttgaactgggcagaaactcaagaagttggctctgttattgaaaaagaacttggtattccattcgctattgataatgatgctaatgt ggctgcactgggtgaacgttgggttggtgctggtgctaacaatcggaatgttgtctttgtaacattgggtacaggtgttggtggcgg tgttatcgctgatggtaacttaattcatggtgttgccggtgctggtggggaaattggtcacattattgttgaacctgacacaggatttg agtgtacttgcggaaacaaggggtgtctggaaactgtagcttcagcaacaggtattgtacgtgtagcacatcatttggcagaaaaa tacgaaggaaactcttctattaaagctgctgtagacaatggtgagtttgtgacaagtaaagatattatcgtagctgctactgaaggt gataagtttgctgacagcattgttgataaagtctctaaatacctcggacttgcaacagcaaacatctcaaacattcttaacccagatt ctgtcgttatcggtggtggtgtttctgccgcaggagaattcttgcgtagtcgtgttgaaggatactttacacgttatgcattcccacaa gttcgccgtacaacaaaagtgaaattagcggagcttggaaatgatgcaggaatcattggagctgctagtcttgcttatagtattgac aaataa

[0173] SEQ ID No:20 GlcK amino acid sequence.

[0174] MSKKLLGIDLGGTTVKFGILTADGEVQEKWAIETNTFENGSHIVPDIVESLKHRLELYGLTAEDFIGIGM GSPGAVDRENKTVTGAFNLNWAETQEVGSVIEKELGIPFAIDNDANVAALGERWVGAGANNRNVVFVT LGTGVGGGVIADGNLIHGVAGAGGEIGHIIVEPDTGFECTCGNKGCLETVASATGIVRVAHHLAEKYEG NSSIKAAVDNGEFVTSKDIIVAATEGDKFADSIVDKVSKYLGLATANISNILNPDSVVIGGGVSAAGEFL RSRVEGYFTRYAFPQVRRTTKVKLAELGNDAGIIGAASLAYSIDK*

[0175] SEQ IN No:21 pgm gene sequence. atgtcttacactgaaaattatcaaaaatggctcgattttgctgaattgcctgcttatcttcgtgatgagttggtttctatggatgaaaaa actaaagaagatgccttctatactaatcttgaattcggtacagctggtatgcgtggtttaattggcgctggtaccaaccgtattaacat ttatgttgttcgtcaagcaaccgaaggtttggcgcaattgatcgactcaaaaggtgaagaagctaaaaaacgtggtgttgctatcg cttatgacagccgtcatttctctcctgagtttgcttttgaatctgcgcaagttttagcagctcatggtattaaatcttatgtgttcgagag ccttcgtccaactcctgaattatcattcgcagtacgtcatctccacacatttgctggtatcatgataacggctagccacaacccagctc cattt aacggatacaaagtttatggtgaagacggtggacaaatgccacccgctgatgccgatgcattgacagatt acatccgtgc tatcgataatcctttcactgtcaaattagctgacctcgaagacagcaaggctagcggtctcatcgaaatcatcggtgaaaatgttga tgctgaatacctaaaagaagttaaagacgttaacatcaaccaggacttgattaatgagtatggtcgtgacatgaagattgtataca cttcacttcatggtactggtgaaatgttggttcgccgtgcccttgctcaagctgggtttgatgctgttcaagtcgttgaagctcaagcg gttcctcatgctgacttcttaactgttaaatctcctaacccagaaaaccaagatgcctttgctcttgctgaagaactcggtcgtaatgt agatgccgacgtattggttgcaactgaccctgacgcggaccgtcttggcgttgaaatccgccaaccagatggttcatacctcaacct ttctggtaaccaaattggtgctatgaagctcacaaaacagctggtactctccctgctaatgctgccctttgtaaatcaatcgtatcaac tgaattagttactaagattgcagaaagctacggcgcaacaatgtttaatgtcttgactggcttcaaatttatcggtgaaaagattcat gaatttgaaacacaacacaattacacttacatgtttggttttgaagaaagcttcggttacctcatcaaaccatttgtacgcgataaag acgctatccaagccgttcttatcgttgcagaaattgctgcatactaccgttcacgtggtatgacattggcagatggtatcgaagaaat ctacaaacaatatggttacttctcagaaaagacaatttcagttatgctttcaggtgttgatggtgctgcagaaatcaagaaaatcatg gacaaattccgtcgcaatgctcctaaacaattcaacaacactgatattgctaaaacagaagacttcttggaacaaacagctactact gctgacggcgtagaaaaattgacaactcctccaagtaacgttttgaaatacatcttggctgatgattcatggtttgccgttcgtccttc aggtacagaaccaaaaatcaaattttacattgcaacagttggagaaactgaagcggatgctaaagaaaaaattgctaacatcga agcagaaatcaatgcttttgtaggtgaatag

