Lactic acid bacteria composition for preparing fermented food products with increased natural sweetness and flavor

Abstract

The present invention relates to a composition for producing a fermented dairy product comprising (i) at least one Streptococcus thermophilus (St) strain wherein the St strain is galactose-fermenting wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation deactivates the glucokinase protein or has a negative effect on the expression of the gene, and (ii) at least one Lactobacillus delbrueckii subsp. Bulgaricus (Lb) strain wherein the Lb strain is lactose deficient and is capable of metabolizing non-lactose carbohydrates.

Description

[0001] This application is a divisional application of an application with an application date of March 21, 2018, an application number of 201880021186.5, and an invention title of "Lactic acid bacteria composition for producing fermented foods with increased natural sweetness and flavor". Technical Field

[0002] The present invention relates to a composition for producing fermented dairy products, which comprises at least one Streptococcus thermophilus (St) strain, wherein the St strain ferments galactose, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on the expression of the gene, and (ii) at least one Lactobacillus delbrueckii subsp. bulgaricus (Lb) strain. Such a composition is used for producing fermented dairy products with increased sweetness. Background Art

[0003] Pure fermented dairy products are recognized by their acidity or sour taste because lactic acid bacteria convert lactose into lactic acid during fermentation. Therefore, they are usually sweetened by adding fruits, honey, sugar, or artificial sweeteners to meet consumers' demand for sweeter products.

[0004] To help overcome the overweight and obesity problems that have become so common in the past 20 years, the food industry has an increasing demand for low-calorie sweet foods. Sweetness is generally considered a pleasant sensation produced by the presence of sugar and some other substances. The perception of sugar varies greatly. Using sucrose as 100 for reference, the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74 (Godshall (1988). Food Technology 42(11):71-78). Therefore, although they still have roughly the same calorie level, glucose tastes more than 4 times sweeter than lactose.

[0005] The sugar in fermented foods is often replaced by sweeteners such as aspartame, acesulfame K, sucralose, and saccharin, which can provide sweetness and lower calorie intake. However, the use of artificial sweeteners may cause off-flavors, and some studies have shown that the consumption of artificial sweeteners is associated with drawbacks such as increased hunger, allergies, and cancer, which have prompted consumers to prefer fermented dairy products that contain only natural sweeteners or preferably no added sweeteners.

[0006] Therefore, a particular challenge lies in developing fermented dairy products with high natural (intrinsic) sweetness.

[0007] The acidity of fermented dairy products depends to a large extent on the lactic acid bacteria present and the process parameters used for the preparation of the fermented dairy products.

[0008] The fermentation of the disaccharide lactose has been extensively studied in lactic acid bacteria because lactose is the main carbon source in milk. In many species, lactose is cleaved into glucose and galactose by β-galactosidase after uptake. Glucose is phosphorylated to glucose-6-phosphate by glucokinase and fermented by most lactic acid bacteria via the Embden-Meyerhof-Parnas pathway (glycolysis) ( Figure 1 ).

[0009] Streptococcus thermophilus is one of the most widely used lactic acid bacteria for commercial thermophilic milk fermentation, where the organism is commonly used as part of a mixed starter culture, the other component being a Lactobacillus sp., such as Lactobacillus delbrueckii subsp. bulgaricus for yogurt or Lactobacillus helveticus for Swiss-type cheese.

[0010] In many countries, the legal definition of yogurt requires Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus together. Both species produce the required amount of acetaldehyde, which is an important flavor component in yogurt.

[0011] Lactose and sucrose are more readily fermented by Streptococcus thermophilus than their component monosaccharides. In the presence of excess galactose, when using Streptococcus thermophilus, only the glucose moiety of the lactose molecule is fermented and galactose accumulates in the fermented dairy product. In yogurt where fermentation is limited by high acid concentration, free galactose remains, while free galactose produced in the early stages of Swiss cheese manufacture is subsequently fermented by Lactobacillus helveticus.

