Glucose-hyperfermenting lactic acid bacteria

Abstract

Streptococcus thermophilus is highly adapted to grow on lactose and its main role in dairy fermentation is the rapid conversion of lactose into lactate. Glucose is known as a poor substrate for growth and only some strains of S. thermophilus are known to ferment galactose. The present invention aims to provide S. thermophilus strains that are better adapted to grow and acidify in low-lactose solutions such as lactose-hydrolyzed milk.

Description

Glucose-hyperfermenting lactic acid bacteriaTechnical fieldThe present invention relates to the development of novel lactic acid bacteria strains for use in the food industry. The novel strains ferment glucose more effectively and can be used to produce fermented products from, for example, lactose-hydrolyzed milk.Background artStreptococcus thermophilus is highly adapted to grow on lactose and its main role in dairy fermentation is the rapid conversion of lactose into lactate. Glucose is known as a poor substrate for growth and only some strains of .S', thermophilus are known to ferment galactose (Hols etal., 2005. FEMS Microbiol Rev. 29(3):435-63).It is therefore expected that .S', thermophilus will acidify lactose-hydrolyzed milk slowly because lactose- hydrolyzed milk contains high concentrations of glucose and galactose, and only low concentrations of lactose (if any). Because Lactobacillus delbrueckii subsp. bulgaricus (hereinafter “Z. bulgaricus") and .S', thermophilus grow differently on glucose and galactose, fermentation of lactose-hydrolyzed milk can cause an undesired change in the balance between these two species than what is observed in normal milk.The inventors have observed poor fermentation of lactose-hydrolyzed milk using .S', thermophilus strains of the prior art. The present invention aims to provide .S', thermophilus strains that are better adapted to grow and acidify in low-lactose solutions such as lactose-hydrolyzed milk.Summary of the inventionThe aim set out by the present invention has been solved by the provision of a glucose-fermenting .S'. thermophilus strain that carries a mutation in the glcU gene. A homologue of the glcU gene was identified in .S'. thermophilus (Soerensen et al., 2016. Appl Environ Microbiol. 82(12): 3683-3692). However, it was not known whether this gene is functional in .S', thermophilus given the large number of pseudogenes identified in the genomes of .S', thermophilus (see, for example, Alexandraki etal., 2019. Front Microbiol. 10: 2916). Surprisingly, a mutation in the glcU gene results in a higher acidification rate when grown on glucose (in comparison to the mother strain) and / or lower pH at the end of fermentation when grown on glucose (in comparison to the mother strain). In some instances, a mutation in the glcU gene results in glucose-hyperfermenting strains capable of acidifying on glucose as the carbon source at substantially the same rate as on lactose as the carbon source. In some instances, amutation in the glcU gene results in a strain that is capable of reaching a lower pH or substantially the same pH when grown on glucose as the carbon source when compared to lactose as the carbon source.The aim set out by the present invention has also been solved by the provision of a glucose-fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster. Surprisingly, a mutation in a sucrose gene cluster results in a higher acidification rate when grown on glucose (in comparison to the mother strain) and / or lower pH at the end of fermentation when grown on glucose (in comparison to the mother strain). In some instances, a mutation in a sucrose gene cluster results in glucose- hyperfermenting strains capable of acidifying on glucose as the sole carbon source at substantially the same rate as on lactose as the sole carbon source. In some instances, a mutation in a sucrose gene cluster results in a strain that is capable of reaching substantially the same pH when grown on glucose as the sole carbon source when compared to lactose as the sole carbon source.Exposing the strains of the invention to sucrose further stimulates growth in glucose. Thus, the present invention also provides a composition (e.g., a preculture) comprising sucrose and an S. thermophilus strain of the present invention.The present invention also provides a method of preparing a fermented product comprising inoculating a milk base with the .S', thermophilus strain of the present invention, or the composition of the present invention. The present invention also provides a fermented product obtained by or obtainable by the method of preparing a fermented product of the present invention. In another aspect, the present invention provides a fermented product comprising an .S', thermophilus strain of the present invention.It is possible to obtain further strains according to the present invention by adapted laboratory evolution (ALE) approaches. In particular, serial passaging a glucose-fermenting .S', thermophilus mother strain (e.g., a strain of the prior art) in high glucose-containing media results in spontaneous mutations in a sucrose gene cluster (e.g., in the scrR gene and / or scrA gene). Thus, the present invention provides a method for producing a .S', thermophilus strain according to the present invention comprising serial passaging a glucose-fermenting .S', thermophilus mother strain in medium supplemented with glucose, and identifying and isolating from a pool of mutant .S', thermophilus strains derived from the mother strain a glucose-fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster. The present invention also provides a .S', thermophilus strain obtained by or obtainable by the method of the present invention.Further aspects of the present invention are detailed below.FiguresFigure 1: Sequence alignment of the glcU gene (including 100 bp upstream of the start codon) of the glucose-hyperfermenting mutants and the sequence of the respective mother strains. A) Alignment of DSM 24012 (query sequence) and DSM 24010 (subject sequence). DSM 24010 has a T302C base pair substitution in the coding region of the glcU gene. B) Alignment of DSM 22934 (query sequence) and DSM 33572 (subject sequence). DSM 33572 contains a base pair insertion of a single thymidine at position -81 of the glcU gene if, as is conventionally done in the art, the first nucleotide of the start codon (in bold) is counted as position +1 and the first nucleotide of the non-coding region of the gene attached to the 5’ end of the start codon is counted as position -1. Position -81 thus refers to the 81stnucleotide before the start codon.Figure 2: Protein alignment of the GlcU expressed by DSM 24012 (query sequence) and DSM 24010 (subject sequence). The base pair substitution in the coding region of the glcU gene of DSM 24010 results in a V101A substitution in the expressed GlcU.Figure 3: Comparison between DSM 24010 and DSM 33572 and their respective mother strains when grown on M17 broth supplemented with glucose. A) DSM 24012 (dashed line) and DSM 24010 (solid line) were precultured in M17 broth supplemented with 2 % (w / v) lactose. B) DSM 24012 (dashed line) and DSM 24010 (solid line) were precultured in M17 broth supplemented with 2 % (w / v) sucrose. C) DSM 22934 (dashed line) and DSM 33572 (solid line) were precultured in M17 broth supplemented with 2 % (w / v) lactose. D) DSM 22934 (dashed line) and DSM 33572 (solid line) were precultured in M17 broth supplemented with 2 % (w / v) sucrose.Figure 4: Comparison between DSM 33572 and its mother strain when grown on M17 broth supplemented with lactose or glucose. DSM 22934 (solid lines) and DSM 33572 (dashed lines) were inoculated into Ml 7 broth