[0176] SEQ ID No:22 Pgm amino acid sequence.

[0177] MSYTENYQKWLDFAELPAYLRDELVSMDEKTKEDAFYTNLEFGTAGMRGLIGAGTNRINIYVVRQATEG LAQLIDSKGEEAKKRGVAIAYDSRHFSPEFAFESAQVLAAHGIKSYVFESLRPTPELSFAVRHLHTFAGIM ITASHNPAPFNGYKVYGEDGGQMPPADADALTDYIRAIDNPFTVKLADLEDSKASGLIEIIGENVDAEYL KEVKDVNINQDLINEYGRDMKIVYTSLHGTGEMLARRALAQAGFDAVQVVEAQAVPHADFLTVKSPNPE NQDAFALAEELGRNVDADVLVATDPDADRLGVEIRQPDGSYLNLSGNQIGAIIAKYILEAHKTAGTLPAN AALCKSIVSTELVTKIAESYGATMFNVLTGFKFIGEKIHEFETQHNYTYMFGFEESFGYLIKPFVRDKDAIQ

[0178] AVLIVAEIAAYYRSRGMTLADGIEEIYKQYGYFSEKTISVTLSGVDGAAEIKKIMDKFRRNAPKQFNNTDI AKTEDFLEQTATTADGVEKLTTPPSNVLKYILADDSWFAVRPSGTEPKIKFYIATVGETEADAKEKIANIE AEINAFVGE.

[0179] DEPOSITS AND EXPERT SOLUTION

[0180] The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

[0181] Table 4. Deposits were made according to the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

[0182] The strains DSM 33677 and DSM 33719 have been described in WO2022 / 106418 and W02022 / 180071 respectively.

[0183] EXAMPLES MATERIAL & METHODS

[0184] Streptococcus thermophilus strains were grown in the following media.

[0185] M17 medium with the following composition per litre H2O: agar, 12.75 g ascorbic acid, 0.5 g casein peptone (tryptic), 2.5 g disodium p-glycerophosphate penta hydrate, 19 g magnesium sulfate hydrate, 0.25 g meat extract, 5 g meat peptone (peptic), 2.5 g soyapeptone (papainic), 5 g yeast extract, 2.5 g final pH 7.1 ±0.2 (25°C)

[0186] M17 broth has the following composition per litre H2O: ascorbic acid, 0.5 g magnesium sulfate, 0.25 g meat extract, 5 g meat peptone (peptic), 2.5 g sodium glycerophosphate, 19 g soya peptone (papainic), 5 g tryptone, 2.5 g yeast extract, 2.5 g final pH 7.0±0.2 (25°C)

[0187] Carbon sources added sterile:

[0188] Sucrose + 0,2 g / l lactose 20 g / l, galactose 20 g / l or 100g / L CasHy: Casein hydrolysate 2%.

[0189] 9.5% B-milk was prepared from reconstituted low fat skim milk powder adjusted to a level of dry matter of 9.5% and pasteurized at 99°C for 30 min. followed by cooling to 40°C or 43°C.

[0190] EXAMPLE 1

[0191] Generation of DSM 35264. Development of the galactose fermenting strain DSM 33719 has been described in W02022 / 180071. Further development of DSM 33719 to DSM 35264 was initiated to obtain faster growing galactose fermenting mutants under aerobic conditions. The method used was manual adaptive laboratory evolution (ALE) where transfers of culture were done daily manually into fresh M17+galactose medium.