[0012] However, some researchers (Hutkins et al. (1986) J. Dairy Sci. 69(1):1-8; Vail-lancourt et al. (2002) J. Bacteriol. 184(3); 785-793) have reported galactose-fermenting strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and WO 2011 / 026863 (Chr. Hansen) describes a method for obtaining Streptococcus thermophilus strains that ferment galactose.

[0013] To meet the requirements of the food industry, it is of great significance to develop new strains that can directly provide more natural sweetness without additional calories (internal sweetness) to the fermentation product through glucose secretion, especially Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus strains.

[0014] Pool et al. (2006. Metabolic Engineering 8(5); 456 - 464) disclosed Lactococcus lactis strains in which the deletion of genes encoding glucokinase EII (man / glc) and the newly discovered glucose - PTS EII (cel) completely disrupted glucose metabolism. The construction method was genetic recombination for generating all mutations, and thus the resulting strains were genetically modified organisms (GMOs) that are currently not suitable for use in food.

[0015] Thompson et al. (1985. J Bacteriol. 162(1); 217–223) studied lactose metabolism in Streptococcus lactis (now renamed Lactococcus lactis). In this work, mutants were obtained using 2 - deoxyglucose in the mannose - PTS system. Subsequently, this mutant was mutagenized using UV mutagenesis, and then glucose - negative colonies were screened by replica plating. In this way, double mutants (mannose PTS and glucokinase) were isolated. These double mutants were used to study the mechanisms related to the regulation of lactose fermentation by "starter" organisms. Compared with their parental strains, these mutants had several disadvantages that made them unsuitable for inclusion in commercial starter cultures. For every mole of lactose fermented, the cell yield of the mutants was half that of the parental strain, and when growing on lactose, the doubling time of the mutants increased significantly. Also, for every mole of lactose fermented, the lactic acid yield was half that of the parental strain. The behavior of these strains in milk was not analyzed, but it was expected that the acidification rate would be significantly reduced.

[0016] In addition, Lactococcus lactis is generally not selected for acetaldehyde production, and it does not contribute to meeting the requirements of the legal definition of yogurt.

[0017] Chervaux et al. (2000. Appl. And Environ. Microbiol., 66, 5306 - 5311) studied the physiology of Lactobacillus delbrueckii subsp. bulgaricus strains in a new chemically defined medium and isolated 2 - deoxyglucose - resistant mutants that were defective in glucose fermentation. Several different phenotypes were observed, and strain - specific effects were reported.

[0018] WO2013 / 160413 discloses Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus strains having enhanced properties for natural sweetening of foods and reducing the lactose content of fermented milk, and methods for screening and isolating these strains.

[0019] WO2015 / 193449 discloses a method for producing a fermented dairy product having a very low lactose concentration using a combination of lactase and lactic acid bacteria having a glucose metabolism defect.

[0020] WO2015 / 193459 discloses a method for producing a fermented dairy product with reduced post-acidification level, wherein one embodiment uses a mixture of four lactic acid bacteria strains having a glucose metabolism defect, and wherein another embodiment uses a mixture of lactose-deficient lactic acid bacteria strains.

[0021] The object of the present invention is to provide a composition for producing a fermented dairy product, which comprises at least one glucose-deficient Streptococcus thermophilus strain and at least one Lactobacillus delbrueckii subsp. bulgaricus strain, wherein the composition produces a fermented dairy product having an improved flavor. Summary of the Invention

[0022] This object is achieved by the present invention, which relates to a composition for producing a fermented dairy product, which comprises (i) at least one Streptococcus thermophilus (St) strain, wherein the St strain is galactose-fermenting, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on the expression of the gene, and (ii) at least one Lactobacillus delbrueckii subsp. bulgaricus (Lb) strain, wherein the Lb strain is lactose-deficient and capable of metabolizing non-lactose carbohydrates.