supplemented with 2 % (w / v) glucose (A), or 2 % (w / v) lactose (B). The strains were precultured in M17 broth supplemented with 2 % (w / v) sucrose or M17 broth supplemented with 2 % (w / v) lactose. The parentheses in each figure legend indicate whether the strain was precultured in the presence of sucrose or lactose.Figure 5: Protein alignment of the ScrR encoded by DSM 22934 (query sequence) and DSM 34852 (subject sequence). A C725 A base pair substitution in the coding region of the scrR gene of DSM 22934 results in a A242D substitution in the protein sequence.Figure 6: Protein alignment of the ScrR encoded by DSM 32503 (query sequence) and DSM 34853 (subject sequence). A T131G base pair substitution in the coding region of the scrR gene of DSM 32503 results in a L44R substitution in the protein sequence.Figure 7: Protein alignment of the ScrA encoded by DSM 32503 (query sequence) and DSM 34853 (subject sequence). A G853A base pair substitution in the coding region of the scrA gene of DSM 32503 results in a V285I substitution in the protein sequence.Figure 8: Protein alignment of the ScrA encoded by DSM 32503 (query sequence) and DSM 34854 (subject sequence). A C385T base pair substitution in the coding region of the scrA gene of DSM 32503 results in a R129C substitution in the protein sequence.Figure 9: Exemplary sucrose gene clusters (adapted from Gasser et al„ 2022, Food Microbiol. 107: 104080, Figure 2). Two gene clusters were identified by Gasser et al. According to Gasser et al., the first cluster corresponded to genes encoding a putative PEP-PTS transporter (scrA) and a fructokinase (scrK) and, the second cluster corresponded to a putative hydrolase -encoding gene (scrB) and a potential regulator of these genes (scrR).Figure 10: Comparison of growth in M17 broth supplemented with lactose or glucose between mother strain DSM 22934 or DSM 32503 and mutant strains DSM 34852, DSM 34853 and DSM 34854, (A) DSM 22934 (solid line) and DSM 34852 (dashed line) were inoculated into M17 broth supplemented with 2 % (w / v) lactose. (B) DSM 22934 (solid line) and DSM 34852 (dashed line) were inoculated into M17 broth supplemented with 2 % (w / v) glucose. (C) DSM 32503 (solid line),p DSM 34853 (dotted line) and DSM 34854 (dashed line) were inoculated into M17 broth supplemented with 2 % (w / v) lactose. (D) DSM 32503 (solid line), DSM 34853 (dotted line) and DSM 34854 (dashed line) were inoculated into Ml 7 broth supplemented with 2 % (w / v) glucose.Figure 11: Acidification curves of (A) a mutant derived from DSM 22934 containing an A242D substitution in the scrR gene and (B) DSM 34853, The mutant strains were inoculated into M17 broth supplemented with lactose, galactose, glucose or sucrose. The M17 broth cultures were tested twice, and the graphs depict the results of the repeat experiments.Figure 12: Acidification curves in soy milk, (A) DSM 22934 (solid line) and DSM 34852 (dashed line) inoculated into soy milk. (B) DSM 32503 (solid line), DSM 34853 (dotted line) and DSM 34854 (dashed line) inoculated into soy milk.Detailed description of the inventionIn .S'. thermophilus, the growth on glucose is less efficient than on lactose. Since glucokinase-negative .S'. thermophilus strains with mutations in the man / glu PTS operon are unable to grow on glucose (see Soerensen et al., 2016. Appl Environ Microbiol. 82(12):3683-3692 and WO 2013 / 160413 Al), this operon encodes the only active PTS capable of transporting glucose in these strains. A homologue of glcU (found in Staphylococcus xylosus, Streptococcus pyogenes and Lactococcus lactis) is present in .S'. thermophilus but, since the glucose-secreting mutants are glucokinase deficient, potential transport of glucose by GlcU would not lead to glucose catabolism because GlcU is believed to be a non-PTS glucose transporter.In Soerensen et al., 2020. Front Bioeng Biotechnol. 8: 623700 we described a cycloserine-resistant mutant with a mutation in the glcU gene, i.e., DSM 24010 (also described in WO 2012 / 052557 Al). The mutant strain contained a T302C point mutation in the coding region resulting in a V101A substitution in the gene product. We have described how this strain results in fermented products with improved texture but have not previously shown that the mutation resulted in an improved ability to ferment glucose (see Example 1).We identified a further strain with a mutation in the promoter of glcU. The strain, i.e., DSM 33572, is a P-thio-o-nitrophenylgalactoside- (TONPG-) resistant mutant of the previously described DSM 22934 strain (see WO 2011 / 026863 Al). Also, this mutant was able to utilize glucose more effectively (see Figure 3C).In order to identify further glcU mutants, we developed an adapted laboratory evolution approach to select for further mutants that have adapted to growth on glucose. Surprisingly, the method consistently selected for strains that carried one or more mutations in a sucrose gene cluster without carrying a mutation in the glcU gene. These mutants, however, also had more desirable acidification profiles in glucose than their respective mother strains.The aspects of the present invention will be explained in greater detail below.DefinitionsThe expression “that carries a mutation in a sucrose gene cluster” means that the strain has a mutation in an endogenous sucrose gene cluster that increases the strain’s ability to ferment glucose. As is understood in the art, a mutation may be a base substitution, deletion of one or more bases or insertion of one or more bases. The sucrose gene cluster may be in accordance with one of the clusters depicted in Figure 9. In some embodiments, the coding region of the scrA gene or a non-coding regulatoryelement of the scrA gene is mutated. Without being bound by a particular theory, it is believed that the scrA gene, which encodes a phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) known to transport sucrose across the cell membrane (Hols et al., 2005. FEMS Microbiol Rev. 29(3):435-63, Figure 3), is able to transport glucose across the cell membrane. It was observed by the present inventors that a strain carrying an R129C mutation in the coding sequence of the scrA gene (i.e., DSM 34854) was able to acidify better in glucose than its mother strain but grew worse in sucrose than its mother strain (see Figures 10B and 12B). This suggests that the PEP -PTS encoded by scrA can be mutated to improve glucose transport across the cell membrane. Thus, mutations that increase the expression of scrA (e.g., mutations in the non-coding regulatory elements of the scrA gene) or increase the ability of the encoded PEP-PTS to transport glucose across the cell membrane (e.g., mutations in the coding region of the scrA gene) are preferred. Alternatively, or in addition to a mutated scrA gene, in some embodiments, the scrR gene is mutated. The scrR gene encodes a repressor and without being bound by a particular theory, it is believed that reducing the capability of the repressor to regulate gene expression results in more effective glucose utilization. Thus, mutations that reduce the expression of scrR (e.g., mutations in the non-coding regulatory elements of the scrR gene or in the coding region of the scrR gene) or reduce the activity of the encoded repressor (e.g., mutations in the coding region of the scrR gene) are preferred.The expression “that carries a mutation in the glcU gene" means that the strain has a mutation in the coding region of the glcU gene or a non-coding regulatory element of the glcU gene that increases the strain’s ability to ferment glucose. As is understood in the art, a mutation may be a base substitution, deletion of one or more bases or insertion of one or more