Lactic acid bacteria composition for producing fermented products

Abstract

The present invention relates to compositions comprising one or more novel Streptococcus thermophilus strains and to the use of said compositions for producing fermented products, such as sweetened dairy products. The present invention also relates to the novel Streptococcus thermophilus strains themselves.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to compositions comprising one or more novel Streptococcus thermophilus strains and the use of said compositions for producing fermented products, such as sweetened dairy products. The present invention also relates to the novel Streptococcus thermophilus strains themselves. [Background technology]

[0002] Purely fermented dairy products are perceived as having a sour or sour taste as a result of the conversion of milk sugar (lactose) to lactic acid by lactic acid bacteria during fermentation, and therefore such dairy products are often sweetened with the addition of fruit, honey, sugar, or artificial sweeteners to meet consumer demand for a sweeter taste.

[0003] In the food industry, there is an increasing demand for low-calorie yet sweet foods to overcome the widespread problem of overweight and obesity that has existed for the last 20 years. Sweetness is usually considered to be satisfying and is produced by the presence of sugars and other substances. The perceived sweetness of sugars varies widely. Using sucrose as the standard of 100, the sweetness of lactose is 16, that of galactose is 32, and that of glucose is 74 (Godshall (1988). Food Technology 42(ll): 71-78). Thus, glucose is perceived as more than four times sweeter than lactose, despite having roughly the same calorie level.

[0004] Sugar in fermented foods is often replaced by sweeteners such as aspartame, acesulfame K, sucralose, and saccharin, which can provide sweetness with a lower calorie intake. However, the use of artificial sweeteners can cause unpleasant tastes, and several studies have shown that the consumption of artificial sweeteners is associated with disadvantages such as increased hunger, allergies, and cancer, contributing to consumers' preference for fermented dairy products that contain only natural sweeteners or preferably no added sweeteners. Therefore, the development of fermented dairy products with increased natural (intrinsic) sweetness poses special challenges.

[0005] The acidity (acidity) of fermented milk products depends largely on the lactic acid bacteria present and the process parameters used to produce the fermented milk product.

[0006] Since lactose is the main carbon source in milk, the fermentation of the disaccharide lactose has been widely studied in lactic acid bacteria. In many species, lactose is taken up and then degraded to glucose and galactose by β-galactosidase. Glucose is phosphorylated to glucose-6-phosphate by glucokinase and fermented by most lactic acid bacteria via the Embden-Meyerhof-Parnas pathway (glycolysis).

[0007] Streptococcus thermophilus (also called S. thermophilus) is one of the most widely used lactic acid bacteria commercially for thermophilic lactic acid fermentation, and this organism is usually used as part of a mixed starter culture, the other components of which are Lactobacillus sp., in the case of yogurt Lactobacillus delbrueckii subsp bulgaricus (L. bulgaricus), and in Swiss-type cheese Lactobacillus helveticus (L. helveticus).

[0008] The legal definition of yogurt in many countries requires S. thermophilus along with L. bulgaricus, while others require S. thermophilus along with L. acidophilus, all of which produce desirable amounts of acetaldehyde, an important flavor component of yogurt.

[0009] Lactose and sucrose are more readily fermented by C. thermophilus than are their component monosaccharides.

[0010] In the presence of excess galactose, when L. thermophilus is used, only the glucose portion of the lactose molecule is fermented, leading to the accumulation of galactose in fermented dairy products. Free galactose remains in yogurt, where high acid concentrations limit fermentation, but free galactose generated in the early stages of Swiss cheese production is later further fermented by L. helveticus.

[0011] However, galactose-fermenting strains of S. thermophilus have been reported by several researchers (Hutkins et al. (1986) J. Dairy Sci. 69(1): 1-8; Vaillancourt et al. (2002) J. Bacteriol. 184(3): 785-793) and WO 2011 / 026863 (Chr. Hansen) describes a method for obtaining galactose-fermenting S. thermophilus strains. The present applicant, Chr. Hansen, has subsequently described other galactose-fermenting strains in several publications.

[0012] To meet the requirements of the food industry, it has become important to propose new strains, especially S. thermophilus strains, that provide more natural sweetness (inherent sweetness) to fermented products without directly introducing extra calories through glucose excretion. It has been observed that galactose strains often exhibit a slower acidification profile, and therefore it is an industry imperative to provide rapid acidifying strains that can ferment galactose or that work well with galactose-fermenting strains. Rapid acidification can reduce fermentation times, which may improve process economics and / or interactions between microbial species. As texture-improving properties may also be affected in galactose-fermenting strains, it is also desirable to develop galactose-fermenting strains with enhanced texture-improving properties or strains with enhanced texture-improving properties that work well with galactose-fermenting strains. Summary of the Invention [Problem to be solved by the invention]

[0013] The above objectives are achieved by the present invention, which in particular is directed to a composition comprising one or more novel galactose-fermenting strains of S. thermophilus strains. The novel galactose-fermenting Thermophilus strains contain one or more mutations in the manM gene encoding glucose / fructose subunit IIC, in the galK gene encoding galactokinase, in the pgm gene encoding phosphoglucomutase, and / or in the galR gene encoding galactose. In addition to their ability to ferment galactose, these novel Thermophilus strains exhibit rapid acidification, acidification to low pH and / or improved texture-improving properties during fermentation when applied alone and / or in combination with other Thermophilus and / or Lactobacillus strains. [Means for solving the problem]

[0014] Thus, in one aspect, the present invention relates to a composition comprising a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11.

[0015] A further aspect of the invention is a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) a glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11 The present invention also relates to a method for producing a fermented product, which comprises fermenting a substrate using a composition according to the present invention.

[0016] Therefore, one aspect of the present invention relates to a fermented product obtainable by the method of the present invention.

[0017] A further aspect of the present invention relates to the use of a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11.

[0018] Importantly, one aspect of the present invention relates to a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the lactation profile of strain DSM 33762 and the parent strain of DSM 33762.

[0020] [Diagram 2] FIG. 2 shows the lactation profile of strain DSM 33720.

[0021] [Diagram 3] FIG. 3 shows the course of laboratory adaptive evolution of the DSM 33719 strain.

[0022] [Figure 4] FIG. 4 shows the lactation profile of strain DSM 33719 and the parent strain of DSM 33719. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Before describing the present invention in more detail, a set of terms and conventions are defined.

[0024] The term "genus" refers to a genus as defined at the website: www.ncbi.nlm.nih.gov / taxonomy. A bacterial "strain" as used herein refers to a bacterium that remains genetically unchanged when grown or propagated. It includes many overlapping identical bacteria.

[0025] In the present context, the term "mutant" or "variant" is to be understood as a strain derived or derivable from the strain of the invention (or mother strain), for example by genetic engineering, radiation and / or chemical treatment. The mutant is preferably a functionally equivalent mutant of the original strain from which it is derived, for example a mutant having substantially the same or improved properties (for example in terms of viscosity, gel hardness, mouth-coating (smooth aftertaste), flavor, post acidification, acidification rate and / or phage fastness). Such mutants are part of the present invention. In particular, the term "mutant" refers to a strain obtained by subjecting the strain of the invention to any conventionally used mutagenic treatment, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), UV light, or a spontaneous mutant. The mutant may have been subjected to several mutagenesis treatments (a single treatment should be understood as one mutagenesis step followed by a screening / selection step), but currently it is preferred that it is no more than 20, or no more than 10, or no more than 5 treatments (or screening / selection steps). In currently preferred mutants, less than 5%, or no more than 1%, or even no more than 0.1% of the oligonucleotides in the bacterial genome are replaced by another nucleotide or deleted compared to the parent strain. As will be clear to the skilled artisan, the mutant of the present invention can also be the parent strain.

[0026] In the present context, the term "variant" or "variant strain" should be understood as a strain that is functionally equivalent to the strain of the invention, e.g. a strain that has substantially the same or improved properties or characteristics, such as texture, acidification rate, viscosity, gel hardness, mouth-coating (smooth aftertaste), flavor, after acidification, and / or phage fastness. Such variants can be identified using appropriate screening techniques, and such mutants are part of the present invention.

[0027] For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman & Wunsch (1970) J. Mol. Biol. 48: 443-453), preferably implemented with the EMBOSS package (EMBOSS: The E european M Olecular B iology O pen S Oftware S The Needle program from uite, an open source analytical software package developed for molecular biology users; Rice et al. (2000) Trends in Genetics 16: 276-277, is implemented in this program. Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "Longest Identity" (obtained using the -no Brief option) is used as the percent identity, calculated as follows:

[0028] (number of identical residues × 100) / (length of alignment – ​​total number of gaps in alignment)

[0029] In the present specification and claims, conventional single-letter and three-letter codes for amino acid residues are used. For ease of reference, the amino acid changes in the mutants and variants of the present invention are described using the following nomenclature: amino acid residue in the parent enzyme; position; substituted amino acid residue. According to this nomenclature, for example, the substitution of an alanine residue at position 30 with a glycine residue is indicated as Ala20Gly or A20G. The deletion of an alanine at the same position is indicated as Ala20* or A20*. The insertion of an additional amino acid residue (e.g., glycine) is indicated as Ala20AlaGly or A20AG. The deletion of consecutive amino acid residues (e.g., between alanine at position 20 and glycine at position 21) is indicated as Δ(Ala20-Gly21) or Δ(A20-G21) (Δ:DELTA). If the parent enzyme sequence contains a deletion compared to the enzyme sequence used to number the insertion at such position (e.g., an alanine at deleted position 20), it is indicated as *20Ala or *20A. Multiple mutations are separated by a plus sign or a slash. For example, two mutations at positions 20 and 21, which replace alanine and glutamic acid with glycine and serine, respectively, are indicated as A20G+E21S or A20G / E21S. If an amino acid residue at a particular position is replaced with two or more alternative amino acid residues, these residues are separated by a comma or a slash. For example, if an alanine at position 20 is replaced with glycine or glutamic acid, it is indicated as A20G,E or A20G / E, or A20G,A20E. When a position suitable for modification is identified herein without suggesting any particular modification, it should be understood that the amino acid residue present at that position can be replaced with any amino acid residue. Thus, for example, when a modification of an alanine at position 20 is mentioned but not specified, the alanine may be deleted or substituted with another 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).