[0023] It is well known that for traditional starter cultures for producing fermented dairy products, which comprise Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus strains, there is a risk of forming unwanted off-flavors, such as free amino acids, such as glutamic acid. It is also well known that the off-flavors are formed by the Lactobacillus delbrueckii subsp. bulgaricus strains, and the level of off-flavors formed depends on the growth level of the strains, which varies according to many factors, such as the inoculation level of the Lactobacillus delbrueckii subsp. bulgaricus strains, the type of growth medium and the growth conditions of fermentation, and the specific Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus strains used in the starter culture. It has now been found that for starter cultures for producing fermented dairy products having increased sweetness, which comprise at least one glucose-deficient Streptococcus thermophilus strain and at least one Lactobacillus delbrueckii subsp. bulgaricus strain, the formation of unwanted off-flavors is at a particularly high level. This is consistent with the fact that the Lactobacillus delbrueckii subsp. bulgaricus strains grow to a particularly high cell number in such starter cultures.

[0024] Surprisingly, it has been found that the use of lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strains, such as lactose-deficient, glucose-positive Lactobacillus delbrueckii subsp. bulgaricus strains, can solve the off-flavor problem of starter cultures using glucose-deficient Streptococcus thermophilus strains. It has also been found that the lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus strains used in the compositions of the present invention do not cause any post-acidification. Finally, it has been found that, compared to conventional, i.e., lactose-positive, glucose-positive Lactobacillus delbrueckii subsp. bulgaricus strains, the combination of lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus and glucose-deficient Streptococcus thermophilus strains results in very high cell counts.

[0025] In view of the fact that Lactobacillus delbrueckii subsp. bulgaricus strains are glucose-positive and are present in fermented dairy products containing glucose, it is very surprising that it is possible to avoid the formation of off-flavors and post-acidification from, for example, lactose-deficient glucose-positive Lactobacillus delbrueckii subsp. bulgaricus strains, where theoretically the strain is capable of continued growth during storage of the fermented dairy product.

[0026] Furthermore, it is even more surprising that it is possible to avoid the formation of off-flavors and post-acidification from Lactobacillus delbrueckii subsp. bulgaricus strains while obtaining high cell counts of Lactobacillus delbrueckii subsp. bulgaricus strains, since it is generally expected that both off-flavors and post-acidification will be caused by the growth of Lactobacillus delbrueckii subsp. bulgaricus strains.

[0027] Without being bound by theory, it is believed that the surprising effects of the present invention are attributed to the metabolic differences between the lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus of the present invention and conventional lactose-positive Lactobacillus delbrueckii subsp. bulgaricus.

[0028] The application discloses the following technical solutions:

[0029] 1. A composition for producing a fermented dairy product, comprising (i) at least one Streptococcus thermophilus (St) strain, wherein the St strain ferments galactose, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on the expression of the gene, and (ii) at least one Lactobacillus delbrueckii subsp. bulgaricus (Lb) strain, wherein the Lb strain is lactose-deficient and is capable of metabolizing non-lactose carbohydrates.

[0030] 2. The composition according to embodiment 1, wherein the St strain is resistant to 2-deoxyglucose.

[0031] 3. The composition according to embodiment 1 or 2, wherein the St strain carries a mutation that reduces the transport of glucose into the cell.

[0032] 4. The composition according to any one of the foregoing embodiments, wherein when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B - milk and grown at 40 °C for 20 hours, the St strain increases the amount of glucose in the 9.5% B - milk to at least 5 mg / ml.

[0033] 5. The composition according to any one of the foregoing embodiments, wherein when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B - milk containing 0.05% sucrose and grown at 40 °C for 20 hours, the St strain increases the amount of glucose in the 9.5% B - milk containing 0.05% sucrose to at least 5 mg / ml.

[0034] 6. The composition according to any one of the foregoing embodiments, wherein the Lb strain is capable of metabolizing non - lactose carbohydrates selected from the group consisting of sucrose, galactose, and glucose.