bases. Without being bound by a particular theory, it is believed that the glcU gene, which is thought to encode a homologue of a known glucose permease, is able to transport glucose across the cell membrane. It was observed by the present inventors that a strain carrying an V101A mutation in the coding sequence of the glcU gene as well as a strain carrying a mutation in the promoter region of the glcU gene was able to acidify better in glucose than its mother strain (see Figures 3 and 4). This suggests that the glcU gene encodes a glucose permease that can be mutated to improve glucose transport across the cell membrane. Thus, mutations that increase the expression of glcU (e.g., mutations in the non-coding regulatory elements of the glcU gene) or increase the ability of the encoded gene product to transport glucose across the cell membrane (e.g., mutations in the coding region of glcU gene) are preferred.The expression “inactivating mutation" refers to any mutation that partially or totally reduces the translation or transcription of agene, or partially ortotally reduces the function ofthe gene product (e.g., the gene product of the scrR gene partially or totally loses its ability to regulate gene expression). The expression “inactivating mutation" may refer to a single mutation or a combination of several mutationsthat cumulatively result in a reduction in expression and / or function. In some embodiments, a noncoding regulatory element of the scrR gene comprises an inactivating mutation and / or the coding region of the scrR gene comprises an inactivating mutation. In some embodiments, the inactivating mutation reduces the expression of the gene product encoded by the scrR gene below detectable or measurable levels, or results in a gene product that cannot regulate gene expression.The expression “activating mutation" refers to any mutation that increases the translation or transcription of a gene or increases the function of the gene product (e.g., an increased glucose influx if the gene product is encoded by the scrA gene or glcU gene). The expression “activating mutation" may refer to a single mutation or a combination of several mutations that cumulatively result in an increase in expression and / or function. In some embodiments, a non-coding regulatory element of the scrA gene or glcU gene comprises an activating mutation and / or the coding region of the scrA gene or glcU gene comprises an activating mutation. In some embodiments, the activating mutation increases the expression of the gene product encoded by the glcU gene or scrA gene, or results in a gene product that increases the influx of glucose.The term “gene ” encompasses the coding region and non-coding regulatory elements. The expression “non-coding regulatory element” is given its normal meaning in the art and refers to any non-coding element that modulates the transcription or translation of a particular gene. Exemplary non-coding regulatory elements include promoter regions, ribosome binding sites and cis -regulatory elements. Promoter regions include a binding site for an RNA polymerase and may further include a cA-regulatory element (e.g., a binding site for a repressor protein and / or a binding site for an activator protein). Ribosome binding sites allow the ribosome to bind to the mRNA to initiate translation.The expression “acidify at substantially the same rate” means that the two measured maximum acidification rates differ by 50% or less, 49% or less, 48% or less, 47% or less, 46% or less, 45% or less, 44% or less, 43% or less, 42% or less, 41% or less, 40% or less, 39% or less, 38% or less, 37% or less,36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31% or less, 30% or less, 29% or less,28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less,20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less,12% or less, 11% or less, or 10% or less. For example, if the maximum acidification rate of a strain grown in medium supplemented with glucose is 0.7 pH units / hour and the maximum acidification rate of the same strain grown in medium supplemented with lactose is 1.4 pH units / hour, the strain acidifies at substantially the same rate in both conditions because the acidification rate in medium supplemented with glucose is 50% of the acidification rate in medium supplemented with lactose.The expression “substantially the same pH” means that two pH values differ by 0. 1 or less pH units, 0.2 or less pH units, 0.3 or less pH units, 0.4 or less pH units, or 0.5 or less pH units. Preferably, the expression “substantially the same pH” means that the two pH values differ by 0.3 or less pH units, 0.2 or less pH units, or 0. 1 or less pH units.The expressions “acidification rate” and “maximum acidification rate ” are used interchangeably and refer to the maximum slope of the pH curve (dpH / dt). The maximum acidification rate can be calculated using, for example, the CINAC v2.07 software (see Dandoy et al., 2011. Microb Cell Fact. 10(Suppl 1): S21).The expression “glucose -fermenting” means that a strain is capable of growth on / in M17 medium supplemented with 2 % (w / v) glucose. The glucose-fermenting .S', thermophilus strains are defined herein as .S', thermophilus strains which lower the pH of Ml 7 broth containing 2 % (w / v) glucose as sole carbohydrate to 5.5 or lower when inoculated from an overnight culture at 1 % (v / v) and incubated for 24 hours at 37 °C.The expression “galactose-fermenting” means that a strain is capable of growth on / in M17 medium supplemented with 2 % (w / v) galactose. The galactose-fermenting .S', thermophilus strains are defined herein as .S', thermophilus strains which lower the pH of M17 broth containing 2 % (w / v) galactose as sole carbohydrate to 5.5 or lower when inoculated from an overnight culture at 1 % (v / v) and incubated for 24 hours at 37 °C. Galactose-fermenting strains may be obtained by the methods described in WO 2011 / 026863 Al.The expression “sucrose -fermenting” means that a strain is capable of growth on / in M17 medium supplemented with 2 % (w / v) sucrose. The sucrose-fermenting .S', thermophilus strains are defined herein as .S', thermophilus strains which lower the pH of Ml 7 broth containing 2 % (w / v) sucrose as sole carbohydrate to 5.5 or lower when inoculated from an overnight culture at 1 % (v / v) and incubated for 24 hours at 37 °C.The expression “milk base ” refers to any milk or milk alternative (e.g., plant-based milk alternatives) that can be fermented by the strains of the present invention. A milk base can be fermented by a strain of the present invention if it comprises sucrose and / or glucose. The sucrose and / or glucose may be naturally present in the milk base or added to the milk base prior to fermentation.The term “milk” is given its normal meaning in the art and refers to a white liquid food produced by the mammary glands of mammals. Mammal sources of milk include, but are not limited to cow, sheep, goat,buffalo, camel, llama, mare and deer. The milk may have been processed and the term includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-reduced milk, or concentrated milk. The term also encompasses reconstituted milk powders. 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. Lactose-reduced milk is 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 (also known as lactose-hydrolyzed milk), or by nano-filtration, electrodialysis, ion exchange chromatography and centrifugation. In a preferred embodiment, the milk is lactose-hydrolyzed milk. Methods of preparing lactose-hydrolyzed milk are known in the art (see, for example, Schulz and Rizvi, 2021. Food Rev Int. 39(5): 2875-2894).The expression "plant-based milk alternative" is given its normal meaning in the art and refers to suspensions of plant-based materials and their extracts in water. Methods for obtaining plant-based milk alternatives are known in the art (see, for example, Sethi et al., 2016. J Food Scie Technol. 