[0030] In the context of the present invention, a mutation in a gene (genetic mutation) should be understood as a change in the nucleotide sequence of the genome of an organism, which leads to a change in the phenotype of the organism, the change can 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 should be understood as a genetic mutation that leads to the removal of one or more nucleotides of the nucleotide sequence of the genome of an organism. An insertion should be understood as the addition of one or more nucleotides to a nucleotide sequence. A substitution (or point mutation) should be understood as a genetic mutation in which a nucleotide in a nucleotide sequence is replaced by another nucleotide. A frameshift should be understood as a genetic mutation caused by the insertion or deletion of a number of nucleotides not divisible by three in a nucleotide sequence, which results in a change in the reading frame and a translation that is completely different from the translation from the original reading frame. The introduction of a stop codon should be understood as a point mutation in a DNA sequence that results in a premature stop codon. The inhibition of binding of an encoded protein to a substrate should be understood as any mutation in a nucleotide sequence that leads to a change in the protein sequence that is responsible for preventing the binding of a substrate to the catalytic site of the protein. Furthermore, a knockout mutation should be understood as a genetic mutation that results in the removal or deletion of a gene, such as an entire gene or an entire open reading frame (ORF), from the genome of an organism.

[0031] In this specification and claims, the conventional single-letter symbols for nucleotides are used according to principles similar to those described above for the amino acid nomenclature.

[0032] Algorithms for aligning sequences and determining the degree of sequence identity between those sequences are well known in the art. For the purposes of the present invention, the process for aligning nucleotide sequences can be carried out using the blastn program, such as that provided by the National Center for Biotechnology Information (NCBI) at https: / / blast.ncbi.nlm.nih.gov, applying standard parameters.

[0033] The acidification profile is measured as disclosed in Example 1.

[0034] The shear stress is measured as disclosed in Example 4.

[0035] With respect to strains of the genus Bacillus, the term "CFU" refers to colony forming units as determined by growth (forming colonies) on MRS agar plates upon incubation at 37° C. for 3 days under anaerobic conditions. The composition of MRS agar medium is as follows (g / L): Bactoproteose peptone No.3: 10.0 Bactobeef extract: 10.0 Bacto Yeast Extract: 5.0 Glucose: 20.0 Sorbitan monooleate complex: 1.0 Ammonium citrate: 2.0 Sodium acetate: 5.0 Magnesium sulfate: 0.1 Manganese sulfate: 0.05 Dipotassium hydrogen phosphate: 2.0 Bacto agar: 15.0 Milli-Q water: 1000mL.

[0036] Adjust pH to 5.4 or 6.5: For L. rhamnosus, L. casei and L. paracasei, adjust pH to 6.5. For all other Lactobacillus species, adjust pH to 5.4. Specifically for L. delbrueckii subsp. bulgaricus, L. acidophilus and L. helveticus, adjust pH to 5.4. For L. rhamnosus, L. casei and L. paracasei, adjust pH to 6.5.

[0037] With respect to S. thermophilus, the term "CFU" refers to colony forming units as determined by growth (forming colonies) on M17 agar plates upon incubation at 37° C. for 3 days under aerobic conditions. The composition of M17 agar medium is as follows (g / L): Tryptone: 2.5g Peptic digest of meat: 2.5g Papain digest of soybean meal: 5.0g Yeast extract: 2.5g Meat extract: 5.0g Lactose: 5.0g Sodium glycerophosphate: 19.0g Magnesium sulfate 7H2O: 0.25g Ascorbic acid: 0.5g Agar: 15.0g Milli-Q water: 1000mL. The final pH was adjusted to 7.1 ± 0.2 (25°C).

[0038] The term "mutations that inactivate glucokinase protein" as used herein refers to mutations that result in "inactivated glucokinase protein", i.e., mutations that result in glucokinase protein that cannot perform its normal function when present in a cell, as well as mutations that prevent the formation of the glucokinase protein or cause its degradation. In particular, an inactivated glucokinase protein is a protein that cannot promote the phosphorylation of glucose to glucose-6-phosphate or promotes the phosphorylation of glucose to glucose-6-phosphate at a significantly reduced rate compared to a functional glucokinase protein. Compared to a gene encoding a functional glucokinase protein, such a gene encoding an inactivated glucokinase protein contains a mutation in the open reading frame (ORF) of the gene, including, but not limited to, a mutation that causes a deletion, a frameshift mutation, the introduction of a stop codon, or an amino acid substitution to change the functional properties of the protein, or a promoter mutation that reduces or abolishes the transcription or translation of the gene.

[0039] As used herein, the term "functional glucokinase protein" refers to a glucokinase protein that, when present in a cell, promotes the phosphorylation of glucose to glucose-6-phosphate.

[0040] As used herein, a "mutant" or "mutant strain" refers to a naturally occurring or induced mutant bacteria that contains one or more mutations in its genome (DNA) that are not present in the wild-type DNA. An "induced mutant" refers to a bacterium in which mutations have been induced by artificial treatment, such as treatment with chemical mutagens, ultraviolet light or gamma radiation, etc. In contrast, a "spontaneous mutant" or "naturally occurring mutant" has not been artificially induced to mutate. As used herein, a mutant bacteria is non-GMO (non-genetically modified organism), i.e., not modified by recombinant DNA technology.

[0041] As used herein, the term "mutation that reduces glucose transport into the cell" refers to a mutation in a gene encoding a protein involved in glucose transport that results in the accumulation of glucose in the cellular environment. Glucose levels in the culture medium of a Thermophilus strain can be readily measured by methods known to those skilled in the art.

[0042] As used herein, the term "mutations that inactivate glucose transporters" refers to mutations that result in an "inactivated glucose transporter," i.e., a glucose transporter protein that is unable to perform its normal function when present in a cell, as well as mutations that prevent the formation of or cause degradation of the glucose transporter protein.

[0043] The term "functional glucose transporter protein" as used herein refers to a glucose transporter protein that, when present in a cell, facilitates the transport of glucose through the cell membrane. The term "glucose-deficient" is used in the context of the present invention to characterize lactic acid bacteria (LAB) that have partially or completely lost the ability to consume glucose as an (energy) source for cell growth or maintaining cell viability. Each deficiency in glucose metabolism can be caused, for example, by a mutation in a gene that inhibits or inactivates the expression or activity of the glucokinase protein and / or the glucose transporter protein involved in glucose uptake.

[0044] LAB with defects in glucose metabolism can increase the glucose concentration in the medium when they grow using lactose as a carbohydrate source. The increase in glucose is caused by glucose secretion by the glucose-deficient LAB. The increase in glucose concentration in the medium can be achieved by, for example, using Dionex TM (Trademark) CarboPac TMThe amount of acetylcholine in the hydroxyl group can be measured by HPLC analysis using a 3×150 mm column of PA20™ (Thermo Fisher Scientific, product number 060142).

[0045] The term "glucose positive" is used in the context of the present invention to characterize LAB that partially or completely maintain the ability to use glucose as a source for cell growth or to maintain cell viability.

[0046] The inventors have surprisingly identified several Thermophilus strains that meet the needs of the industry. The novel galactose fermenting strain(s) exhibit, for example, improved rheological properties (e.g., texture) when applied as part of a mixed (starter) culture in a dairy substrate. Similarly, the novel galactose fermenting strain(s) exhibit, for example, improved acidification properties (e.g., acidification rate) when applied alone or as part of a mixed culture in a dairy substrate (Examples 1-2). Also, the novel galactose fermenting strain(s) can acidify the fermentation medium to a lower pH compared to its parent strain, which can be seen, for example, in Example 2.

[0047] The novel Thermophilus strains have the potential to be used in dairy starter cultures, e.g. yoghurt starter cultures, to improve the rheological parameters of the final product, such as enhanced sweetness and shear stress. Rheology is closely related to the organoleptic quality of the product, and therefore the interplay between rheology and taste in the final product is of utmost importance. The novel Thermophilus strains also have an improved acidification profile, allowing for shorter fermentation times.

[0048] composition Thus, one aspect of the present invention relates to a composition comprising a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11.

[0049] In one embodiment, the mutation causes an alteration in the encoded protein selected from the group consisting of: a) the PTS mannose / glucose / fructose subunit IIC at a position corresponding to position 57 of SEQ ID NO:2; b) galactokinase at a position corresponding to position 47 of SEQ ID NO:4; c) phosphoglucomutase at a position corresponding to position 242 of SEQ ID NO: 6 or 14; d) phosphoglucomutase at a position corresponding to position 164 of SEQ ID NO:8; e) the galactose operon repressor at a position corresponding to position 28 of SEQ ID NO:10; and f) A glucose kinase at a position corresponding to position 268 of SEQ ID NO:12.

[0050] In a further embodiment, the mutation is as follows: (a) a C to T substitution at a position corresponding to position 169 of SEQ ID NO:1; (b) a G to A substitution at a position corresponding to position 139 of SEQ ID NO:3; (c) an A to C substitution at a position corresponding to position 726 of SEQ ID NO:5 or 13; (d) a C to T substitution at a position corresponding to position 490 of SEQ ID NO:7; (e) a C to T substitution at a position corresponding to position 82 of SEQ ID NO:9; and (f) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:11.

[0051] In a further embodiment, the alteration in the encoded protein is as follows: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; (b) an Ile to Val substitution at a position corresponding to position 47 of SEQ ID NO:4; (c) a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO: 6 or 14; (d) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; (e) a Leu to Phe substitution at a position corresponding to position 28 of SEQ ID NO:10; and (f) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12.

[0052] In certain embodiments, the Streptococcus thermophilus strain comprises the following mutation: (a) a substitution of C to T at a position corresponding to position 169 of SEQ ID NO:1; a substitution of nucleotide G to A at a position corresponding to position 139 of SEQ ID NO:3; and a substitution of nucleotide A to C at a position corresponding to position 726 of SEQ ID NO:5 or 13; (b) a substitution of C to T at a position corresponding to position 490 of SEQ ID NO:5 or 13; and a substitution of nucleotide C to T at a position corresponding to position 82 of SEQ ID NO:7; or (c) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:9.

[0053] In terms of amino acid profile, the Streptococcus thermophilus strain appears to contain the following changes in the encoded protein: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; a substitution of Ile to Val at a position corresponding to position 47 of SEQ ID NO:4; a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO:6 or 14; (b) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; a substitution of Leu to Phe at a position corresponding to position 28 of SEQ ID NO:10; or (c) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12.