[0035] 7. The composition according to embodiment 6, wherein the Lb strain is capable of metabolizing glucose.

[0036] 8. The composition according to any one of the foregoing embodiments, wherein the Lb strain is selected from the group consisting of the strain deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D - 38124 Braunschweig, Germany under the deposit number DSM 28910 on June 12, 2014 and mutant strains derived from DSM28910, wherein the mutant strains are further characterized by the ability to form white colonies on a medium containing lactose and X - Gal.

[0037] 9. The composition according to any one of embodiments 1 - 8, wherein the St strain is selected from the group consisting of: Streptococcus thermophilus strain CHCC19216 deposited at the German Collection of Microorganisms with the deposit number DSM 32227; strain Streptococcus thermophilus CHCC16731 which has been deposited at the German Collection of Microorganisms (DSMZ), Inhoffenstrasse 7B, D - 38124 Braunschweig, Germany, with the deposit number DSM28889 on June 4, 2014; Streptococcus thermophilus strain CHCC15757 deposited at the German Collection of Microorganisms with the deposit number DSM 25850; Streptococcus thermophilus strain CHCC15887 deposited at the German Collection of Microorganisms with the deposit number DSM 25851; Streptococcus thermophilus strain CHCC16404 deposited at the German Collection of Microorganisms with the deposit number DSM 26722; and mutant strains derived therefrom, wherein the mutant strain is obtained by using one of the deposited strains as a starting material, and wherein the mutant has retained or further improved the glucose - secreting property of the deposited strain.

[0038] 10. A method for producing a fermented dairy product, which comprises inoculating and fermenting a milk matrix with the composition according to any one of embodiments 1 - 9.

[0039] 11. A fermented dairy product, which comprises the composition according to any one of embodiments 1 - 9.

[0040] 12. Use of the composition according to any one of embodiments 1 - 9 for the preparation of a fermented dairy product. Description of the Drawings

[0041] Figure 1 is a schematic diagram of lactose catabolism in Streptococcus thermophilus. GlcK, glucokinase; LacS, lactose transporter; LacZ, β - galactosidase; GalM, mutarotase; GalK, galactokinase; GalT, galactose - 1 - phosphate uridylyltransferase; GalE, UDP - glucose 4 - epimerase; Gal1P, galactose - 1 - phosphate. Detailed Description

[0042] Definition

[0043] As used herein, the term "lactic acid bacteria" refers to Gram-positive, microaerophilic or anaerobic bacteria that ferment sugars with the simultaneous production of acids, including lactic acid as the major acid produced, acetic acid, and propionic acid. The most industrially useful lactic acid bacteria are found in the "Lactobacillales", which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp, Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp., and Propionibacterium spp. Lactic acid bacteria, including Lactobacillus spp. and Streptococcus thermophilus species, are typically supplied to the dairy industry as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures for direct inoculation into fermentation vessels or vats to produce dairy products, such as fermented dairy products. Such cultures are commonly referred to as "starter cultures" or "starters".

[0044] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a", "an", "the", and similar indicators should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. Unless otherwise specified, the terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the recitation of numerical ranges herein is merely intended to be a shorthand method for separately referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were recited herein individually. Unless otherwise specified herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and is not intended to limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential for the practice of the invention.

[0045] For the present invention, the expression "increased sweetness" means an increase in sweetness compared to the sweetness produced by the parental strain that does not carry a mutation in the DNA sequence of the glcK gene encoding glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on the gene expression.

[0046] The expression "CFU" means Colony Forming Unit.

[0047] Streptococcus thermophilus strains of the composition of the present invention

[0048] In some countries, the legal definition of yogurt requires the presence of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Both of these species produce the desired amount of acetaldehyde, an important flavor component in yogurt.