53(9): 3408- 3423). In some embodiments, the plant-based milk alternative is oat milk, rice milk, com milk, spelt milk, soy milk, peanut milk, lupin milk, cowpea milk, almond milk, coconut milk, hazelnut milk, pistachio milk, walnut milk, sesame milk, flax milk, hemp milk, sunflower milk, quinoa milk, teff milk, amaranth milk or a mixture of two or more of the listed plant-based milk alternatives. In a preferred embodiment, the plant-based milk alternative is soy milk.The expression "serial passaging” is given its normal meaning in the art and refers to a process of growing bacteria in iterations. For example, a bacterium is grown in one environment and then a portion of the bacterial population is removed and put into a new environment. This process can be repeated until a desired spontaneous mutation is detected.The term “medium” is given its normal meaning in the art and refers to any substance in which an organism is cultured. The term encompasses solid and liquid media. An exemplary solid medium is M17 agar and an exemplary liquid medium is M17 broth. The expression “liquid medium” is used synonymously with the term “broth”. The expression “M17 broth” refers to a liquid medium with a pH of 7 at 25 °C consisting of the following ingredients that are dissolved into one liter of water:The expression “supplemented with” defines any additional ingredient that is added to the M17 broth. The expression "supplemented with” does not allow for further undefined ingredients and should be construed as including and limited to. Thus, for example, the expression “Ml 7 broth supplemented with 2% (w / v) glucose” means that the broth consists essentially of the M17 broth recipe provided above and 2 % (w / v) glucose and does not allow for the presence of a further carbohydrate such as, for example, lactose, galactose and sucrose.The expression “mother strain” may refer to any strain of the prior art that is glucose-fermenting. In a preferred embodiment, the mother strain is DSM 24012, DSM 22934 or DSM 32503. DSM 24012 was described previously in WO 2012 / 052557 Al. DSM 22934 was described previously in WO 2011 / 026863 Al. DSM 32503 was described previously in WO 2018 / 220104 Al.■S', thermophilus strains with mutated glcU geneIn a first aspect, the present invention provides a glucose-fermenting .S', thermophilus strain that carries a mutation in the glcU gene. In some embodiments, the mutation is in the coding region of the glcU gene or in a non-coding regulatory element of the glcU gene. In some embodiments, the mutation is an activating mutation. In some embodiments, the strain is not the strain that was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 24010.The nucleotide sequence of the glcU gene of DSM 24012 and DSM 22934 (including 100 bp upstream) are provided below and are examples of non-mutated wild-type sequences (start codon is underlined):DSM 24012 glcUGTTTATATTTACCTCTTTAAAATAATTTTTTACCTAAAAAATTAAGCATAGCTCTATATTA CTCTTATTTCGGGAAAAAACAAGAGATAAAAAAATACACATGCAAGGAGTTCTTTTCGCG CTTGTTCCACTTTTTGCTTGGGGTTCTATCGGATTGGTAGCTAATATACTTGGTGGTGATG CTAATCAACAAACACTTGGAATGACTTTGGGCGCTTTTGTTGTTGCACTTATTGTTTCCTT ATTCCGCATGCCAACGTTGACATGGCAAATTTTCTTAATTGGATTTATTGGTGGATTGTTT TGGGCAATTGGACAATTTGGTCAGTTTAATTCAATGAAATACATGGGTGTTTCAGTAGCG AGTCCACTTTCAGCAGGAAGTCAATTAGTATTTGGTGTATTGCTTGGGGTTTTTGCTTTCC ACGAATGGACAAAACAAATTCAATTTATTATCGGATTTATTGCGATGGCTGTTTTGGTAGTTGGGTTCTATTTCTCAGCTAAAAGTGACCCAGAAAATGCAGTTGTTAAAGAAGGACGTAA TTATACTAAAGGATTGATTGCTTTAACTTACTCAACTTTGGGATATGTTCTCTATGTTATTC TTTTTAATAACTTAGCAGTACTTTGGTTCAATGTTCATTTTGATACACTGACAATTATCTTG CCAATGTCAGTTGGAATGATCTTTGGAGCACTTGTGATGGGTCGTTTCAAAATTAAAATG GAAAAATATGTTTATCAAAATATGATTGATGGAGTAATGTTTGGTGTAGGTAATATCTTT ATGCTTATGGCTGCAAGTGCTGCTGGTAACGCAATTGCCTTTTCATTCTCACAATTAGGTG TTTTCATTTCAACTATTGGAGGAATTCTCTTCCTTGGTGAAAAGAAAACCAAAAAAGAAT TGGTTTATGTTGGAATTGGAATTGTTCTGTTCGTAACAGGTGCAATTTTACTTGCAATTGTAAAATCTAAAGGATAA (SEQ ID NO: 1)DSM 22934 glcUTGTATTTTTTTATCTCTTGTTTTTTCCCGAAATAAGAGTAATATAGAGCTATGCTTAATTTT TTAGGTAAAAAATTATTTTAAAGAGGTAAATATAAACATGCAAGGAGTTCTTTTCGCGCT TGTTCCAATTTTTGCTTGGGGTGCTGTCGGATTGGTAGCTAATATACTTGGTGGTGATCCT AATCAACAAACACTGGGAATGACTTTGGGCGCTTTTGTTGTTGCACTTATTGTTTCCTTAT TCCGCATGCCAACGTTGACATGGCAAATTTTCTTAATTGGATTTATTGGTGGATTGTTTTG GGTAATTGGACAATTTGGTCAGTTTAATTCAATGAAATACATGGGTGTTTCAGTAGCGAG TCCACTTTCAGCAGGAAGTCAATTAGTATTTGGTGTATTGCTTGGGGTTTTTGCTTTCCAC GAATGGACAAAACAAATTCAATTTATTATCGGATTTATTGCGATGGCTCTTTTGGTAGTTG GGTTCTATTTCTCAGCTAAACGTGACCCAGAAAATGCAGTTGTTAAAGAAGGACGTAATT ATACTAAAGGATTGATTGCTTTAACTTACTCAACTTTGGGATATGTTCTCTATGTTATTCTT TTTAATAACTTAGCAGTACTTTGGTTCAATGTTCATTTTGATACACTGACAATTATCTTGC CAATGTCAGTTGGAATGATCTTTGGAGCACTTGTGATGGGTCGTTTCAAAATTAAAATGG AAAAATATGTTTATCGAAATATAATTGATGGAGTAATGTTTGGTGTAGGTAATATCTTTAT GCTTATGGCTGCAAGCGCTGCTGGTAACGCAATTGCCTTTTCATTCGCACAATTAGGTGTT ATCATTTCAACTATTGGAGGAATTCTCTTCCTTGGTGAAAAGAAAACCAAAAAAGAATTG GTTTATGTTGGAATTGGAAGTGTTCTGTTCGTAACAGGTGCAATTTTACTTGCAATTGTAAAATCTAAAGGATAA (SEQ ID NO: 2)In some embodiments, the mutation causes the strain to reach substantially the same pH or a lower pH in Ml 7 broth supplemented with 2 % (w / v) glucose as in Ml 7 broth supplemented with 2 % (w / v) lactose after 24 hours at 40 °C (i.e., at the 24 hour timepoint after inoculation when grown at 40 °C). In a preferred embodiment, the mutation causes the strain to reach a lower pH in M17 broth supplemented with 2 % (w / v) glucose as in Ml 7 broth supplemented with 2 % (w / v) lactose after 24 hours at 40 °C.In some embodiments, the mutation causes the strain to acidify faster in Ml 7 broth supplemented with 2 % (w / v) glucose than the mother strain from which the mutant is derived. In some embodiments, the mutation causes the strain to acidify faster in M17 broth supplemented with 2 % (w / v) glucose than DSM 24012, DSM 22934 or DSM 32503.In some embodiments, the strain carries a mutation in the coding region of the glcU gene. In some embodiments, the mutation consists of a substitution at position 101 of the translated amino acid sequence (numbering is defined with respect to SEQ ID NO: 3 as depicted in Figure 2 and below). In some embodiments, the protein encoded by the glcU gene has at least 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity with residues 2-100 and 102-295 of SEQ ID NO: 3 and the amino acid residue at position 101 is substituted. In a preferred embodiment, the residue at position 101 is not a valine.MQGVLFALVPLFAWGSIGLVANILGGDANQQTLGMTLGAFVVALIVSLFRMPTLTWQIFLIGF IGGLFWAIGQFGQFNSMKYMGVSVASPLSAGSQLVFGVLLGVFAFHEWTKQIQFIIGFIAMAV LVVGFYFSAKSDPENAVVKEGRNYTKGLIALTYSTLGYVLYVILFNNLAVLWFNVHFDTLTII LPMSVGMIFGALVMGRFKIKMEKYVYQNMIDGVMFGVGNIFMLMAASAAGNAIAFSFSQLG VFISTIGGILFLGEKKTKKELVYVGIGIVLFVTGAILLAIVKSKG (SEQ ID NO: 3, position 101 has been underlined)In some embodiments, the mutation consists of a V101A substitution. In some embodiments, the strain is not the strain that was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 24010.In some embodiments, the strain carries a mutation in a non-coding regulatory element of the glcU gene. In some embodiments, the mutation is in the promoter region of the glcU gene. For example, in a preferred embodiment of the invention, the mutation consists of a T insertion at position -81 of the glcU gene (i.e., the 81stnucleotide before the start codon as depicted in Figure IB).In some embodiments, the promoter region of the glcU gene of the strain of the present invention comprises the nucleotide sequence TTTTTCCCGA (SEQ ID NO: 4) or TTTTTTCCCGAAA (SEQ ID NO: 5).In some embodiments, the strain is the strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession number DSM 33572, or a mutant strain derived therefrom that has retained or further improved on the glucose fermenting property (e.g., theacidification rate when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C is the same or higher and / or the pH after 24 hours is the same or lower when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C) of the DSM 33572 strain.In some embodiments, the strain according to the first aspect of the invention is galactose-fermenting. Strains that are galactose-fermenting are preferred for the fermentation of compositions comprising galactose, such as lactose-hydrolyzed milk.In some embodiments, the strain according to the first aspect of the invention further carries a mutation in a sucrose gene cluster. The mutation in the sucrose gene cluster may be in accordance with any one of the embodiments described below.