[0192] From overnight cultures in M17+2% galactose (OD~4), 1 ml was first transferred into 100 ml M17+2% galactose in 250 ml Erlenmeyer flasks and placed in a Thermoshaker (Innova® 42R Eppendorf) at 40°C. The shaking intensity was gradually changed during the ALE experiment starting with 40 rpm and ending with 100 rpm for the last 2 growths. The galactose concentration was kept at 2% until the last two passes, 10 to 11, where the flasks contained M17+10% galactose. The transfers were done every morning by inoculating 1% culture from the surface of the flask (no aggregation) into to fresh prewarmed media. Before every new pass, a sample of each culture was frozen and stored, optical density (ODeoo) was measured, and morphology was inspected by microscopy.

[0193] Cells from the last passages were diluted, plated on M17+2% galactose, and then incubated under both anaerobic and aerobic conditions. Only colonies appearing from aerobic conditions were used for further characterization. A total of 79 single isolates were examined together with the mother strain DSM 33719 for growth in M17+2% lactose, M17+2% galactose and M17+ 10% galactose in deep well microtiter plates and incubated at 40°C ON. The optical density was measured in a BioTek LogPhase 600 Microbiology Reader. From the candidates showing improved growth in M17+10% galactose and on M17+2% lactose, we selected DSM 35264.

[0194] Sequence analysis of the DSM 35264 gene as compared to the mother strain DSM 33719 revealed presence of a C543A mutation in the nucleotide sequence of the lacS gene leading to an amino acid change from proline to histidine a position 148 (P148H) in the lactose permease (LacS) as shown in the gene sequence SEQ ID No:3 and the amino acid sequence SEQ ID No:4. The difference between DSM 35264 as compared to DSM 33719 indicated that this combination of mutations enables the strain to utilize lactose in a more rational way and monosaccharides coming from lactose, galactose and glucose, can be utilize simultaneously and little is wasted into the milk.

[0195] EXAMPLE 2

[0196] Acidification of profile of DSM 35264.

[0197] Subsequently, the acidification profiles of DSM 35264 was compared to DSM 33719 by fermenting 200mL B-milk+0.05% sucrose 40°C in baby bottles using / Cinac. The milk was inoculated with 1% from a pre-culture (OD~4) pregrown in M17+2% galactose and 2% CasHy 40°C. The acidification profiles for DSM 35264 and DSM 33719 are shown in figure 1.

[0198] Acidification to pH 5.0 was 5 hours for DSM 35264 and 10 hours for DSM 33719. Time to pH 4.5 was 14 hours for DSM 35264 and 19 hours for DSM 33719. It is apparent that DSM 35264 acidifies milk significantly faster than the mother strain DSM 33719.

[0199] EXAMPLE 3

[0200] Carbohydrate analysis DSM 35264.

[0201] Acidification of B-milk was set up as described in example 2 and analysis of carbohydrates present in the milk at the indicated time points was conducted. Fermented milk samples were prepared for carbohydrate analysis using HPLC by weighing Ig + / - 0.5g into lOmL centrifuge tubes. 2mL ice cold 96% ethanol was added, the samples where whirly-mixed and placed at -50°C until analyzed by HPLC. B-milk alone was included as control and showed limited change in concentration of lactose and no detectable concentrations of glucose and galactose.

[0202] From the table below it is apparent that during fermentation with the mother strain the concentration of lactose is reduced and the amounts of glucose, galactose and allolactose are increased. When fermenting with the lacS mutant the concentration of lactose is reduced to a lesser degree and the amounts of glucose, galactose are very low and allolactose is not detectable.

[0203] Table 5. Concentration of carbohydrates at different time points during fermentation with DSM33719 or DSM 35264. EXAMPLE 4

[0204] Texturizing properties of DSM 35264.