[0054] In a particular embodiment, the Thermophilus strain is: (a) DSM 33719 or a variant or variant thereof; (b) DSM 33720 or a variant or variant thereof; or (c) DSM 33762 or a variant or variant thereof.

[0055] Further details of the thermophilus strains and mutations disclosed above in (a)-(f) can be found below, namely in the section entitled "Novel Streptococcus thermophilus Mutants."

[0056] The composition of the present invention can be provided in several forms. It can be a powder, pellet or tablet. The composition can be in frozen, dry, lyophilized or liquid form. Thus, in one embodiment, the composition is in frozen, dry, lyophilized or liquid form.

[0057] The composition of the present invention may further comprise cryoprotectant, cryoprotectant, antioxidant, nutrient, bulking agent, flavoring, or mixture thereof.Preferably, the composition comprises one or more of cryoprotectant, cryoprotectant, antioxidant and / or nutrient, more preferably cryoprotectant, cryoprotectant and / or antioxidant, most preferably cryoprotectant or cryoprotectant or both.The use of protective agent such as cryoprotectant and any cryoprotectant is known to those skilled in the art. Suitable cryoprotectants or cryoprotectants include mono-, di-, tri-, and polysaccharides (such as glucose, mannose, xylose, milk sugar (lactose), sucrose (sucrose), trehalose, raffinose, maltodextrin, starch, and gum arabic (acacia)), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptone, gelatin, yeast extract, and the like), and inorganic compounds (e.g., sodium tripolyphosphate).

[0058] In one embodiment, the composition of the present invention may comprise one or more cryoprotectants selected from inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine, and derivatives of any such compounds. Suitable antioxidants include ascorbic acid, citric acid and its salts, gallates, cysteine, sorbitol, mannitol, and maltose. Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, and vitamins (such as B vitamins and vitamin C). The composition may optionally contain additional substances including bulking agents (such as lactose, maltodextrin, etc.) and / or flavoring agents.

[0059] In one embodiment of the invention, the cryoprotectant is an agent or mixture of agents that has a booster effect in addition to its cryoprotective properties.

[0060] The expression "booster effect" is used to describe the situation where a cryoprotectant confers an increased metabolic activity (booster effect) to a thawed or reconstituted (starter) culture when it is inoculated into the medium to be fermented or transformed. Viability and metabolic activity are not synonymous concepts. Commercially available frozen or lyophilized cultures can retain their viability, even though they may have lost a significant portion of their metabolic activity. For example, cultures can lose their acidogenic (acidifying) activity when stored in storage, even for a short period of time. Therefore, viability and booster effect need to be evaluated by different assays. Viability is evaluated by viability assays, such as measuring colony forming units, while booster effect is evaluated by quantifying the relative metabolic activity of the thawed or reconstituted culture relative to the viability of the culture. The term "metabolic activity" refers to the deoxygenating activity of the culture, its acidogenic activity, i.e. the production of, for example, lactic acid, acetic acid, formic acid and / or propionic acid, or the production of its metabolic products, such as aroma compounds, such as acetaldehyde, (α-acetolactate, acetoin, diacetyl and 2,3-butylene glycol (butanediol)).

[0061] In one embodiment, the composition of the invention contains or comprises 0.2% to 20% of the cryoprotectant or mixture of agents measured as a weight percent (w / w) of the raw material. However, it is preferred to add the cryoprotectant or mixture of agents in amounts ranging from 0.2% to 15%, 0.2% to 10%, 0.5% to 7%, and 1% to 6% by weight, such as amounts ranging from 2% to 5% by weight of the cryoprotectant or mixture of agents measured as a weight percent (w / w) of the frozen raw material. In a preferred embodiment, the culture contains about 3% by weight of the cryoprotectant or mixture of agents measured as a weight percent (w / w) of the raw material. An amount of about 3% of the cryoprotectant corresponds to a concentration in the 100 mM range. It should be recognized that for each aspect of the embodiment of the invention, the ranges can be increments of the stated ranges.

[0062] In this context, the term "x% to y%" is meant to be inclusive of the endpoints and is thus equivalent to "from x%, inclusive, to y%, inclusive."

[0063] In a further aspect, the composition of the invention contains or comprises ammonium salts, such as ammonium salts of organic acids (e.g. ammonium formate and ammonium citrate), as boosters (e.g. growth boosters or acidification boosters) for bacterial cells, such as cells belonging to the species Thermophilus, e.g. (substantially) urease negative bacterial cells. The terms "ammonium salts", "ammonium formate" and the like should be understood as a source of salts or combinations of ions. The term "source" (or simply source) of, for example, "ammonium formate" or "ammonium salts" refers to a compound or mixture of compounds that, when added to a culture of cells, provides ammonium formate or ammonium salts. In some embodiments, the ammonium source releases ammonium into the growth medium, while in other embodiments, the ammonium source is metabolized to produce ammonium. In some preferred embodiments, the ammonium source is exogenous. In some particularly preferred embodiments, the ammonium is not provided by the dairy substrate. Of course, it should be understood that ammonia may be added instead of ammonium salts. Thus, the term ammonium salts refers to ammonia (NH3), NH4OH, NH4 + etc.

[0064] In one embodiment, the compositions of the present invention may include thickeners and / or stabilizers such as pectin (e.g., HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginates, and guar gum.

[0065] In one embodiment where the microorganisms produce polysaccharides (such as EPS) that give rise to a thick / sticky texture in the acidified milk (so-called sour milk) product, the acidified milk product is produced substantially free or completely free of added thickeners and / or stabilizers such as pectin (such as HM pectin, LM pectin), gelatin, CMC, soy fiber / soy polymers, starch, modified starch, carrageenan, alginates, guar gum, etc. By substantially free it should be understood that the product comprises 0% to 20% by weight (w / w) (e.g. 0% to 10%, 0% to 5%, or 0% to 2%, or 0% to 1%) of thickeners and / or stabilizers.

[0066] As previously disclosed, interactions between microbial species are important, not only in terms of growth but equally important in terms of rheological properties (e.g. texture) and taste development.

[0067] In one important embodiment the composition comprises one or more Streptococcus thermophilus strains selected from the group comprising DSM 33719, DSM 33719 mutants, DSM 33719 variants, DSM 33720, DSM 33720 mutants, DSM 33720 variants, DSM 33762, DSM 33762 mutants, DSM 33762 variants.

[0068] In a preferred embodiment, the composition comprises (a) DSM 33720, DSM 33719 and DSM 33762; (b) DSM 32227 and DMS 33762; or (c) DSM 33719 and DSM 32227 Includes.

[0069] As can be seen from Examples 4 and 5, the strain combinations were tested for texture and acidification profile in the fermented product.

[0070] The composition may be provided as a mixture or kit of parts comprising: (a) a thermophilus strain selected from the group consisting of DSM33720 or a mutant or variant thereof, DSM33719 or a mutant or variant thereof, DSM33762 or a mutant or variant thereof, or any combination thereof; and (b) A strain belonging to the genus Lactobacillus.

[0071] The composition may be a mixture or kit-of-parts comprising: (a) a strain of Lactobacillus comprising the following genetic mutations: (i) a substitution of Pro to Leu at position corresponding to position 57 of SEQ ID NO:2; a substitution of Ile to Val at position corresponding to position 47 of SEQ ID NO:4 and a substitution of Glu to Asp at position corresponding to position 242 of SEQ ID NO:6 or 14, and / or (ii) a substitution of Pro to Ser at position corresponding to position 164 of SEQ ID NO:8 and a substitution of Leu to Phe at position corresponding to position 28 of SEQ ID NO:10, and / or (iii) a substitution of Gly to Cys at position corresponding to position 268 of SEQ ID NO:12; and (b) one or more strains belonging to the genus Lactobacillus.

[0072] In one embodiment, (i) is DSM 33762 or a mutant or variant thereof, (ii) is DSM 33720 or a mutant or variant thereof, and / or (iii) is DSM 33719 or a mutant or variant thereof.

[0073] For example, when used in the production of yoghurt, the composition preferably comprises one or more strains belonging to the genus Lactobacillus (lactic acid bacteria). The one or more strains of the genus Lactobacillus may be Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus paracasei subsp. paracasei, and Lacticaseibacillus rhamnosus. The bacterium may be selected from the group consisting of Lactobacillus rhamnosus, Limosilactobacillus fermentum, Lactiplantibacillus plantarum subsp. plantarum and Lactobacillus helveticus.

[0074] In a preferred embodiment of the invention, the Lactobacillus strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum subsp. Plantarum and Lactobacillus acidophilus.

[0075] In a particular embodiment, the Lactobacillus strain of the invention is L. bulgaricus, a lactic acid bacterium commonly used for commercial milk fermentation, the organism of which is commonly used as part of a mixed starter culture.

[0076] In a particular embodiment, the Lactobacillus delbrueckii subsp. bulgaricus bacterium is DMS28910 or a mutant or variant thereof.

[0077] In one embodiment of the invention, the Lactobacillus strain of the invention is a glucose deficient strain. In another embodiment of the invention, the Lactobacillus strain of the invention is glucose positive.

[0078] Thus, in a preferred embodiment, the composition and / or mixture or kit of parts of the invention comprises Thermophilus strain DSM 32227 and / or DSM 33762 and a strain belonging to the Lactobacillus species. Thus, in a preferred embodiment, the composition and / or mixture or kit of parts comprises Thermophilus strain DSM 33719 and / or DSM 32227 and a strain belonging to the Lactobacillus species. In a preferred embodiment, the composition and / or mixture or kit of parts comprises Thermophilus strain DSM 33720 and a strain belonging to the Lactobacillus species.

[0079] The composition of the present invention may contain probiotic bacteria. The probiotic strains can be added before or after fermentation. If added before fermentation, the probiotic strains can also act as fermenting bacteria.

[0080] The term "probiotics" refers to live bacteria (probiotics) that are administered in adequate amounts to a consumer with the aim of achieving a health-enhancing effect on the consumer. Probiotics are capable of surviving the environment of the gastrointestinal tract after ingestion and have the ability to colonize the intestine of the consumer.

[0081] It should be understood that the taxonomy of the genus Lactobacillus was revised in 2020. The new taxonomy is disclosed in Zheng et al. (2020) and is summarized herein unless otherwise indicated. For the purposes of the present invention, the following table lists the old and new names of some Lactobacillus species relevant to the present invention.