[0049] Cheeses such as Mozzarella, pizza cheese, and Feta can also be prepared by fermentation using both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus ( et al. (2010), in The Technology of Cheese making, 2 nd Ed. Blackwell Publishing, Oxford; 166 - 192).

[0050] To meet the requirements of the food industry, it is desirable to develop new strains that directly produce more natural sweetness (internal sweetness) in fermented products without contributing additional calories, especially Lactobacillus delbrueckii subsp. bulgaricus strains and Streptococcus thermophilus strains.

[0051] Streptococcus thermophilus is one of the most widely used lactic acid bacteria in commercial milk fermentation, where the organism is commonly used as part of a mixed starter culture, the other component being a Lactobacillus species, such as Lactobacillus delbrueckii subsp. bulgaricus for yogurt, and Lactobacillus helveticus for Swiss - type cheeses.

[0052] When Streptococcus thermophilus is used, only the glucose part of the lactose molecule is fermented by Streptococcus thermophilus, and galactose accumulates in the fermented dairy product. In yogurt where high acid concentration limits fermentation, free galactose remains, while the free galactose produced in the early stages of Swiss cheese manufacture is subsequently fermented by Lactobacillus helveticus. Lactococcus lactis, which is found in many starter cultures for cheese making, is also able to consume the galactose produced by Streptococcus thermophilus.

[0053] In order to ensure a Streptococcus thermophilus strain with the best possible growth performance, the inventors exposed galactose-fermenting strains of Streptococcus thermophilus to the selecting agent 2-deoxyglucose. Typically, 2-deoxyglucose resistant mutants have mutations in the gene encoding glucokinase and in the genes encoding glucose transport. The isolated mutant CHCC16731, which is resistant to 2-deoxyglucose, has a mutation in its glucokinase (glcK) gene. In addition to the mutation in the glucokinase gene, both CHCC16731 and CHCC19216 have mutations that mean secreted glucose is not transported back into the cell again.

[0054] Surprisingly, this mutant alone is still fully capable of acidifying milk, although the time to acidify to pH 5 is delayed by 2 - 5 hours. Thus they are useful in fermented milk applications and they retain the ability of the parent strain to acidify milk, which is characteristic of yogurt. In addition, it was found that when the mutant was inoculated into 9.5% β - milk containing 0.05% sucrose and fermented at 40 °C for at least 20 hours, the mutant secreted high levels of glucose. At the same time, the residual lactose content in the fermented milk was very low. Thus, using these strains to produce fermented dairy products can be of importance for lactose-intolerant people.

[0055] Thus, as calculated by Godshall (1988. Food Technology 42(11):71 - 78), the final fermented milk has an increased internal sweetness index.

[0056] In one aspect of the invention, the Streptococcus thermophilus strain is a galactose-fermenting mutant strain of Streptococcus thermophilus, wherein the mutant strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation inactivates the encoded glucokinase protein or has a negative effect on gene expression. Methods for measuring the level of glucokinase activity or the level of glucokinase gene expression are readily available (Porter et al. (1982) Biochim. Biophys. Acta, 709; 178–186) and include enzyme assays with commercially available kits and transcriptomics or quantitative PCR using readily available materials.

[0057] In a preferred embodiment of the invention, the Streptococcus thermophilus strain of the invention is resistant to 2-deoxyglucose.

[0058] As used herein, a bacterial "strain" refers to bacteria that remain genetically identical when growing or reproducing. It includes a large number of identical bacteria.

[0059] As used herein, the term "Streptococcus thermophilus strain capable of galactose fermentation" refers to a Streptococcus thermophilus strain that is capable of growing on / in M17 medium + 2% galactose. A Streptococcus thermophilus strain capable of galactose fermentation is defined herein as a Streptococcus thermophilus strain that, when inoculated at 1% from an overnight culture and incubated at 37 °C for 24 hours, reduces the pH of M17 culture medium containing 2% galactose as the sole carbohydrate to 5.5 or lower.