[0205] B-milk fermented with DSM 35264 or DSM 33719 from example 2 were tested for texture. Efflux time in triplicates from a 25 mL pipette was measured in seconds and showed in the table below. The result shows that the mutant strain is in average 146.3 sec. to empty the pipette as compared to the mother strain which only need 33.3 sec. Thus DSM 35264 provides more texture than DSM 33719.

[0206] Table 6. Efflux time from a 25 mL pipette

[0207] EXAMPLE 5

[0208] Generation of DSM 35265, acidification profile and carbohydrate analysis.

[0209] Development of DSM 35265 from its mother strain DSM 35263 was done by adaptive laboratory evolution (ALE) on galactose similar to the method as described in example 1.

[0210] Sequence analysis of the DSM 35265 gene as compared to DSM 35263 revealed presence of two mutations C1229T and G1526A in the nucleotide sequence of the lacS gene leading to an amino acid change from Arginine to Cysteine a position 377 (R.377C) and Glycine to Alanine at position 476 (G476K) in the lactose permease (LacS) as shown in the gene sequence SEQ ID No: 13 and the amino acid sequence SEQ ID No: 14.

[0211] Table 7. Acidification profile Table 8. Concentration of carbohydrates in milk fermented with DSM 35265.

[0212] (Original in Electronic Form)

[0213] (This sheet is not part of and does not count as a sheet of the international application)

[0214] (Original in Electronic Form)

[0215] (This sheet is not part of and does not count as a sheet of the international application) 3 / 4

[0216] PCT

[0217] (Original in Electronic Form)

[0218] (This sheet is not part of and does not count as a sheet of the international application)

[0219] FOR RECEIVING OFFICE USE ONLY (Original in Electronic Form)

[0220] (This sheet is not part of and does not count as a sheet of the international application)

[0221] FOR INTERNATIONAL BUREAU USE ONLY

Claims

36CLAIMS1. A method for producing a Streptococcus thermophilus strain exhibiting a low excretion of carbohydrates comprising the steps:(a) providing a mother strain which is able to ferment lactose and glucose;(b) selecting a mutant strain derived from the mother strain which mutant strain has a change in:(i) one or more of the genes encoding a protein involved in the metabolism of galactose wherein said strain is able to ferment galactose; and(ii) the LacS gene encoding a lactose permease which change results in at least one mutation in the amino acid sequence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID NO:2; and wherein the mutant strain has a Lac(+) Glu(+) Gal(+) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain.

2. The method according to claim 1, wherein the amino acid sequence of lactose permease at the position corresponding to position 148 is Histidine (H), position 377 is Cysteine (C), and position 476 is Lysine (K).

3. The method according to any one of the proceeding claims, wherein the carbohydrate is one or more selected from: lactose, allolactose, glucose, and galactose.

4. The method according to any one of the proceeding claims, wherein the reduced secretion of carbohydrates results in a concentration of glucose and / or galactose in a milk base fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0-0.8; 0- 0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL.

5. The method according to any one of the proceeding claims, wherein the genes involved in the metabolism of galactose is selected from galactokinase galK), glucosekinase glcK) and phosphoglucomutase (pgrr).

6. The method according to claim 5, wherein the mutated gene is: ga!K and the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No:18; glcK and the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No:20; and / or pgm and the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22.

7. The method according to any one of the proceeding claims, wherein step l(b)(i) is conducted before step l(b)(ii), or step l(b)(ii) is conducted before step l(b)(i).