[0082] [Table 1]

[0083] In a particular embodiment of the invention, the probiotic strains of the invention are selected from the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri and Lactobacillus johnsonii. johnsonii, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp.lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium pseudocatenulatum and Bifidobacterium infantis.

[0084] In certain embodiments of the invention, the probiotic Lactobacillus strains are selected from the group consisting of Lactobacillus acidophilus, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lacticaseibacillus casei, Lactobacillus delbrueckii, Lactobacillus lactis, Lactiplantibacillus plantarum, Limosilactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus johnsonii).

[0085] In a particular embodiment of the invention, the probiotic strain is Lactobacillus acidophilus deposited under DSM 13241 (LA-5®).

[0086] In a particular embodiment of the invention, the probiotic Lactobacillus strains are Lacticaseibacillus rhamnosus and Lactibacillus paracasei. In a particular embodiment of the invention, the probiotic strain is Lacticaseibacillus rhamnosus LGG® strain deposited under ATCC 53103. In a particular embodiment of the invention, the probiotic strain is Lacticaseibacillus paracasei CRL431 strain deposited under ATCC 55544. In a particular embodiment of the invention, the probiotic Bifidobacterium strain is selected from the group consisting of Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium animalis subsp. lactis, Bifidobacterium dentium, Bifidobacterium catenulatum, Bifidobacterium angulatum, Bifidobacterium magnum, Bifidobacterium spp. magnum, Bifidobacterium pseudocatenulatum, and Bifidobacterium infantis.In a particular embodiment of the invention, the probiotic Bifidobacterium probioticus strain is the Bifidobacterium animalis subsp. lactis BB-12® strain deposited under DSM 15954.

[0087] The mixture or kit of parts described above can be further combined with other lactic acid bacteria, such as but not limited to probiotic bacteria. In one embodiment, the at least one lactic acid bacteria is selected from the group consisting of Bifidobacterium genus, such as Bifidobacterium animalis subsp. lactis (e.g. BB-12®), Lactobacillus acidophilus (e.g. LA-5®), Lacticaseibacillus rhamnosus (e.g. LGG®) and any combination thereof. Which Bifidobacterium genus, Lactobacillus acidophilus and / or Lacticaseibacillus rhamnosus are applied depends on the application and the food to be produced.

[0088] In contrast, a "kit of parts" containing one or more strains means that the strains or cultures of the strains are physically separate but intended to be used together, so that each strain or culture of Thermophilus and Lactobacillus is contained in a separate box or pouch. In one embodiment, the strains and cultures of strains consisting of Thermophilus strains and Lactobacillus strains such as Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophillus, Lacticaseibacillus casei, Lacticaseibacillus paracasei and / or Limosilactobacillus reuteri strains are under the same form, i.e. in the form of frozen form, pellets or frozen pellets, powder form, e.g. in a dried or lyophilized form.

[0089] In a particular embodiment of the present invention, the composition comprises 10 4 ~10 12 CFU (colony forming units) / g of Thermophilus strain, e.g. 10 5 ~10 11 CFU / g, e.g. 10 6 ~10 10 CFU / g, or 10 7 ~10 9 CFU / g of Thermophilus strains.

[0090] In certain embodiments, the composition comprises 10 4 ~10 12 CFU / g Lactobacillus, 10 5 ~10 11 CFU / g, e.g. 10 6 ~10 10 CFU / g, or e.g. 10 7 ~10 9It further contains CFU / g of Lactobacillus strain.

[0091] In certain embodiments, the composition comprises 10 4 ~10 12 CFU / g, 10 5 ~10 11 CFU / g, 10 6 ~10 10 CFU / g, or 10 7 ~10 9 Contains CFU / g of the following strains: Lactobacillus delbrueckii subsp bulgaricus, Lactobacillus acidophillus, Lacticaseibacillus casei, Lacticaseibacillus paracasei and / or Limosilactobacillus reutei.

[0092] Lactobacillus and Lacticaceus bacteria, such as Lactobacillus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei and / or Lactobacillus rhamnosus, and other lactic acid bacteria, are widely used as starter cultures for technical purposes in the production of various foods, such as in the dairy industry for fermented milk products, etc. Thus, in another preferred embodiment, the composition is suitable as a starter culture.

[0093] The composition may be a starter culture (or simply referred to as a starter), such as a yogurt starter culture.

[0094] The composition and / or starter culture may be frozen, spray dried, freeze dried, vacuum dried, air dried (air dried), tray dried, or in liquid form. Typically, the storage stability of the composition and / or starter culture may be extended by formulating a product with low water activity. By controlling the water activity (Aw), it is possible to predict and adjust the effect of moisture migration on the product. Thus, it may be preferred that the water activity (Aw) of the dry composition herein is in the range of 0.01 to 0.8, preferably in the range of 0.05 to 0.4.

[0095] It will be understood that the aspects and embodiments disclosed in the sections entitled "Method for Producing Fermented Products" and "Novel Streptococcus thermophilus Strains" are equally relevant to this section of the specification. In particular, it will be apparent to one of skill in the art that the disclosures relating to specific strains of the invention under the section entitled "Novel Streptococcus thermophilus Strains" are applicable to this section of the specification.

[0096] Methods for producing fermented products The term "milk" should be understood as the lacteal secretion obtained by milking any mammal, such as a cow, sheep, goat, buffalo or camel. In a preferred embodiment, the milk is cow's milk.

[0097] The term "dairy substrate" can be any raw milk and / or processed dairy raw material that can be subjected to fermentation according to the method of the present invention. Useful dairy substrates thus include, but are not limited to, any dairy or dairy-like product containing protein, such as full or low fat milk, skimmed milk, buttermilk, reconstituted milk powder, condensed milk, dry milk, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream, or other solutions / suspensions. Obviously, the dairy substrate can be any animal-derived product, such as substantially pure mammalian milk, or reconstituted milk powder, or the dairy substrate may be derived in part from plant material. Preferably, at least a portion of the proteins in the dairy substrate are (i) proteins naturally occurring in mammalian milk, such as casein and whey proteins, or (ii) proteins naturally occurring in plant milk. However, a portion of the proteins may be proteins not naturally occurring in milk.

[0098] Prior to fermentation, the dairy substrate may be homogenized and pasteurized according to methods known in the art.

[0099] "Homogenization" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, it can be performed to disperse the milk fat into smaller sizes so that it no longer separates from the milk. This can be achieved by forcing the milk through small holes (orifices) at high pressure.

[0100] As used herein, "pasteurization" refers to the treatment of a dairy substrate to reduce or eliminate the presence of viable organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specific temperature for a specific time. Typically, the specific temperature is achieved by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. This may be followed by a rapid cooling step.

[0101] The fermentation processes used to produce fermented milk products are well known and the skilled person knows how to select the appropriate process conditions such as temperature, oxygen, amount and characteristics of microorganisms as well as process time. Obviously, the fermentation conditions are selected to aid in the achievement of the invention, i.e. to obtain a fermented product such as a dairy or non-dairy product (fermented milk product) in solid or liquid form.

[0102] The term "fermented dairy product" as used herein refers to a food or feed product where the preparation of the food or feed product involves the fermentation of a milk substrate by lactic acid bacteria. As used herein, "fermented dairy product" includes, but is not limited to, products such as yogurt and cheese. Examples of cheeses prepared by fermentation with Lactobacillus thermophilus and Lactobacillus bulgaricus include mozzarella and pizza cheese (Hoier et al. (2010), The Technology of Cheesemaking, 2nd ed., Blackwell Publishing, Oxford; 166-192). Preferably, the fermented dairy product is yogurt.

[0103] In the present context, the term "starter culture" (or simply starter) refers to a culture that is a preparation (composition) of one or more bacterial strains (such as lactic acid bacteria strains) to help initiate the fermentation process in the preparation of fermented products such as various food, feed and beverage products.

[0104] In the present context, a "yoghurt starter culture" is a bacterial culture comprising at least one L. bulgaricus and / or L. acidophilus strain and at least one S. thermophilus strain. According to this specification, "yoghurt" refers to a fermented milk product obtained by inoculating a milk substrate with a composition comprising a Lactobacillus strain, such as L. bulgaricus and / or L. acidophilus, and a Thermophilus strain, and fermenting it.

[0105] A further aspect of the invention is a method for producing a fermented product comprising the step of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) a glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11 Or a method comprising fermenting a substrate using the composition of the present invention.

[0106] The substrate will preferably be a dairy substrate. The dairy substrate may be of animal origin, but it does not have to be of purely animal origin, but may further comprise substrates of plant origin. The fermented product may be a food product, as well as a dairy product.

[0107] Depending on the product to be produced, the substrate may be a dairy substrate, which is particularly preferred when the final product is a fermented dairy product such as yoghurt, buttermilk or kefir.

[0108] The dairy substrate may be an animal or plant-based product. Thus, in one embodiment, the fermented product is a food product, such as a dairy product. The dairy product may be selected from the group consisting of fermented dairy products, such as, but not limited to, yogurt, buttermilk and kefir, or cheese, such as, but not limited to, fresh cheese or pasta filata.

[0109] Even though the fermented product and / or food product itself may contain acids and flavors produced during fermentation, it may be desirable for the fermented product and / or dairy product to contain an ingredient selected from the group consisting of fruit concentrates, syrups, probiotic strains or cultures, colorants, thickeners, flavoring agents, preservatives, and mixtures thereof.

[0110] Similarly, enzymes may be added to the substrate, e.g., a milk substrate, before, during and / or after fermentation, such enzymes being selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, lactase, chymosin, rennet and mixtures thereof.

[0111] In one embodiment, the fermented product may be in the form of a stirred product, a stationary product, or a drinkable product.

[0112] Another obvious aspect of the present invention relates to a fermented product obtainable by the method of the present invention. One aspect of the present invention therefore relates to a fermented food product comprising a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11.

[0113] Also, one aspect of the present invention relates to the use of a Streptococcus thermophilus strain having a mutation in one or more genes selected from the following group for the production of a fermented product: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) a glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO: 11. The fermented product can be a food product such as a dairy product or a dairy-like product.

[0114] It will be appreciated that aspects and embodiments disclosed in the sections entitled "Compositions" and "Novel Streptococcus thermophilus Strains" may be equally relevant to that portion of the specification. In particular, the disclosures regarding specific strains of the invention under "Novel Streptococcus thermophilus Strains" are applicable to that portion of the specification, as would be apparent to one of skill in the art.