■S', thermophilus strains carrying a mutation in a sucrose gene clusterIn a second aspect, the present invention provides a glucose-fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster. In some embodiments, the strain is not the LMG18311 strain and / or CNRZ1066 strain described in Gasser etal., 2022. Food Microbiol. 107: 104080. LMG18311 has been deposited at the American Type Culture Collection under accession number ATCC BAA-250. CNRZ1066 is part of the Institut National de la Recherche Agronomique (INRA) strain collection.In some embodiments, the mutation in the sucrose gene cluster causes the strain to acidify at substantially the same rate in M17 broth supplemented with 2 % (w / v) glucose at 40 °C as in M17 broth supplemented with 2 % (w / v) lactose at 40 °C. In some embodiments, the mutation causes the strain to acidify faster (i.e., has a higher maximum acidification rate) in Ml 7 broth supplemented with 2 % (w / v) glucose at 40 °C than the mother strain from which the mutant is derived. In some embodiments, the mutation causes the strain to acidify faster in M17 broth supplemented with 2 % (w / v) glucose at 40 °C than DSM 24012, DSM 22934 and / or DSM 32503.In some embodiments, the mutation in the sucrose gene cluster causes the maximum acidification rate of the strain when grown in M17 broth supplemented with 2 % (w / v) glucose at 40 °C to be at least 40% (e.g., at least 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60%) of the maximum acidification rate of the strain when grown in M17 broth supplemented with 2 % (w / v) lactose at 40 °C. In a preferred embodiment, the mutation in the sucrose gene cluster causes the maximum acidification rate of the strain when grown in M17 broth supplemented with 2 % (w / v) glucose at 40 °C to be at least 50% (e.g., at least 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60%) of the maximum acidification rate of the strain when grown in M17 broth supplemented with 2 % (w / v) lactose at 40 °C.In some embodiments, the mutation in the sucrose gene cluster causes the maximum acidification rate of the strain when grown in M17 broth supplemented with 2 % (w / v) glucose at 40 °C to be at least 0.5, 0.6, 0.7 or 0.8 pH units / hour.In some embodiments, the mutation in the sucrose gene cluster causes the strain to reach substantially the same or lower pH in M17 broth supplemented with 2 % (w / v) glucose as in M17 broth supplemented with 2 % (w / v) lactose after 24 hours at 40 °C (i.e., at the 24 hour timepoint after innoculation when grown at 40 °C). In some embodiments, the mutation in the sucrose gene cluster causes the strain to reach a lower pH in M17 broth supplemented with 2 % (w / v) glucose after 24 hours at 40 °C than the mother strain from which the mutant is derived. In some embodiments, the mutation in the sucrose gene cluster causes the strain to reach a lower pH in M17 broth supplemented with 2 % (w / v) glucose after 24 hours at 40 °C than DSM 24012, DSM 22934 or DSM 32503.In some embodiments, the mutation in the sucrose gene cluster can be induced by growing a mother strain in a glucose-rich medium wherein glucose is the sole fermentable carbohydrate until the mutation is induced spontaneously. In some embodiments, the mutation is obtainable by serial passaging a glucose-fermenting .S', thermophilus mother strain in medium supplemented with glucose. The medium may be a broth (e.g., M17 broth). Further, the medium may be supplemented with at least 1% (w / v), at least 2% (w / v), at least 3% (w / v), at least 4% (w / v), or at least 5% (w / v) glucose. Preferably, the mutation is obtainable by serial passaging a glucose-fermenting .S', thermophilus mother strain in Ml 7 broth supplemented with at least 5% (w / v) glucose.In some embodiments, the strain according to the first or second aspect of the invention comprises a mutation in the scrR gene and / or the scrA gene.The nucleotide sequence of the scrR gene of DSM 22934 and DSM 32503 (including 100 bp upstream) are provided below and are examples of non-mutated wild-type sequences (start codon is underlined):DSM 22934 scrRTCACTAGCCGCGTCTTCCCAAGTGCTGAACAAACTGGTATCAAAGTAGCATCAGGGAACG TTTCTGGTCACTACTTCGACTTAAAATACTAGGGTGATTTATGGTCGCAAAGCTTACTGAC GTTGCCGAACTGGCTGGTGTGAGCCCAACCACAGTTTCCCGCGTCATAAACAAAAAGGGC TACCTCTCACAAAAAACCATTGACAAAGTACACGCTGCCATGCAGGAGCTGAATTACAAG CCTAATAATTTGGCTCGAGGTCTCAAAGGAAAATCAGCCAAGCTCGTTGGCTTGATTTTTC CAAATATTTCCAACATTTTTTACGCTGAGCTTATCGAACGACTTGAGGACGAGCTCTTTAA GCAGGGTTATAAAACCATCATTTGTAACAGTGAGCATGATCCCAACAAGGAAAAGGACTATTTGGAAATGCTTTCCGCCAACCAATGTGATGGTATCATCTCGTCTTCTCACAATCTTGGTATAGAAGATTATGAAAAGGTTGAAGCTCCTATCATTGCCTTCGACCGTAATCTGGCCCCAAAGATTCCTATTATCTCATCTGATAACTTTGAAGGAGGAAAATTAGCCGCAAAAACCTTGGTCAAGGCCGGTTGTCAGCGTATTGTCATGATTACAGGTAATGATAATTCAGATTCCCCAACTGGACTCCGTCAACTTGGATTTAATTACGAATTGAATAAACAGGGTATTGTCTGTCCTGTCCCAAACGATTTATCTATCATGCGACGAGAATTAGAAATCAAATCTATTATTTGTCGTGAAAAGCCGGATGGTATCTTTGTCTCAGATGATTTAACAGCTATCTTAGTTATAAAAGTAGCTAGACAACTAGAACTTTCCATCCCAAATGACTTAAAAATCATCGGTTACGACGGTACTGCCTTTATCCGACACTTCTATCCAGAGCTAACAACCATCCAACAGCCTTTGGATGAAATTGCTAAACTTTGTGTCGAAATTCTATTGAAGAAAATCAAAGGCGAAATAGTCAGTCGTGATTATGTGCTACCAATAAATTTGATTTCTGGTTCCAGTATATAA (SEQ ID NO: 6)DSM 32503 scrRTCACTAGCCGCGTCTTCCCAAGTGCTGAACAAACTGGTATCAAAGTAGCATCAGGGAACGTTTCTGGTCACTACTTCGACTTAAAATACTAGGGTGATTTATGGTCGCAAAGCTTACTGACGTTGCCGAACTGGCTGGTGTGAGCCCAACCACAGTTTCCCGCGTCATAAACAAAAAGGGCTACCTCTCACAAAAAACCATTGACAAAGTACACGCTGCCATGCAGGAGCTGAATTACAAGCCTAATAATTTGGCTCGAGGTCTCAAAGGAAAATCAGCCAAGCTCGTTGGCTTGATTTTTCCAAATATTTCCAACATTTTTTACGCTGAGCTTATCGAACGACTTGAGGACGAGCTCTTTAAGCAGGGTTATAAAACCATCATTTGTAACAGTGAGCATGATCCCAACAAGGAAAAGGACTATTTGGAAATGTTTTCCGCCAACCAATGTGATGGTATCATCTCGTCTTCTCACAATCTTGGTATAGAAGATTATGAAAAGGTTGAAGCTCCTATCATTGCCTTCGACCGTAATCTGGCCCCAAAGATTCCTATTATCTCATCTGATAACTTTGAAGGAGGAAAATTAGCCGCAAAAACCTTGGTCAAGGCCGGTTGTCAGCGTATTGTCATGATTACAGGTAATGATAATTCAGATTCCCCAACTGGACTCCGTCAACTTGGATTTAATTACGAATTGAATAAACAGGGTATTGTCTGTCCTGTCCCAAACGATTTATCTATCATGCGACGAGAATTAGAAATCAAATCTATTATTTGTCGTGAAAAGCCGGATGGTATCTTTGTCTCAGATGATTTAACAGCTATCTTAGTTATAAAAGTAGCTAGACAACTAGAACTTTCCATCCCAAATGACTTAAAAATCATCGGTTACGACGGTACTGCCTTTATCCGACACTTCTATCCAGAGCTAACAACCATCCAACAGCCTTTGGATGAAATTGCTAAACTTTGTGTCGAAATTCTATTGAAGAAAATCAAAGGCGAAATAGTCAGTCGTGATTATGTGCTACCAATAAATTTGATTTCTGGTTCCAGTATATAA (SEQ ID NO: 7)The above nucleotide sequences encode the following polypeptides:DSM 22934 ScrRMVAKLTDVAELAGVSPTTVSRVINKKGYLSQKTIDKVHAAMQELNYKPNNLARGLKGKSAKLVGLIFPNISNIFYAELIERLEDELFKQGYKT1ICNSEHDPNKEKDYLEMLSANQCDGIISSSHNLGIEDYEKVEAPIIAFDRNLAPKIPIISSDNFEGGKLAAKTLVKAGCQRIVMITGNDNSDSPTGLRQLGFNYELNKQGIVCPVPNDLSIMRRELEIKSIICREKPDGIFVSDDLTAILVIKVARQLELSIPNDLKIIGYDGTAFIRHFYPELTTIQQPLDEIAKLCVEILLKKIKGEIVSRDYVLPINLISGSSI (SEQID NO: 8, positions 44 and 242 