[0060] As used herein, the term "mutation that inactivates the glucokinase protein" refers to a mutation that gives rise to an "inactivated glucokinase protein", which, if present in a cell, is a glucokinase protein that cannot perform its normal function, and a mutation that prevents the formation of the glucokinase protein or causes degradation of the glucokinase protein.

[0061] In particular, an inactivated glucokinase protein is a protein that, compared to a functional glucokinase protein, 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 gene encoding a functional glucokinase protein, the gene encoding such an inactivated glucokinase protein contains a mutation in the open reading frame (ORF) of the gene, wherein the mutation may include, but is not limited to, deletions, frameshift mutations, introduction of a stop codon, or a mutation that causes an amino acid substitution (that alters the functional properties of the protein), or a promoter mutation that reduces or eliminates gene transcription or translation.

[0062] In a preferred embodiment, the mutation reduces the activity of the glucokinase protein (the rate of phosphorylation of glucose to glucose-6-phosphate) by at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%.

[0063] Glucokinase activity can be determined by the glucokinase enzymatic assay described by Pool et al. (2006. Metabolic Engineering 8; 456-464).

[0064] As used herein, the term "functional glucokinase protein" refers to a glucokinase protein that, if present in a cell, promotes the phosphorylation of glucose to glucose-6-phosphate. In particular, a functional glucokinase protein can be encoded by a gene containing an ORF having a sequence corresponding to positions 1-966 in SEQ ID NO.1, or a sequence having at least 85% identity, such as at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the sequence corresponding to positions 1-966 of SEQ ID NO.1.

[0065] The percent identity between two sequences can be determined using mathematical algorithms, e.g., the algorithms of Karlin and Altschul (1990. Proc. Natl. Acad. Sci. USA 87; 2264), the modified algorithm of Karlin and Altschul (1993. Proc. Natl. Acad. Sci. USA 90; 5873-5877); the algorithm of Myers and Miller (1988. CABIOS 4; 11-17); the algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48; 443-453); and the algorithm of Pearson and Lipman (1988. Proc. Natl. Acad. Sci. USA 85; 2444-2448). Computer software based on these mathematical algorithms for determining nucleic acid or amino acid sequence identity is also available. For example, nucleotide sequences can be compared using the BLASTN program, score = 100, wordlength = 12. Amino acid sequences can be compared using the BLASTX program, score = 50, wordlength = 3. For the remaining parameters of the BLAST program, default parameters can be used.

[0066] Many countries do not accept the use of genetically modified organisms (GMOs) in fermented dairy products. Instead, the present invention provides naturally occurring or induced mutant strains that are capable of providing the desired glucose accumulation in fermented dairy products.

[0067] Thus, in a highly preferred embodiment of the present invention, the mutant strain is a naturally occurring mutant or an induced mutant.

[0068] As used herein, the terms "mutant bacterium" or "mutant strain" refer to natural (spontaneous, naturally occurring) mutant bacteria or induced mutant bacteria that contain one or more mutations in the genome (DNA) that are not present in the wild-type DNA. An "induced mutant" is a bacterium in which the mutation is induced by artificial treatment, e.g., treatment with chemical mutagens, UV- or γ-radiation, etc. In contrast, a "spontaneous mutant" or "naturally occurring mutant" has not been mutagenized by man. The mutant bacteria herein are non-GMOs (non-genetically modified organisms), i.e., not modified by recombinant DNA technology.

[0069] A "wild-type strain" refers to the non-mutated form of a bacterium as found in nature.

[0070] Terms such as "strain with sweetening properties", "strain capable of providing the desired glucose accumulation in fermented dairy products", and "strain with enhanced natural sweetening properties of food" are used interchangeably herein to characterize the advantageous aspects of using the strains of the present invention in dairy fermentation.

[0071] In a preferred embodiment, when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B - milk and grown at 40°C for at least 20 hours, the mutant strain of Streptococcus thermophilus of the present invention increases the amount of glucose in 9.5% B - milk to at least 5 mg / mL.