[0115] A novel Streptococcus thermophilus strain The present inventors have surprisingly found Streptococcus thermophilus strains having mutations in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11.

[0116] The Thermophilus strain comprises a nucleotide sequence that is at least 50% identical to SEQ ID NO:1, 3, 5, 7, 11 and / or 13. Preferably, the Thermophilus strain comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:1, 3, 5, 7, 11 and / or 13.

[0117] Thus, at the amino acid level, it can be further expected that the mutation causes a change in the encoded protein selected from the group consisting of: a) the PTS mannose / glucose / fructose subunit IIC at a position corresponding to position 57 of SEQ ID NO:2; b) galactokinase at a position corresponding to position 47 of SEQ ID NO:4; c) phosphoglucomutase at a position corresponding to position 242 of SEQ ID NO: 6 or 14; d) phosphoglucomutase at a position corresponding to position 164 of SEQ ID NO:8; e) the galactose operon repressor at a position corresponding to position 28 of SEQ ID NO:10; and f) A glucose kinase at a position corresponding to position 268 of SEQ ID NO:12.

[0118] The galK gene encodes galactokinase, an enzyme in the Leloir pathway of galactose metabolism that converts α-galactose to galactose-1-phosphate.

[0119] The pgm gene encodes phosphoglucomutase, an enzyme that converts the reversible reaction of β-glucose-1-phosphate (G1P) to glucose-6-phosphate (G6P).

[0120] The galR gene encodes the galactose operon repressor that regulates the galactose operon of Streptococcus at the transcriptional level.

[0121] In a particular embodiment of the use of the present invention, the Thermophilus strain is galactose-fermenting and carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein (more specifically at the position corresponding to position 805 in SEQ ID NO: 11), wherein said mutation inactivates the glucokinase protein or has a negative effect on the expression of said gene.

[0122] In preferred embodiments, the mutation reduces the activity of the glucokinase protein (rate of phosphorylation of glucose to glucose-6-phosphate) by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0123] Glucokinase activity can be determined by the glucokinase enzyme assay as described by Pool et al. (2006. Metabolic Engineering 8; 456-464).

[0124] In a particular embodiment of the use of the present invention, the Thermophilus strain carries a mutation that reduces the transport of glucose into the cell, hi a particular embodiment, the Thermophilus strain carries a mutation in a gene encoding a component of a glucose transporter, which mutation inactivates the glucose transporter or has a negative effect on the expression of said gene.

[0125] In a more particular embodiment, the thermophilus strain is a PTS mannose / glucose / fructose subunit IIC (IIC of the glucose / mannose phosphotransferase system). Man The IIC protein is also referred to as a genomic DNA sequence, and the two terms are used interchangeably herein, wherein the mutation is Man Inactivates the protein or has a negative effect on expression of the gene. In preferred embodiments, the mutation reduces glucose transport into the cell by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% when compared to a cell not carrying such a mutation.

[0126] The transport of glucose into the cells can be measured by the glucose uptake assay described by Cochu et al. (2003) Appl. Environ Microbiol. 69(9); 5423-5432).

[0127] Preferably, the Thermophilus strain has a mutation in a gene encoding a component of a glucose transporter, which mutation inactivates the glucose transporter protein or has a negative effect on the expression of said gene.

[0128] The component can be any component of a glucose transporter protein that is important for glucose transport, for example, activation of any component of the glucose / mannose phosphotransferase system of Thermophilus will cause functional inactivation of the glucose transporter.

[0129] In particular, an inactivated glucose transporter protein is a protein that is unable to facilitate transport of glucose across the plasma membrane or facilitates transport of glucose across the plasma membrane at a significantly reduced rate when compared to a functional glucose transporter protein. A gene encoding such an inactivated glucose transporter protein contains a mutation in the open reading frame (ORF) of the gene compared to a functional glucose transporter protein, including, but not limited to, a mutation resulting in a deletion, a frameshift mutation, the introduction of a stop codon, or an amino acid substitution that alters the functional properties of the protein, or a promoter mutation that reduces or abolishes the transcription or translation of the gene.

[0130] In preferred embodiments, the mutation reduces the activity of the glucose transporter protein (rate of glucose transport) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. Glucose transporter activity can be measured by a glucose uptake assay such as that described by Cochu et al. (2003, Appl. Environ. Microbiol. 69 (9); 5423-5432).

[0131] In certain embodiments, the Thermophilus strain is capable of increasing the amount of glucose in 9.5% B-milk to at least 5 mg / mL when inoculated into 9.5% B-milk at a concentration of 1.0E06-1.0E07 CFU / mL and grown at 40° C. for 20 hours.

[0132] In a particular embodiment of the use of the present invention, Lactobacillus thermophilus strains DSM 33719 and / or DSM 33762 are capable of increasing the amount of glucose in 9.5% B milk to at least 5 mg / mL when inoculated at a concentration of 1.0E06-1.0E07 CFU / mL into 9.5% B milk containing 0.05% sucrose and grown at 40° C. for 20 hours.

[0133] In the present context, 9.5% B milk is heat-treated milk made by reconstitution with low-fat skim milk powder to a dry weight level of 9.5% and pasteurization at 99°C for 30 minutes followed by cooling to 40°C.

[0134] In more preferred embodiments of the invention, the mutant strain provides an increase in glucose to at least 4 mg / mL, at least 5 mg / mL, at least 6 mg / mL, at least 7 mg / mL, at least 8 mg / mL, at least 9 mg / mL, at least 10 mg / mL, at least 11 mg / mL, at least 12 mg / mL, at least 13 mg / mL, at least 14 mg / mL, at least 15 mg / mL, at least 20 mg / mL, or at least 25 mg / mL.

[0135] In a further embodiment, Lactobacillus thermophilus DSM 33719 and / or DSM 33762, when inoculated with one or more additional strains of lactic acid bacteria to a concentration of 1.0E06-1.0E08 CFU / mL and grown for 20 hours at 40° C., are capable of increasing the amount of glucose to at least 5 mg / mL in a milk substrate comprising 0.1% sucrose, 4% protein (adjusted with skim milk powder) and 1.5% fat (adjusted to 1.5% fat milk with 9% cream). In a more preferred embodiment of the invention, the amount of glucose is increased to at least 4 mg / mL, at least 5 mg / mL, at least 6 mg / mL, at least 7 mg / mL, at least 8 mg / mL, at least 9 mg / mL, at least 10 mg / mL, at least 11 mg / mL, at least 12 mg / mL, at least 13 mg / mL, at least 14 mg / mL, at least 15 mg / mL, at least 20 mg / mL or at least 25 mg / mL.

[0136] As used herein, the term "mutations that reduce glucose transport into cells" refers to mutations in genes that encode proteins involved in glucose transport, which cause the accumulation of glucose in the environment surrounding the cell.

[0137] The level of glucose in the culture medium of a Thermophilus strain can be readily determined by methods well known to those skilled in the art.

[0138] It will be appreciated that the Thermophilus strain comprises an amino acid sequence that is at least 50% identical to SEQ ID NO:2, 4, 6, 8, 10 and / or 12. Preferably, the Thermophilus strain comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2, 4, 6, 8, 10 and / or 12.

[0139] In certain embodiments, the mutation at the nucleic acid level is: (a) a C to T substitution at a position corresponding to position 169 of SEQ ID NO:1; (b) a G to A substitution at a position corresponding to position 139 of SEQ ID NO:3; (c) an A to C substitution at a position corresponding to position 726 of SEQ ID NO: 5 or 13; (d) a C to T substitution at a position corresponding to position 490 of SEQ ID NO:7; (e) a C to T substitution at a position corresponding to position 82 of SEQ ID NO:9; and (f) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:11.

[0140] In certain embodiments, the encoded protein is: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; (b) an Ile to Val substitution at a position corresponding to position 47 of SEQ ID NO:4; (c) a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO: 6 or 14; (d) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; (e) a Leu to Phe substitution at a position corresponding to position 28 of SEQ ID NO:10; and (f) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12.

[0141] In a preferred embodiment, the Streptococcus thermophilus strain comprises the following mutations: (d) a substitution of C to T at a position corresponding to position 169 of SEQ ID NO:1; a substitution of nucleotide G to A at a position corresponding to position 139 of SEQ ID NO:3; and a substitution of nucleotide A to C at a position corresponding to position 726 of SEQ ID NO:5 or 13; (e) a substitution of C to T at a position corresponding to position 490 of SEQ ID NO: 5 or 13; a substitution of nucleotide C to T at a position corresponding to position 82 of SEQ ID NO: 7; or (f) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:9.

[0142] In a preferred embodiment, the Streptococcus thermophilus strain contains the following changes in the encoded protein: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; a substitution of Ile to Val at a position corresponding to position 47 of SEQ ID NO:4; and a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO:6 or 14; (b) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; and a substitution of Leu to Phe at a position corresponding to position 28 of SEQ ID NO:10; or (c) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12.

[0143] [Table 2]

[0144] All identified strains have been deposited. In a preferred embodiment, the present invention relates to the following Thermophilus strains: (a) DSM 33719 or a variant or variant thereof; (b) DSM 33720 or a variant or variant thereof; or (c) DSM 33762 or a variant or variant thereof.

[0145] In a preferred embodiment, the present invention relates to Thermophilus strain DSM 33719 or a mutant or variant thereof, Thermophilus strain DSM 33720 or a mutant or variant thereof and / or Thermophilus strain DSM 33762 or a mutant or variant thereof. In a further preferred embodiment of the invention, the mutant strain is a naturally occurring mutant or an induced mutant.

[0146] To ensure applicability in industry, it is contemplated that mutants or variants of DSM 33719 will exhibit the same or similar characteristics as DSM 33719, such as acidification profile, texture improving properties, etc. Similarly, it is contemplated that mutants or variants of DSM 33720 will exhibit the same or similar characteristics as DSM 33720, such as acidification profile, texture improving properties, etc. Similarly, it is contemplated that mutants or variants of DSM 33719 will exhibit the same or similar characteristics as DSM 33719, such as acidification profile.