have been underlined)DSM 32503 ScrRMVAKLTDVAELAGVSPTTVSRVINKKGYLSQKTIDKVHAAMQELNYKPNNLARGLKGKSAKLVGLIFPNISNIFYAELIERLEDELFKQGYKTIICNSEHDPNKEKDYLEMFSANQCDGIISSSHNLGIEDYEKVEAPIIAFDRNLAPKIPIISSDNFEGGKLAAKTLVKAGCQRIVMITGNDNSDSPTGLRQLGFNYELNKQGIVCPVPNDLSIMRRELEIKSIICREKPDGIFVSDDLTAILVIKVARQLELSIPNDLKIIGYDGTAFIRHFYPELTTIQQPLDEIAKLCVEILLKKIKGEIVSRDYVLPINLISGSSI (SEQID NO: 9, positions 44 and 242 have been underlined)The nucleotide sequence ofthe scrA gene of DSM 32503 (including 100 bp upstream) is provided below and is an example of a non-mutated wild-type sequence (start codon is underlined):DSM 32503 scrAACGTTTTCAAAAACAAATTTTGTTAAAGATGTTAAAATTGATATCGTAAAGAAAAGCGAAACGTTTTCAAAAACAAATTTTATTAAGGAGAATTTTGCAAATGGATTACAAACAAATTGCAAAAGAAGTCATCGAAGCCCTCGGTGGACGTGAAAATGTTAACAGTGTTGCTCACTGTGCGACACGTCTACGTGTTATGGTTAAAGATGAAAACAAAATCAATAAAGAAAAAGCTGAGAATATTGAAAAAGTTCAAGGTGCTTTCTTTAACTCAGGTCAATACCAAATGATCTTTGGTACAGGTACTGTTAACAAGATTTACGACGAAATCGTTGCTCAAGGTCTTCCAACAGCATCTAAAGACGAACAAAAAGCAGAAGCTGCTAAACAAGGGAACTGGTTCCAACGTGCCATCCGTTCATTCGGTGACGTTTTCGTTCCATTGCTTCCAGCTATCGTAGCGACTGGTCTTTTCATGGGTATCCGTGGAGCCATCAATAATGATACAGTTCTTGCTCTTTTTGGTACAACATCTAAAGCCTTCGCTGCTACTGATTTCTACACTTATACAGTTGTATTGACAGATACAGCCTTTGCCTTCTTCCCAGCCTTGATCTGTTGGTCAGCCTTTAACATTTTCGGTGGTTCACCACTTCTTGGTTTGGTTCTTGGTTTGATGATGGTTAACAATGCTCTTCCAAATGCTTGGGATGTTGCATCAGGTGCTGCAAAACCAATTTACTTCTTTGACTTTATTCCAGTAGTTGGTTACCAAAACTCAGTCCTTCCAGCCTTCTTCGTAGGTTTGATTGGTGCTAAGTTTGAACAATGGGTTCGTAAATGGGTTCCAGATGTTCTTGACCTTCTCTTGCGTCCACTTGTTGTCTTTGCTGTGATGTCAGCTTTGGCCCTCTTTATCATTGGTCCTGTCTTCCACACAGTTGAGAGCTACGTACTTGCTGGTACAGAGTGGATTCTTGCCTTGCCATTTGGTCTTGCAGGTCTTGTTCTAGGTGGTATTCACCAAATTATCGTCGTTACAGGGGTTCACCACGTTTTCAACTTGCTTGAAGCTAACCTTATTTCTAATACAGGTAAAGACCCACTTAACGCTATCATCACAGCTGCGATGACTGCTCAAGCTGGTGCAACACTTGCTGTTGGTGTTAAAACTAAAGACTCTAAGTTGAAAGCTCTCGCTTTCCCTGCAAGTCTTTCAGCAGTACTTGGTATCACTGAACCAGCTATCTTCGGGGTTAACCTTCGTTTCGGTAAACCATTTATCATGGGTCTTATCGCTGGTGCTGCTGGTGGTTGGTTGGCATCAATCCTTAACCTTGCTGGTACAGGATTTGGTGTAACAATCGTTCCTGGTACTCTTCTCTACCTAAATGGTCAAGTACTTAAGTATGTAATCATGGTACTTGTAACACTTGCTCTTGGTTTCGCTCTTACTTGGATCTTTGGATATAAAGAGGAAGAGGTTGAAGCTCAAACAGAAGTTGTTGCTGAAGATATCGCCTCAGCAGGTTCTGCTCCAGTTGCATTGCAAGCTGAAACAATTGCTGCACCACTTAAAGGTGAAGTTGTAGCTTTGGAAAATGTAAATGACCCAGTCTTCTCTTCAGGAGCTATGGGTAAAGGTGCCGCTATCAAACCTTCAGGTAACCGAGTTGTTGCACCATTTGATGGTGAAGTGCAAATTGCCTTCCCAACAGGTCACGCTTACGGTCTTAAATCTGATAAAGGTGCTGAAGTGCTTATCCACATCGGTATCGATACTGTCTCACTTGACGGTAAAGGATTTGATGCTAAGGTTCAAGCAAATCAACGTGTTAAAAAAGGTGATGTCTTGGCTACCTTCGATAGCTCAGTTATCACTGAAGCAGGTCTTGACGATACAACTATGGTTATCGTTACAAACACTGCAGACTACGAAGATGTTTCATTAGTAGCAACTGGTTCAGTTGCCGAGGGTGATGACTTCATCGCAGTTAAATAA (SEQ ID NO: 10)The above nucleotide sequence encodes the following polypeptide:DSM 32503 ScrAMDYKQIAKEVIEALGGRENVNSVAHCATRLRVMVKDENKINKEKAENIEKVQGAFFNSGQYQMIFGTGTVNKIYDEIVAQGLPTASKDEQKAEAAKQGNWFQRAIRSFGDVFVPLLPAIVATGLFMGIRGAINNDTVLALFGTTSKAFAATDFYTYTVVLTDTAFAFFPALICWSAFNIFGGSPLLGLVLGLMMVNNALPNAWDVASGAAKPIYFFDFIPVVGYQNSVLPAFFVGLIGAKFEQWVRKWVPDVLDLLLRPLVVFAVMSALALFIIGPVFHTVESYVLAGTEWILALPFGLAGLVLGGIHQIIVVTGVHHVFNLLEANLISNTGKDPLNAIITAAMTAQAGATLAVGVKTKDSKLKALAFPASLSAVLGITEPAIFGVNLRFGKPFIMGLIAGAAGGWLASILNLAGTGFGVTIVPGTLLYLNGQVLKYVIMVLVTLALGFALTWIFGYKEEEVEAQTEVVAEDIASAGSAPVALQAETIAAPLKGEVVALENVNDPVFSSGAMGKGAAIKPSGNRVVAPFDGEVQIAFPTGHAYGLKSDKGAEVLIHIGIDTVSLDGKGFDAKVQANQRVKKGDVLATFDSSVITEAGLDDTTMVIVTNTADYEDVSLVATGSVAEGDDFIAVK (SEQ ID NO: 11, positions 129 and 285 have been underlined)In some embodiments, the mutation in the scrR gene is an inactivating mutation. In some embodiments, the inactivating mutation is a base substitution. In some embodiments, the inactivating mutation is a base substitution and the encoded polypeptide is 315-327 amino acid residues in length, preferably 321amino acid residues in length (the length of SEQ ID NOs: 8 and 9 is 321 amino acid residues). In some embodiments, the mutation is in the coding region of the scrR gene or a non-coding regulatory element of the scrR gene. In some embodiments, the mutation consists of a substitution at position 242 and / or 44 of the translated amino acid sequence (numbering is defined with respect to SEQ ID NO: 8 or 9 as depicted in Figure 5 or 6). In some embodiments, the mutation in the coding region of the scrR gene results in a A242D or L44R substitution in the gene product encoded by the scrR gene. In other words, in some embodiments, the mutation consists of a A242D or L44R substitution.In some embodiments, the protein encoded by the scrR gene has at least 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity with residues 2-43 and 45-321 of SEQ ID NO: 8 or 9 and the amino acid residue at position 44 is substituted. In a preferred embodiment, the residue at position 44 is not a leucine. In some embodiments, the protein encoded by the scrR gene has at least 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity with residues 2-241 and 243-321 of SEQ ID NO: 8 or 9 and the amino acid residue at position 242 is substituted. In a preferred embodiment, the residue at position 242 is not an alanine.In some embodiments, the mutation in the scrA gene is an activating mutation. In some embodiments, the mutation is in the coding region of the scrA gene or a non-coding regulatory element of the scrA gene. In some embodiments, the mutation consists of a substitution at position 285 and / or 129 of the translated amino acid sequence (numbering is defined with respect to SEQ ID NO: 11 as depicted in Figure 7 or 8). In some embodiments, the mutation in the coding region of the scrA gene results in a V285I or R129C substitution in the gene product encoded by the scrA gene. In other words, in some embodiments, the mutation consists of a V285I or R129C substitution.In some embodiments, the protein encoded by the scrA gene has at least 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity with residues 2-128 and 130-633 of SEQ ID NO: 11 and the amino acid residue at position 129 is substituted. In a preferred embodiment, the residue at position 129 is not an arginine. In some embodiments, the protein encoded by the scrA gene has at least 95%, 96%, 97%, 98% or 99% sequence identity or 100% sequence identity with residues 2-284 and 286-633 of SEQ ID NO: 11 and the amino acid residue at position 285 is substituted. In a preferred embodiment, the residue at position 285 is not a valine.In some embodiments, the strain carries a V285I substitution mutation in the scrA gene and an L44R substitution in the scrR gene.In some embodiments, the strain is the strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession number DSM 34852, or a mutant strain derived therefrom that has retained or further improved on the glucose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C is the same or higher and / or the pH after 24 hours is the same or lower when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C) and / or sucrose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) sucrose at 40 °C is the same or higher) of the DSM34852 strain.In some embodiments, the strain is the strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession number DSM 34853, or a mutant strain derived therefrom that has retained or further improved on the glucose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C is the same or higher and / or the pH after 24 hours is the same or lower when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C) and / or sucrose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) sucrose at 40 °C is the same or higher) of the DSM34853 strain.In some embodiments, the strain is the strain that was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession number DSM 34854, or a mutant strain derived therefrom that has retained or further improved on the glucose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C