8. The method according to any one of the preceding claims, wherein the mother strain is Gal(+ ) .

9. The method according to any one of the preceding claims, said method comprising the steps of:(a) Providing a mother strain which is able to ferment lactose and glucose;(b) Selecting a mutant strain derived from the mother strain, wherein the selection comprises:(i) Cultivating the mother strain under selective conditions in a medium comprising galactose, preferably where galactose is the primary or sole carbon source, thereby applying selective pressure for the emergence or enhancement of galactose fermentation capability;(ii) Serially transferring or sub-culturing the culture over multiple generations, maintaining the selective environment to favor the growth of mutants with improved galactose utilization;(iii) Optionally monitoring growth and adaptation by measuring relevant parameters such as optical density or substrate consumption;(iv) After a defined number of passages or upon observation of improved growth, isolating individual clones or populations;(v) Screening the isolates for the desired Gal(+) phenotype, for example by assessing their ability to grow in galactose-containing media;(vi) Optionally determining that the mutant has a change in one or more of the genes encoding a protein involved in the metabolism of galactose ;(vii) Determining that the mutant has a change in the LacS gene encoding a lactose permease which change results in at least one mutation in the amino acid sequence of lactose permease selected from a position corresponding to position 148, 377 and 476 in SEQ ID No:2, preferably by sequencing; and(viii) Confirming that the selected mutant strain exhibits a Lac(+), Glu(+), Gal(+) phenotype and has a reduced secretion of carbohydrates as compared to the mother strain, for example as determined by carbohydrate analysis of fermented milk (e.g., using HPLC).

10. The method according to any one of the proceeding claims, wherein the mother strain is selected from: DSM 33677; DSM33719; DSM 35263; or any mutants or variants thereof.

11. A Streptococcus thermophilus strain obtained by the method according to any one of the proceeding claims.

12. A Streptococcus thermophilus strain with a Lac(+) Glu(+) Gal(+) phenotype and at least one mutation in the lacS gene encoding a lactose permease wherein the mutation leads to an amino acid substitution at a position corresponding to position 148, 377 and / or 476 in SEQ ID No:2.

13. The strain according to claim 12, wherein the amino acid substitution is 148H, 377C and / or 476K.

14. The strain according to any one of claims 11-13, wherein the strain comprises a lactose permease with a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID No:4; SEQ ID No:6; SEQ ID No:8; SEQ ID No: 10; SEQ ID No: 12; SEQ ID No: 14; or SEQ ID No: 16.

15. The strain according to any one of claims 11-14, wherein the strain comprises a LacS gene with a sequence identity of at least 95%, 96%, 97%, 98%, 99% or 100% to any of SEQ ID No:3; SEQ ID No:5; SEQ ID No:7; SEQ ID No:9; SEQ ID No: 1l; SEQ ID No:13; or SEQ ID No: 15.

16. The strain according to any one of claims 11-15, wherein the strain has a mutation in one or more of the genes selected from galactokinase galK), glucosekinase {glcK) and phosphoglucomutase pgrrf).

17. The strain according to claim 16, wherein the mutated gene is: ga!K and the mutation leads to a Valine (V) at a position corresponding to position 47 in SEQ ID No: 18; glcK and the mutation leads to a Glutamic acid (E) at a position corresponding to position 16 in SEQ ID No:20; and / or pgm and the mutation leads to a Aspartic acid (D) at a position corresponding to position 242 in SEQ ID No:22.

18. The strain according to any one of claims 11-17, wherein the amount of glucose and / or galactose in milk fermented with the strain is in the range of 0-1.0 mg / mL; 0-0.9; 0- 0.8; 0-0.7; 0-0.6; 0-0.5; 0-0.4; 0-0.3; 0-0.2; 0-0.1; or 0 mg / mL.

19. The strain according to any one of claims 11-18, wherein the strain is (a) derived from a mother strain selected from: DSM 33677; DSM 33719 or DSM 35263; or (b) selected from: DSM 35264; DSM 35265; and mutants or variants thereof.

20. A composition, as a mixture or as a kit-of-part, comprising the strain according to any one of claims 11-19.

21. Method for manufacturing a fermented product comprising the steps:(a) adding the strain according to any one of claims 11-19 or the composition according to claim 20 to a milk base; and(b) fermenting the milk base until a target pH is reached to obtain a fermented product, preferably a fermented dairy product.

22. A fermented product comprising the strain according to any one of claims 11-19 or the composition according to claim 20.

23. The fermented product according to claim 22, wherein the fermented product is obtained by the method according to claim 21.

24. The fermented product according to any one of claims 22 to 23. wherein the strain is present in a concentration of at least 107CFU / g product.

25. Use of the strain according to any one of claims 11-19 or the composition according to claim 20 for: reducing the content of carbohydrates in a fermented product; reducing the fermentation time; and / or increasing the texture in a fermented product.

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