[0147] To ensure applicability in industry, it is contemplated that mutants or variants of DSM 33719 will exhibit the same or similar characteristics (e.g., acidification profile, texture improving properties, etc.) as DSM 33719. Similarly, it is contemplated that mutants or variants of 33720 will exhibit the same or similar characteristics (e.g., acidification profile, texture improving properties, etc.) as, for example, DSM 33720.

[0148] In one embodiment, the mutant or variant of DSM 33719 comprises a nucleotide sequence which is at least 50% identical to SEQ ID NO: 1, 3 and / or 5. Preferably, the mutant or variant of DSM 33719 comprises a nucleotide sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 3 and / or 5.

[0149] In one embodiment, a mutant or variant of DSM 33720 comprises a nucleotide sequence that is at least 50% identical to SEQ ID NO: 7 and / or 9. Preferably, a mutant or variant of DSM 33720 comprises a nucleotide sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7 and / or 9.

[0150] A mutant or variant of DSM 33762 comprises a nucleotide sequence which is at least 50% identical to SEQ ID NO: 11. Preferably, a mutant or variant of DSM 33762 comprises a nucleotide sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11.

[0151] In one embodiment, the mutant or variant of DSM 33719 comprises an amino acid sequence that is at least 50% identical to SEQ ID NO: 1, 3 and / or 5. Preferably, the mutant or variant of DSM 33719 comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 3 and / or 5.

[0152] In one embodiment, a mutant or variant of DSM 33720 comprises an amino acid sequence that is at least 50% identical to SEQ ID NO: 7 and / or 9. Preferably, a mutant or variant of DSM 33720 comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7 and / or 9.

[0153] In one embodiment, a mutant or variant of DSM 33762 comprises an amino acid sequence that is at least 50% identical to SEQ ID NO: 11. Preferably, a mutant or variant of DSM 33762 comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:11.

[0154] As mentioned above, there is a need in the industry to develop new Thermophilus strains with sweetening properties, and thus, in a preferred embodiment, the Thermophilus strains of the present invention are galactose fermenting.

[0155] In this context, the term "galactose fermenting" as defined herein means at least 10 4 This means that when the strain is inoculated at cells / mL, the pH is reduced by at least 1.0 after 16 hours of incubation at 37°C in M17 medium containing 2% galactose (galactose is added as the only carbohydrate).

[0156] Some strains can be galactose and glucose fermentative. This is the case, for example, of DSM 33720, which can ferment both glucose and galactose. DSM 33720 carries a mutation such that glucose is not transported outside the cell at all or is transported only to a limited extent. Glucose is therefore not accumulated in the fermentation product to the same extent as with other galactose fermenting strains. DSM 33720 is therefore a strain with mild sweetening properties.

[0157] The terms "strain with sweetening properties", "strain capable of providing a desirable glucose accumulation in fermented dairy products" and "strain with properties to enhance the natural sweetness of foods" are used interchangeably herein to characterize advantageous aspects of the use of the strains of the invention in the fermentation of dairy products.

[0158] In one embodiment of the present invention, a fermentation product produced by the addition of a composition (mixed culture) comprising DSM 33720, DSM 33762 and DSM 33719 and one or more additional lactic acid bacteria is subjected to a fermentation test for 300 seconds. -1 (s -1 ), which produces a stress of greater than 45 Pa, 50 Pa, 60 Pa, 70 Pa, 80 Pa, 90 Pa, or 100 Pa. Such stresses would be desirable as they mimic the perceived stickiness / mouth thickness that is preferred by a sensory panel. Shear stress is measured as described in Example 4.

[0159] The Thermophilus strain(s) of the present invention are 2-deoxyglucose resistant. The term "2-deoxyglucose resistant" as used herein with respect to Thermophilus is defined by the ability of a particular mutant strain, when streaked on a panel of M17 medium containing 20 mM 2-deoxyglucose, to grow into colonies after 20 hours of incubation at 40°C. The presence of 2-deoxyglucose in the medium will inhibit the growth of non-mutant strains, while not affecting or not significantly affecting the growth of mutant strains. Thus, for selection purposes, 2-deoxyglucose can be applied in the selection step.

[0160] In one embodiment, the S. thermophilus DSM 33720 strain carries a mutation that renders the strain highly galactose fermenting.

[0161] It will be understood that aspects and embodiments disclosed in the sections entitled "Compositions" and "Method of Producing a Fermented Product" are equally relevant to this section of the specification.

[0162] The use of the terms "a", "an", "the" and similar articles in the context of describing the present invention (particularly in the context of the claims below) should be construed as including both the singular and the plural unless otherwise indicated. The terms "comprise", "have", "include" and "contain" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise indicated. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value falling within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually referred to herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "etc.") are merely intended to facilitate a better understanding of the present invention, and do not impose limitations on the scope of the present invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0163] Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0164] Preferences, options and embodiments of a given aspect, feature or parameter of the invention should be considered as disclosed in combination with all options, options and embodiments of all other aspects, features and parameters of the invention, unless the context indicates otherwise. This is particularly true for the description of the microencapsulated bacterial cultures and all their features that may be part of the final composition obtained by the methods described herein. The embodiments and features of the invention are also outlined in the following paragraphs and illustrated by the following non-limiting examples.

[0165] Microorganism Depository and Expert Solutions Applicant requests that samples of the deposited microorganisms set forth in the table below be maintained available exclusively to the public until the date a patent is issued.

[0166] [Table 3] EXAMPLES

[0167] material and method

[0168] [Table 4]

[0169] Carbon source added aseptically: Lactose: 20g / L Glucose: 20g / L, 25g / L or 50g / L Galactose: 20g / L, 25g / L or 50g / L.

[0170] As known in the art, M17 medium is considered suitable for the growth of Thermophilus. The skilled artisan will understand that in this context, M17 concentrates are supplied by various suppliers and independent of any particular supplier, and will give the same results regarding 2-deoxyglucose resistance in the context of the present invention (within standard measurement uncertainties) for the cells of interest in the context of the present invention.

[0171] Streptococcus thermophilus strain DSM 33762 is a 2-deoxyglucose-resistant mutant, and strain DSM 33720 is a high galactose-fermenting strain, both derived from the same galactose-fermenting mother strain (MS-1). DSM 33719 is a fast glucose-secreting strain derived from a high lactose-fermenting and glucose-secreting mother strain (MS-2).

[0172] Lactic acidification profile and carbohydrate analysis B milk was used as the substrate. B milk consists of skim milk powder at a dry weight level of 9.5% (w / v) that was reconstituted with distilled water, pasteurized at 99°C for 30 min, and then cooled to 30°C.

[0173] Acidification was followed using CINAC conventional technology hardware and software, or using the iCINAC system (from AMS Alliance), which was developed to monitor the acidification activity of lactic acid fermentation and uses electrodes to simultaneously track and record changes in pH and temperature.

[0174] Carbohydrate content was examined using HPLC analysis. Aliquots of acidified B milk were collected after approximately 20 hours of acidification at 40° C. The amount of carbohydrate is expressed in g / L.

[0175] Example 1 - Use of 2-deoxyglucose to isolate glucose kinase mutants of Streptococcus thermophilus with increased glucose excretion To isolate mutants, MS-1 cells obtained from growth of a single colony were inoculated into 10 mL of M17 broth containing 2% lactose and grown overnight at 40°C.

[0176] The next day, the cultures were plated in serial dilutions on M17 agar plates containing 2% galactose and 30 mM 2-deoxyglucose and incubated at 40°C for 20 h. Resistant colonies were restreaked on the same type of agar plate originally used for selection. Surviving bacteria were inoculated into fresh M17 broth containing either 2% lactose, 2% galactose or 2% glucose and growth was measured. Among the selected mutants, DSM 33762 was identified. This isolate secretes glucose when grown in milk (Figure 1) and genome sequencing identified a mutation in the gene encoding glucokinase.

[0177] From figure 1 it is clear that DSM 33762 shows a slower acidification profile compared to MS-1. Furthermore, carbohydrate analysis of DSM 33762 shows a decreased lactose content, whereas the secretion of both galactose and glucose is increased, compared to the fermented milk prepared with MS-1.

[0178] Example 2 - Use of adaptive laboratory evolution methods to isolate Streptococcus thermophilus mutants with faster growth rates on galactose-containing media Using adaptive laboratory evolution (ALE), we generated galactose-fermenting mutants that grew faster in media containing galactose as the sole carbohydrate (sugar) source compared to the parent strain.

[0179] ALE was performed with three parallel cultures of MS-1. The cultures were grown for 4 weeks in M17 medium with 5% galactose + 2% casein hydrolysate. ALE was performed essentially following the protocol described in Troy E. Sandberg, Michael J. Salazar, Liam L. Weng, Bernhard O. Palsson, Adam M. Feist, "The emergence of adaptive laboratory evolution as an efficient tool for biological discovery and industrial biotechnology", Metabolic Engineering, Vol. 56, 2019, pp. 1-16 (Sandberg et al., 2019).

[0180] The adapted cultures were tested weekly for improved growth rate. This was performed by testing an aliquot of the adapted culture directly in the same medium used for the ALE method and then plating dilutions of the adapted culture on galactose-containing M17 agar plates. Single colonies were purified from these plates and tested for optimized capacity in M17 medium with galactose + casein hydrolysate. The majority of the isolates showed a significant improvement in growth rate compared to MS-1. DSM 33720 was isolated after 3 weeks of ALE.

[0181] In milk, DSM 33720 was found to significantly improve texture and accelerate acidification rates (Figure 2).

[0182] Example 3 - Use of Adaptive Laboratory Evolution (ALE) to Isolate Glucose-Excreting Mutants of Streptomyces thermophilus with Faster Growth Rates on Galactose- and Lactose-Containing Media As described in WO2013 / 160413, MS-2 is a strain with high glucose secretion due to a double mutation (glcK, Glu / Man PTS). However, such mutants often show sluggish performance in milk, so we attempted to isolate faster derivatives using ALE. Three parallel cultures of MS-2 were grown for 3 weeks in M17 medium containing 5% galactose + 2% casein hydrolysate. At week 4, the medium was changed to M17 containing 2.5% galactose + 2.5% lactose + 2% casein hydrolysate. ALE was performed essentially according to the protocol described in Sandberg et al. (2019).

[0183] The graph in Figure 3 represents growth over a full four weeks when the culture was diluted and regrown once each day or several times each day until more rapid growth was achieved. Starting with a low basal growth rate, the growth rate increased significantly after four weeks. After four weeks, diluted cultures were plated from each of three parallel cultures and single colonies were selected from these agar plates. DSM 33719 is one isolate from the grey curve in Figure 3 (culture 3).