is the same or higher and / or the pH after 24 hours is the same or lower when grown on M17 broth supplemented with 2 % (w / v) glucose at 40 °C) and / or sucrose fermenting property (e.g., the acidification rate when grown on M17 broth supplemented with 2 % (w / v) sucrose at 40 °C is the same or higher) of the DSM34854 strain.In some embodiments, the strain according to the second aspect of the invention is galactose-fermenting. Strains that are galactose-fermenting are preferred for the fermentation of compositions comprising galactose, such as lactose-hydrolyzed milk.The present invention also provides compositions comprising a strain according to the first and / or second aspect of the present invention, and, optionally, sucrose.In some embodiments, the composition comprises 104to 1012cfu / g of a strain according to the first and / or second aspect of the invention. In some embodiments, the composition further comprises one or more lactic acid bacteria strains. The one or more lactic acid bacteria strains may belong to the genus Lactococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Brevibacterium, Enterococcus or Propionibacterium. In a preferred embodiment, the composition further comprises an L. bulgaricus strain.In some embodiments, the composition is a starter culture. In some embodiments, the starter culture comprises 104to 1012cfu / g of a strain according to the first and / or second aspect of the invention. In some embodiments, the starter culture further comprises one or more lactic acid bacteria strains. The one or more lactic acid bacteria strains may belong to the genus Lactococcus, Lactobacillus, Leuconostoc, Pseudoleuconostoc, Pediococcus, Brevibacterium, Enterococcus or Propionibacterium. In a preferred embodiment, the starter culture further comprises a L. bulgaricus strain. In some embodiments, the starter culture may comprise a cryopreservant.In some embodiments, the composition is a preculture comprising a strain according to the first and / or second aspect of the invention and sucrose. It was surprisingly found that preculturing the strains of the present invention in sucrose stimulates subsequent growth on glucose.Methods of preparing a fermented productThe present invention also provides a method of preparing a fermented product comprising inoculating a milk base with (a) the .S', thermophilus strain of the first and / or second aspect of the invention, or (b) the composition of the present invention.In some embodiments, the milk base is milk or a plant-based milk alternative. In some embodiments, the milk is lactose-hydrolyzed milk. In some embodiments, the milk is lactose-hydrolyzed milk and the .S', thermophilus strain is galactose-fermenting. In some embodiments, the plant-based milk alternative comprises sucrose. Sucrose may be present naturally in the plant-based milk alternative or may be added to the plant-based milk alternative. In some embodiments, the plant-based milk alternative comprises sucrose and the .S', thermophilus strain comprises a mutation in the scrR gene. In some embodiments, the plant-based milk alternative is soy milk and the .S', thermophilus strain comprises a mutation in the scrR gene.In some embodiments, the .S', thermophilus strain is cultured in a medium (e.g., broth) comprising sucrose prior to inoculating the milk base. In a preferred embodiment, the .S', thermophilus strain is cultured in a medium comprising sucrose prior to inoculating the lactose-hydrolyzed milk or a plant-based milk alternative comprising glucose. Glucose may be present naturally in the plant-based milk alternative or may be added to the plant-based milk alternative.Fermented productsThe present invention also provides a fermented product comprising the .S', thermophilus strain according to the first and / or second aspect of the present invention. The present invention also provides a fermented product obtained or obtainable by the methods of preparing a fermented product of the present invention.Methods for producing a .S', thermophilus strain according to the second aspect of the inventionA mutation in the sucrose gene cluster can be induced by growing a mother strain in a glucose-rich medium wherein glucose is the sole fermentable carbohydrate until the mutation is induced spontaneously. Thus, the present invention also provides a method for producing a .S', thermophilus strain according to the second aspect of the invention comprising (1) serial passaging a glucose-fermenting mother strain in medium supplemented with glucose, and (2) identifying and isolating a glucose- fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster from a pool of mutant .S', thermophilus strains derived from the mother strain. The serial passaging step results in a pool of mutant s, thermophilus strains. These mutants arise spontaneously.The medium may be a broth (e.g., M17 broth). Further, the medium may be supplemented with at least 1% (w / v), at least 2% (w / v), at least 3% (w / v), at least 4% (w / v), or at least 5% (w / v) glucose. Preferably, the medium is supplemented with at least 5% (w / v) glucose.In some embodiments, a glucose-fermenting S. thermophilus strain that carries a mutation in a sucrose gene cluster (e.g., a mutation in the scrR gene and / or a mutation in the scrA gene) is identified by screening the pool of mutant .S', thermophilus strains for a strain that grows faster in broth supplemented with glucose than the mother strain. In some embodiments, a strain produced by the method of the present invention has a higher maximum acidification rate in M17 broth supplemented with 2 % (w / v) glucose than the mother strain.In some embodiments, a glucose-fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster is identified by sequencing the pool of mutant .S', thermophilus strains.In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 serial passages are performed. Preferably, at least 5 serial passages are performed. More preferably, at least 10 serial passages are performed. Most preferably, at least 15 serial passages are performed. The identificationand isolation step can be performed after each serial passage until a S thermophilus strain that carries a mutation in a sucrose gene cluster is obtained.Items of the inventionThe present invention also provides the following items which can be combined with the relevant aspects and embodiments provided above.[1] A glucose-fermenting Streptococcus thermophilus strain that carries a mutation in the glcU gene.[2] The .S', thermophilus strain of item [1], wherein the mutation in the glcU gene causes the strain to reach a lower pH in M17 broth supplemented with 2 % (w / v) glucose than in M17 broth supplemented with 2 % (w / v) lactose after 24 hours at 40 °C.[3] The .S', thermophilus strain of item [1] or [2], wherein the strain carries a mutation in the coding region of the glcU gene.[4] The .S', thermophilus strain of any one of items [1] to [3], wherein the mutation consists of a V101A mutation.[5] The .S', thermophilus strain of any one of items [1] to [4], wherein the strain is not the strain that was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 24010.[6] The .S', thermophilus strain of any one of items [1] to [5], wherein the strain carries a mutation in a non-coding regulatory element of the glcU gene.[7] The .S', thermophilus strain of item [6], wherein the mutation consists of a T insertion at position -81 of the glcU gene.[8] The .S', thermophilus strain of any one of items [l]-[7], wherein the strain further carries a mutation in a sucrose gene cluster.[9] The .S', thermophilus strain of any one of items [l]-[8], wherein the strain is galactose- fermenting.