[0072] In another preferred embodiment, when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B - milk containing 0.05% sucrose and grown at 40°C for at least 20 hours, the mutant strain of Streptococcus thermophilus of the present invention increases the amount of glucose in 9.5% B - milk containing 0.05% sucrose to at least 5 mg / mL.

[0073] In the context of the present invention, 9.5% B - milk is milk that has been prepared from reconstituted low - fat skim milk powder to a dry matter content of 9.5%, pasteurized at 99°C for 30 min (minutes), and then cooled to 40°C.

[0074] In a more preferred embodiment of the present invention, the mutant strain causes the amount of glucose to increase to at least 6 mg / mL, such as at least 7 mg / mL, such as at least 8 mg / mL, such as at least 9 mg / mL, such as at least 10 mg / mL, such as at least 11 mg / mL, such as at least 12 mg / mL, such as at least 13 mg / mL, such as at least 14 mg / mL, such as at least 15 mg / mL, such as at least 20 mg / mL, such as at least 25 mg / mL.

[0075] In another embodiment of the present invention, the mutant strain of Streptococcus thermophilus is resistant to 2 - deoxyglucose.

[0076] As used herein, the term "resistant to 2 - deoxyglucose" is defined as the ability of a particular mutant bacterial strain to grow into colonies when streaked on an M17 medium plate containing 20 mM 2 - deoxyglucose and incubated at 40°C for 20 hours. The presence of 2 - deoxyglucose in the medium prevents the growth of non - mutant strains, while the growth of the mutant strain is not affected or is not significantly affected. Non - mutant strains that can be used as sensitive reference strains for evaluating resistance preferably include strains CHCC14994 and CHCC11976.

[0077] When using 10 6 -10 7When the Streptococcus thermophilus strain of the present invention at 10 - 10 CFU / ml is inoculated into 9.5% B - milk and fermented with the Streptococcus thermophilus strain of the present invention at 40°C for at least 20 hours, the mutant Streptococcus thermophilus strain of the present invention secretes glucose into the milk. Preferably, when inoculated with 10 6 -10 7 CFU / ml of the Streptococcus thermophilus strain of the present invention into 9.5% B - milk and fermented with said Streptococcus thermophilus strain at 40°C for at least 20 hours, this mutant strain alone will secrete at least 5 mg / ml of glucose into the B - milk. Although the time to acidify to pH 5 is delayed by 2 - 5 hours, the strain can still completely acidify the milk. The final fermented milk contains less than 15 mg / ml of lactose in the fermented milk. Therefore, the final fermented milk has a higher internal sweetness index of about 2 - fold or more.

[0078] In yet another embodiment, the mutant strain of the present invention can be characterized by its growth pattern. This can be illustrated by the finding that the mutant strain has a higher growth rate in M17 medium + 2% galactose than in M17 medium + 2% glucose. The growth rate is measured as the development of the optical density of an exponentially growing culture at 600 nanometers (OD 600 ) over time.

[0079] In a preferred embodiment, the growth rate is at least 5% higher in M17 medium + 2% galactose than in M17 medium + 2% glucose, such as at least 10% higher, such as at least 15% higher, such as at least 20% higher.

[0080] Mutation in the glcK gene

[0081] In a preferred embodiment, the mutation results in the replacement of the codon encoding glycine at position 249 of SEQ ID NO.2 with a codon encoding arginine. Preferably, the mutation in the glcK gene results in the replacement of G with A at position 745 of SEQ ID NO.1. The strain CHCC16731 has this mutation.

[0082] In a preferred embodiment, the mutation results in the replacement of the codon encoding serine at position 72 of SEQ ID NO.2 with a codon encoding proline (not shown). Preferably, the mutation in the glcK gene results in the replacement of T with C at position 214 of SEQ ID NO.1 (not shown).