[0184] Figure 4 shows the results for DSM 33719. Acidification with DSM 33719 was much faster compared to MS-2 and DSM 33719 acidified to a lower pH. Carbohydrate analysis of the fermented milk revealed why DSM 33719 was faster: glucose secretion was reduced by half and it was also observed that DSM 33719 was well adapted to growing on peptides (casein hydrolysate). Further analysis of DSM 33719 revealed that in addition to showing faster acidification, especially when casein hydrolysate was added or in precultures, it also conferred an improved texture to the milk when it was part of a mixed culture (see example 4).

[0185] Example 4 - Acidification and carbohydrate analysis of milk fermented with different combinations and ratios of strains

[0186] [Table 5]

[0187] The yogurt starter cultures Plemium 1.0 and YF-L904 sold by the applicant Chr. Hansen were included in the experiments as references.

[0188] Prior to fermentation, two different milk substrates were prepared: skim milk powder and sucrose were added to the milk, which was then hydrated for 2 h without stirring, homogenized, and pasteurized at >80°C for 1 min.

[0189] Dairy substrate 1 contained 0.1% sucrose, 4% protein (adjusted with skim milk powder) and 1.5% fat (1.5% fat milk adjusted with 9% cream). Dairy substrate 2 contained 5% sucrose, 4% protein (adjusted with skim milk powder) and 1.5% fat (1.5% fat milk adjusted with 9% cream).

[0190] The milk substrate was transferred to a 200 mL feeding bottle. The bottle was inoculated with a 1:100 dilution prepared from frozen pellets dissolved in B milk (F-DVS). Separate dilutions were prepared for each strain. To formulate the blends, several strains were inoculated in different ratios and volumes into one CINAC bottle. The final inoculum of the blends corresponded to 1E+6–1E+8 CFU / mL.

[0191] All strains were from the first dilution made from F-DVS, except for DSM 33762, which was used in culture 2. For preparation of DSM 33762 inoculum (seed), 1 mL of M-17 BK medium containing 4% sucrose and excess casein peptone was inoculated with a scrap of DSM 33762 and incubated at 43°C for at least 18 h.

[0192] The inoculated bottles were placed in a water bath and heated to 43°C.

[0193] The pH was monitored at 43 °C according to conventional techniques using CINAC hardware and software until the mixture reached a final pH of 4.55. To obtain a homogeneous gel of the fermented milk, each bottle was stirred and processed at 25 °C and 2 bar in a Micro Application Platform (MAP), a small laboratory-scale device equivalent to conventional post-treatment units (PTUs) used on dairy farms.

[0194] Fermented milk samples were prepared for carbohydrate analysis using HPLC by weighing 1 g ± 0.5 g into a 10 mL centrifuge tube. 2 mL of ice-cold 96% ethanol was added, the sample was mixed vigorously and placed at -50°C until analysis by HPLC.

[0195] The baby bottles containing the fermented milk samples were stored at 5°C for 7 days before texture analysis (rheology) was performed. For rheological measurements, the samples were transferred to bobbins and the shear stress was analyzed.

[0196] The shear stress was measured using an Anton Paar ASC rheometer, model DSR502. -3 s -1 From 300s -1 Continue to 10 -3 s -1 A rotation step is used based on the rotational deformation of the sample, which returns to the flow rate. The corresponding shear stress is measured. In these results, one shear rate (300 s -1 ) was extracted. Samples were placed at 13°C for 1 hour before measurement. Each sample was gently stirred with a spoon from bottom to top five times to ensure a homogenous sample. The rheology cup was filled to the mark and placed in the sample magazine. Samples were measured in duplicate using two separate yogurt cups. Measurements were performed on day +7 and the measurement temperature was set at 13°C. Samples were stored at 5°C until the day of measurement.

[0197] [Table 6]

[0198] Testing was performed using dairy substrate 1. The results for Culture 3 and Culture 4 show that different blends, i.e., each of the eight Culture 3 blends contains the same strains but in different amounts and ratios.

[0199] Fermentation time, texture and glucose levels are culture / blend dependent. Cultures 3 and 4 provide excellent texture equivalent to one of the best texture enhancing starter cultures Plemium 1.0, while culture 1 provided a much poorer texture in the fermented milk product. Different strain ratios between blend variants may result in variations in fermentation time, texture or glucose levels. Thus, culture and blend composition can be adjusted according to the specific needs required for the final fermented product.

[0200] [Table 7]

[0201] The above table shows the results obtained with culture 1 and culture 2 (blends 1-6), which are six blend variants containing the same strains in different amounts and ratios. It can be seen that culture 2 gives similar textures in the two dairy substrates compared to culture 1. At the same time, culture 2 results in shorter fermentation times and lower glucose concentrations.

[0202] Example 5 - Varying the ratio of strains in the blend alters the properties of fermented dairy products Fermented milk was produced and analyzed as described in Example 4.

[0203] [Table 8]

[0204] [Table 9]

[0205] [Table 10]

[0206] Different strain ratios in the blended variants of culture 4 (S03b, S03 and P07) significantly affected the fermentation time, texture (300 1*s(s -1 ), shear stress), and glucose levels are varied. In both dairy substrate 1 and dairy substrate 2, each blend variant had better texture compared to culture 1. In dairy substrate 2, culture 4 blend variant showed faster fermentation compared to culture 1. Thus, the characteristics of the cultures / blends are determined by the properties of the individual strains and the abundance of the strains, resulting in different fermented products. Thus, the composition of the cultures / blends can be tailored to address the specific needs of the final fermented product.

[0207] item [Item Y1] A composition comprising a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of: (a) the manM gene encoding the PTS mannose / glucose / fructose subunit IIC located at a position corresponding to position 169 of SEQ ID NO:1; (b) the galK gene encoding galactokinase at a position corresponding to position 139 of SEQ ID NO:3; (c) a pgm gene encoding phosphoglucomutase at a position corresponding to position 490 of SEQ ID NO: 5 or 13; (d) a pgm gene encoding phosphoglucomutase at a position corresponding to position 726 of SEQ ID NO:7; (e) a galR gene encoding a galactose operon repressor located at a position corresponding to position 82 of SEQ ID NO:9; and (f) The glcK gene encoding glucose kinase located at a position corresponding to position 805 of SEQ ID NO:11. [Item Y2] The composition according to item Y1, wherein the mutation results in a change in the encoded protein selected from the group consisting of: a) the PTS mannose / glucose / fructose subunit IIC at a position corresponding to position 57 of SEQ ID NO:2; b) galactokinase at a position corresponding to position 47 of SEQ ID NO:4; c) phosphoglucomutase at a position corresponding to position 242 of SEQ ID NO: 6 or 14; d) phosphoglucomutase at a position corresponding to position 164 of SEQ ID NO:8; e) the galactose operon repressor at a position corresponding to position 28 of SEQ ID NO:10; and f) A glucose kinase at a position corresponding to position 268 of SEQ ID NO:12. [Item Y3] The composition according to any one of the preceding items, wherein the mutation is: (a) a C to T substitution at a position corresponding to position 169 of SEQ ID NO:1; (b) a G to A substitution at a position corresponding to position 139 of SEQ ID NO:3; (c) an A to C substitution at a position corresponding to position 726 of SEQ ID NO: 5 or 13; (d) a C to T substitution at a position corresponding to position 490 of SEQ ID NO:7; (e) a C to T substitution at a position corresponding to position 82 of SEQ ID NO:9; (f) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:11. [Item Y4] The composition according to any one of the preceding items, wherein the change in the encoded protein is: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; (b) an Ile to Val substitution at a position corresponding to position 47 of SEQ ID NO:4; (c) a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO: 6 or 14; (d) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; (e) a Leu to Phe substitution at a position corresponding to position 28 of SEQ ID NO:10; and (f) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12. [Item Y5] The composition according to any one of the preceding items, wherein the Streptococcus thermophilus strain comprises the following mutation: (a) a substitution of C to T at a position corresponding to position 169 of SEQ ID NO:1; a substitution of nucleotide G to A at a position corresponding to position 139 of SEQ ID NO:3; and a substitution of nucleotide A to C at a position corresponding to position 726 of SEQ ID NO:5 or 13; (b) a substitution of C to T at a position corresponding to position 490 of SEQ ID NO:5 or 13; and a substitution of nucleotide C to T at a position corresponding to position 82 of SEQ ID NO:7; or (c) a G to T substitution at a position corresponding to position 805 of SEQ ID NO:9. [Item Y6] The composition according to any one of the preceding items, wherein the Streptococcus thermophilus strain comprises the following mutation in the encoded protein: (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; a substitution of Ile to Val at a position corresponding to position 47 of SEQ ID NO:4; a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO:6 or 14; (b) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; and a substitution of Leu to Phe at a position corresponding to position 28 of SEQ ID NO:10; or (c) A Gly to Cys substitution at a position corresponding to position 268 of SEQ ID NO:12. [Item Y7] The composition according to any one of the preceding items, wherein the Streptococcus thermophilus strain is: (a) DSM 33719 or a variant or variant thereof; (b) DSM 33720 or a variant or variant thereof; or (c) DSM 33762, or any variant or variant thereof. [Item Y8] The composition according to any one of the preceding items, wherein the composition comprises one or more Streptococcus thermophilus strains selected from the group comprising DSM 33719, DSM 33719 mutants, DSM 33719 variants, DSM 33720, DSM 33720 mutants, DSM 33720 variants, DSM 33762, DSM 33762 mutants, DSM 33762 variants. [Item Y9] The composition according to any one of the preceding items, wherein the composition comprises: (a) DSM 33720, DSM 33719, and DSM 33762; or (b) DSM 33719 and DSM 32227. [Item Y10] The composition according to any one of the preceding items, further comprising one or more strains belonging to the genus Lactobacillus. [Item Y11] The composition according to item Y10, wherein the one or more Lactobacillus strains are selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus rhamnosus, Limosilactobacillus farmentum, Lactiplantibacillus plantarum subsp. plantarum, and Lactobacillus helveticus. [Item Y12] The composition according to any one of items Y9 to Y10, wherein the Lactobacillus delbrueckii is Lactobacillus delbrueckii subsp. bulgaricus DSM 28910 strain or a mutant or variant thereof. [Item Y13] A composition according to any one of the preceding items, comprising as a mixture or as a kit of parts: (a) a S. thermophilus strain selected from the group consisting of DSM 33720 or a mutant or variant thereof, DSM 33719 or a mutant or variant thereof, DSM 33762 or a mutant or variant thereof, or any combination thereof; and (b) A strain belonging to the genus Lactobacillus. [Item Y14] The composition according to any one of the preceding items, wherein the composition is a starter culture. [Item Y15] The composition according to any one of the preceding items, wherein the composition is in the form of a frozen, spray-dried, lyophilized, vacuum-dried, air-dried, tray-dried, or liquid. [Item Z1] A method for producing a fermented product, comprising fermenting a substrate using a Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of (a) to (f) of Item Y1. [Item Z2] The method according to Item Z1, wherein the substrate is a milk substrate. [Item Z3] The method according to any one of Items Z1 to Z2, wherein the milk substrate is of animal origin. [Item Z4] The method according to any one of Items Z1 to Z3, wherein the dairy substrate further contains a plant-derived substrate. [Item Z5] The method according to any one of Items Z1 to Z4, wherein the fermented product is a food product. [Item Z6] The method according to any one of Items Z1 to Z3, wherein the food is a dairy product. [Item Z7] The method according to Item Z6, wherein the dairy product is selected from the group consisting of fermented dairy products (e.g., yogurt, buttermilk or kefir) or cheese (e.g., fresh cheese or pasta filata). [Item Z8] The method according to any one of items Z1 to Z7, wherein the fermented product further comprises an ingredient selected from the group consisting of fruit concentrates, syrups, probiotic strains or cultures, colorants, thickeners, flavorings, preservatives, antiseptics and mixtures thereof. [Item Z9] The method according to Item Z8, wherein an enzyme is added to the substrate before, during and / or after fermentation, the enzyme being selected from the group consisting of enzymes capable of cross-linking proteins, transglutaminase, aspartic protease, chymosin, rennet, lactase and mixtures thereof. [Item Z10] The method according to any one of items Z1 to Z9, wherein the fermented product is in the form of a stirred type, a fixed type, or a drinkable type. [Item Q1] A fermented product obtained by the method according to any one of items Z1 to Z10. [Item Q2] The fermented product according to Item Q1, wherein the fermented product is a food product. [Item Q3] The fermented product according to any one of items Q1 to Q2, wherein the food is a dairy product. [Item P1] A fermented product comprising one or more Streptococcus thermophilus strains having mutations in one or more genes selected from the group consisting of (a) to (f) of Item Y1. [Item P2] The fermented product according to Item P1, wherein the fermented product is a food product. [Item P3] The fermented product according to Item P1, wherein the food product is a dairy product. [Item W1] Use of one or more S. thermophilus strains having mutations in one or more genes selected from the group consisting of (a) to (f) of Item Y1 for the production of a fermentation product. [Item W2] The use according to Item W1, wherein the fermented product is a food product. [Item W3] The use according to any one of Items W1 to W2, wherein the food is a dairy product. [Item X1] A Streptococcus thermophilus strain having a mutation in one or more genes selected from the group consisting of (a) to (f) of Item Y1. [Item X2] [Item X3] A Streptococcus thermophilus strain according to any one of Items X1 to X2, which is galactose fermenting. [Item X4] A Streptococcus thermophilus strain according to any one of Items X1 to X3, which is resistant to 2-deoxyglucose. [Item X5] The Streptococcus thermophilus strain according to any one of Items X1 to X4, wherein the Streptococcus thermophilus strain carries a mutation that reduces glucose transport into the cell. [Item X6] The Streptococcus thermophilus strain according to any one of Items X1 to X5, wherein the Streptococcus thermophilus strain increases the amount of glucose in 9.5% B milk to at least 5 mg / mL when inoculated into 9.5% B milk at a concentration of 1.0E06 to 1.0E07 CFU / mL and grown at 40°C for 20 hours. [Item X7] The Streptococcus thermophilus strain according to any one of Items X1 to X5, wherein the Streptococcus thermophilus strain increases the amount of glucose in 9.5% B milk containing 0.05% sucrose to at least 5 mg / mL when inoculated into 9.5% B milk containing 0.05% sucrose at a concentration of 1.0E06 to 1.0E07 CFU / mL and grown at 40°C for 20 hours.