[0010] A composition comprising sucrose and the .S'. thermophilus strain of any one of items [ l]-[9] .

[0011] A method of preparing a fermented product comprising inoculating a milk base with the .S'. thermophilus strain of any one of claims [l]-[9], or the composition of item

[0010] ,

[0012] The method of item

[0011] , wherein the milk base is lactose-hydrolyzed milk.

[0013] A fermented product comprising the .S' thermophilus strain of any one of items [ l]-[9] .

[0014] A fermented product obtained or obtainable by the method of item

[0011] or

[0012] ,Deposits and expert solutionThe applicant requests that a sample of the deposited microorganisms stated in Table 1 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.Table 1: 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.ExamplesMaterials and methodsFor .S'. thermophilus, M17 media was used.The M17 agar medium has the following composition per liter water:The final pH of the M17 agar medium was 7. 1 ± 0.2 (measured at 25°C).Ml 7 broth has the following composition per liter water:The final pH of the M17 broth was 7.0 ± 0.2 (measured at 25°C).M17 broth and M17 agar medium were supplemented with 20 g / L of a carbon source (e.g., lactose, glucose, galactose, or sucrose).Example 1: Improved glucose utilization oiglcU mutantsThe .S' thermophilus mother strains (DSM 24012 and DSM 22934) and the respective glcU mutant strains (DSM 24010 and DSM 33572) strain were inoculated into M17 broth supplemented with either 2% (w / v) lactose or 2% (w / v) sucrose and incubated overnight at 40°C under anaerobic conditions.The overnight cultures were used to inoculate 200 m of sterile Ml 7 broth supplemented with 2 % (w / v) glucose in baby bottles. The inoculum size was 1 % (v / v) and the culture was incubated at 40°C in a water bath. Acidification was followed by determination of the changes in pH at 40°C, using an ICINAC pH logger using CINAC software (Alliance Instruments). The results of the acidifications are shown in Figure 3.Example 2: Comparative sugar utilization of DSM 33572 and its mother strainThe .S', thermophilus mother strain (DSM 22934) and the respective glcU mutant strain (DSM 33572) strain were inoculated into M17 broth supplemented with either 2% (w / v) lactose or 2% (w / v) sucrose and incubated overnight at 40°C under anaerobic conditions.The overnight cultures were used to inoculate 200 m of sterile Ml 7 broth supplemented with 2 % (w / v) glucose or lactose in baby bottles. The inoculum size was 1 % (v / v) and the culture was incubated at 40°C in a water bath. Acidification was followed by determination of the changes in pH at 40°C, using an ICINAC pH logger using CINAC software (Alliance Instruments). The results of the acidifications are shown in Figure 4.As shown in Figure 4A, the mutant strain reached a lower pH when precultured in the presence of sucrose or lactose and then cultured in M17 broth supplemented with glucose. When precultured in a broth supplemented with sucrose, the acidification rate of the mutant in M17 broth supplemented with glucose was increased.Further, the mutant strain was able to reach a lower pH when grown in M17 broth supplemented with glucose than when grown in Ml 7 broth supplemented with lactose.Example 3 : Selection of mutants with mutations in a sucrose gene clusterAn .S', thermophilus mother strain was initially inoculated into M17 broth supplemented with 2% (w / v) lactose and incubated overnight at 40°C under anaerobic conditions.The overnight culture was used for an adaptive laboratory evolution method wherein the mother strain underwent serial passaging in Ml 7 broth supplemented with 5 % (w / v) glucose. Once a day, the overnight culture inoculated on the previous day was used to inoculate 10 m of sterile Ml 7 broth supplemented with 5 % (w / v) glucose. The inoculum size was 1 % (v / v) and the culture was incubated overnight at 40°C under anaerobic conditions. This process was repeated a total of 15 times.After 15 days, the adapted cultures were diluted 10'4to 10'8and plated on M17 agar plates supplemented with 2% (w / v) glucose. The agar plates were incubated overnight at 40 °C under anaerobic conditions.On the next day colonies were picked and inoculated into M17 broth supplemented with 2 % (w / v) glucose and M17 broth supplemented with 2% (w / v) lactose in a microtiter plate. The mother strain was also inoculated into Ml 7 broth supplemented with 2 % (w / v) glucose and M17 broth supplemented with2% (w / v) lactose as a control. The growth was measured using a BioTek LogPhase 600 Microbiology Reader at 40 °C.Strains that grew faster than the mother strain in M17 broth supplemented with glucose were selected and found to contain a mutation in a sucrose gene cluster.When the above protocol was performed using DSM 22934 (a galactose-fermenting strain) as the mother strain, we obtained two mutants. Both mutants contained an A242D substitution as depicted in Figure 5 and produced similar acidification curves. One of the mutants was deposited (see DSM 34852).When the above protocol was performed using DSM 32503 as the mother strain, we obtained DSM 34853 and DSM 34854. DSM 34853 contains a mutation in the coding region of the scrR gene (see Figure 6) as well as a mutation in the coding region of the scrA gene (see Figure 7). DSM 34854 contains a mutation in the coding region of the scrA gene (see Figure 8).Example 4: Acidification curves of sucrose gene cluster mutants in glucose or lactoseThe .S', thermophilus mother strains (DSM 22934 and DSM 32503) and the respective mutant strains (DSM 34852, DSM 34853 and DSM 34854) were inoculated into M17 broth supplemented with both 2% (w / v) lactose and 2% (w / v) glucose incubated overnight at 40°C under anaerobic conditions.The overnight cultures were used to inoculate 200 m of sterile Ml 7 broth supplemented with 2 % (w / v) glucose, or lactose in baby bottles. The inoculum size was 1 % (v / v) and the culture was incubated at 40°C in a water bath. Acidification was followed by determination of the changes in pH at 40°C, using an ICINAC pH logger using CINAC software (Alliance Instruments). The results of the acidifications are shown in Figure 10.Example 5: Acidification curves of sucrose gene cluster mutantsDSM 34853 and the mutant derived from DSM 22934 containing an A242D substitution in the scrR gene were inoculated into M17 broth supplemented with 2% (w / v) lactose and incubated overnight at 40°C under anaerobic conditions.The overnight cultures were used to inoculate 200 m of sterile Ml 7 broth supplemented with 2 % (w / v) glucose, galactose, lactose or sucrose in baby bottles. The inoculum size was 1 % (v / v) and the culture was incubated at 40°C in a water bath. Acidification was followed by determination of the changes in pH at 40°C, using an ICINAC pH logger using CINAC software (Alliance Instruments). The results of the acidifications are shown in Figure 11.Example 6: Fermentation of soy milkThe .S'. thermophilus mother strains (DSM 22934 and DSM 32503) and the respective mutant strains (DSM 34852, DSM 34853 and DSM34854) were inoculated into M17 broth supplemented with 2% (w / v) sucrose and incubated overnight at 43°C under anaerobic conditions.The overnight cultures were used to inoculate 150 m Provomel unsweetened soy milk (organic bio). This soy milk contains from 0.5% to 1% sucrose and a few sugar fibers. Only the sucrose is fermentable by .S', thermophilus. The inoculum size was 1 % (v / v) and the culture was incubated at 43°C in a water bath. Acidification of the soy was followed by determination of the changes in pH at 43°C, using an ICINAC pH logger using CINAC software (Alliance Instruments). The results of the acidifications are shown in Figure 12.As shown in Figure 12, mutations in the scrR gene were associated with improved sucrose utilization.A mutation in the scrA gene in DSM 34854 resulted in a slower acidification indicating that, without being bound by a particular theory, this mutation may increase the affinity of the transporter for glucose while at the same time reducing the affinity for sucrose.I28PCT(Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)I29PCT(Original in Electronic Form) (This sheet is not part of and does not count as a sheet of the international application)FOR RECEIVING OFFICE USE ONLYFOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. A glucose-fermenting Streptococcus thermophilus strain that carries a mutation in a sucrose gene cluster.

2. The .S'. thermophilus strain of claim 1, wherein the mutation in the sucrose gene cluster causes the strain to acidify at substantially the same rate in M17 broth supplemented with 2 % (w / v) glucose at 40 °C as in M17 broth supplemented with 2 % (w / v) lactose at 40 °C.

3. The .S'. thermophilus strain of claim 1 or 2, wherein the mutation in the sucrose gene cluster causes the strain to reach substantially the same or lower pH in M17 broth supplemented with 2 % (w / v) glucose as in M17 broth supplemented with 2 % (w / v) lactose after 24 hours at 40 °C.

4. The .S' thermophilus strain of any one of claims 1-3, wherein the mutation is obtainable by serial passaging a glucose-fermenting .S' thermophilus mother strain in M17 broth supplemented with at least 5 % (w / v) glucose.

5. The .S', thermophilus strain of any one of claims 1-4, wherein the strain comprises a mutation in the scrR gene, and / or the strain comprises a mutation in the scrA gene.

6. The .S', thermophilus strain of claim 5, wherein the mutation in the scrR gene is an inactivating mutation and / or the mutation in the scrA gene is an activating mutation.

7. The .S', thermophilus strain of claim 6, wherein the inactivating mutation consists of a L44R or A242D substitution and / or the activating mutation consists of a V285I or R129C substitution.

8. The .S', thermophilus strain of any one of claims 1-7, wherein the strain further carries a mutation in the glcU gene.

9. The .S', thermophilus strain of any one of claims 1-8, wherein the strain is galactose-fermenting.

10. A composition comprising sucrose and the .S', thermophilus strain of any one of claims 1-9.

11. A method of preparing a fermented product comprising inoculating a milk base with the S. thermophilus strain of any one of claims 1-9, or the composition of claim 10.

12. The method of claim 11, wherein the milk base is:(i) milk, preferably lactose-hydrolyzed milk, or(ii) a plant-based milk alternative, preferably soy milk.

13. A fermented product comprising the .S', thermophilus strain of any one of claims 1-9.

14. A fermented product obtained or obtainable by the method of claim 11 or 12.

15. A method for producing a .S', thermophilus strain according to any one of claims 1 -9 comprising:(1) serial passaging a glucose-fermenting .S', thermophilus mother strain in medium supplemented with glucose, and(2) identifying and isolating a glucose-fermenting .S', thermophilus strain that carries a mutation in a sucrose gene cluster from a pool of mutant .S', thermophilus strains derived from the mother strain.

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