[0208] SEQ ID NO: 1: Nucleotide sequence of the ManM gene encoding the PTS mannose / glucose / fructose subunit IIC SEQ ID NO: 2: Amino acid sequence of PTS mannose / glucose / fructose subunit IIC (ManM) SEQ ID NO: 3: Nucleotide sequence of galK encoding galactokinase SEQ ID NO: 4: Amino acid sequence of galactokinase (GalK) SEQ ID NO: 5: Nucleotide sequence of pgm encoding phosphoglucomutase SEQ ID NO: 6: Amino acid sequence of phosphoglucomutase (Pgm) SEQ ID NO: 7: Nucleotide sequence of pgm encoding phosphoglucomutase SEQ ID NO: 8: Amino acid sequence of phosphoglucomutase (Pgm) SEQ ID NO: 9: Nucleotide sequence of galR encoding the galactose operon repressor SEQ ID NO: 10: Amino acid sequence of the galactose operon repressor (GalR) SEQ ID NO: 11: Nucleotide sequence of glcK encoding glucose kinase SEQ ID NO: 12: Amino acid sequence of glucose kinase (GlcK) SEQ ID NO: 13: Nucleotide sequence of pgm encoding phosphoglucomutase SEQ ID NO: 14: Amino acid sequence of phosphoglucomutase (Pgm)

Claims

1. below: a) a PTS mannose / glucose / fructose subunit IIC at a position corresponding to position 57 of SEQ ID NO:2; b) a galactokinase at a position corresponding to position 47 of SEQ ID NO:4; c) phosphoglucomutase at a position corresponding to position 242 of SEQ ID NO: 6 or 14; d) phosphoglucomutase at a position corresponding to position 164 of SEQ ID NO:8; e) the galactose operon repressor at a position corresponding to position 28 of SEQ ID NO:10; and f) a glucose kinase located at a position corresponding to position 268 of SEQ ID NO:12; 20. A composition comprising a Streptococcus thermophilus strain having a mutation causing an alteration in an encoded protein selected from the group consisting of:

2. The alteration in the encoded protein is (a) a Pro to Leu substitution at a position corresponding to position 57 of SEQ ID NO:2; (b) an Ile to Val substitution at a position corresponding to position 47 of SEQ ID NO:4; (c) a Glu to Asp substitution at a position corresponding to position 242 of SEQ ID NO: 6 or 14; (d) a Pro to Ser substitution at a position corresponding to position 164 of SEQ ID NO:8; (e) a Leu to Phe substitution at a position corresponding to position 28 of SEQ ID NO:10; and (f) a substitution of Gly to Cys at a position corresponding to position 268 of SEQ ID NO:

12.

2. The composition of claim 1 ,

3. The Streptococcus thermophilus strain is selected from the group consisting of: (a) DSM 33719 or a mutant or variant thereof; (b) DSM 33720 or a mutant or variant thereof; or (c) DSM 33762, or a mutant or variant thereof; The composition according to any one of claims 1 to 2,

4. The composition (a) DSM 33720, DSM 33719, and DSM 33762; (b) DSM 32227 and DSM 33762; or (c) DSM 33719 and DSM 32227; The composition of claim 1 comprising:

5. The composition of claim 1 further comprising one or more strains belonging to the genus Lactobacillus.

6. As a mixture or kit of parts: (a) DSM 33720 or a mutant or variant thereof, DSM 33719 or a mutant or variant thereof, DSM 33762 or a mutant or variant thereof, or any combination thereof; and (b) Strains belonging to the genus Lactobacillus The composition of claim 1 comprising:

7. 13. A method for producing a fermented product, comprising fermenting a substrate with a Streptococcus thermophilus strain having a mutation causing an alteration in an encoded protein selected from the group consisting of (a)-(f) of claim 1.

8. The method of claim 7 , wherein the substrate is a dairy substrate.

9. A fermented product obtainable by the method according to any one of claims 7 to 8.

10. A fermented product comprising one or more Streptococcus thermophilus strains having a mutation resulting in an alteration in an encoded protein selected from the group consisting of (a)-(f) of claim 1.

11. 13. Use of one or more Streptococcus thermophilus strains carrying a mutation resulting in an alteration in an encoded protein selected from the group consisting of (a) to (f) of claim 1 for the production of a fermented product.

12. A Streptococcus thermophilus strain having a mutation causing an alteration in an encoded protein selected from the group consisting of (a)-(f) of claim 1.

13. The alteration in the encoded protein is (a) a substitution of Pro with Leu at a position corresponding to position 57 of SEQ ID NO:2; (b) an Ile to Val substitution at a position corresponding to position 47 of SEQ ID NO:4; (c) a Glu to Asp substitution at a position corresponding to position 242 of SEQ ID NO: 6 or 14; (d) a Pro to Ser substitution at a position corresponding to position 164 of SEQ ID NO:8; (e) a Leu to Phe substitution at a position corresponding to position 28 of SEQ ID NO:10; and (f) a substitution of Gly to Cys at a position corresponding to position 268 of SEQ ID NO:

12.

13. The Streptococcus thermophilus strain of claim 12,

14. The alteration in the encoded protein is (a) a substitution of Pro to Leu at a position corresponding to position 57 of SEQ ID NO:2; a substitution of Ile to Val at a position corresponding to position 47 of SEQ ID NO:4; and a substitution of Glu to Asp at a position corresponding to position 242 of SEQ ID NO:6 or 14; (b) a substitution of Pro to Ser at a position corresponding to position 164 of SEQ ID NO:8; and a substitution of Leu to Phe at a position corresponding to position 28 of SEQ ID NO:10; or (c) a substitution of Gly to Cys at a position corresponding to position 268 of SEQ ID NO:

12. The Streptococcus thermophilus strain according to any one of claims 12 to 13,

15. The Streptococcus thermophilus strain comprises: (a) DSM 33719 or a mutant or variant thereof; (b) DSM 33720 or a mutant or variant thereof; or (c) DSM 33762 or a mutant or variant thereof; 13. The Streptococcus thermophilus strain of claim 12,

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