[0083] In another preferred embodiment, the mutation results in the replacement of the codon encoding threonine at position 141 of SEQ ID NO.2 with a codon encoding isoleucine (not shown).

[0084] Preferably, the mutation in the glcK gene results in the substitution of C with T at position 422 of SEQ ID NO.1 (not shown).

[0085] It should be emphasized that the glcK gene of Streptococcus thermophilus can be inactivated by other types of mutations at other sites of the glcK gene.

[0086] Mutations that reduce glucose transport into the cell

[0087] In a preferred embodiment, the Streptococcus thermophilus strain carries a mutation that reduces glucose transport into the cell.

[0088] 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.

[0089] When the culture medium is a milk matrix, the level of glucose in the culture medium of the Streptococcus thermophilus strain can be easily measured by methods known to those skilled in the art.

[0090] In a preferred embodiment, the mutation reduces glucose transport into the cell by at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%.

[0091] The transport of glucose into the cell can be determined by the glucose uptake assay described by Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423 - 5432).

[0092] Preferably, the Streptococcus thermophilus strain carries a mutation in a gene encoding a component of the glucose transporter, wherein the mutation inactivates the glucose transporter or has a negative effect on the expression of the gene.

[0093] The component can be any component critical for glucose transport in the glucose transporter. For example, inactivation of any component of the glucose / mannose PTS in Streptococcus thermophilus is expected to result in inactivation of the glucose transporter function.

[0094] As used herein, the term "mutation that inactivates the glucose transporter" refers to a mutation that results in an "inactivated glucose transporter", where an "inactivated glucose transporter" is a glucose transporter protein that, if present in the cell, cannot perform its normal function, and mutations that prevent the formation of the glucose transporter protein or lead to the degradation of the glucose transporter protein.

[0095] In particular, an inactivated glucose transporter is a protein that cannot promote the transport of glucose across the plasma membrane or promotes the transport of glucose across the plasma membrane at a significantly reduced rate, compared to a functional glucose transporter. Compared to a gene encoding a functional glucose transporter, the gene encoding such an inactivated glucose transporter contains a mutation in the open reading frame (ORF) of the gene, wherein the mutation can include, but is not limited to, deletions, frameshift mutations, introduction of a stop codon, or mutations that result in amino acid substitutions that alter the functional properties of the protein, or promoter mutations that reduce or eliminate gene transcription or translation.

[0096] In a preferred embodiment, the mutation reduces the activity (rate of glucose transport) of the glucose transporter protein by at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%.

[0097] Glucose transporter activity can be measured by the glucose uptake assay described by Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423-5432).

[0098] As used herein, the term "functional glucose transporter protein" refers to a glucose transporter protein that, if present in a cell, promotes the transport of glucose across the plasma membrane.

[0099] In a preferred embodiment of the present invention, the Streptococcus thermophilus strain of the present invention carries a mutation in the DNA sequence of the manN gene encoding the IID protein of the glucose / mannose phosphotransferase system, wherein the mutation inactivates the IID Man protein or has a negative effect on the expression of the gene. Man

[0100] CHCC16731 has a change from threonine to proline at position 79 of the manN gene encoding the IID protein of the glucose / mannose phosphotransferase system. Preferably, the mutation in the ManN gene results in the substitution of A at position 235 of SEQ ID NO.3 with C. Man

[0101] Thus, in a preferred embodiment, the Streptococcus thermophilus strain of the present invention carries a mutation in the DNA sequence of the manN gene encoding the IID protein of the glucose / mannose phosphotransferase system, wherein the mutation results in the substitution of threonine at position 79 with proline in SEQ ID No.4. Man

[0102] In another preferred embodiment of the present invention, the Streptococcus thermophilus strain of the present invention is in the IIC of the glucose / mannose phosphotransferase system Man ​​​The DNA sequence of the manM gene of the protein carries a mutation, wherein the mutation inactivates the IIC Man protein or has a negative effect on the expression of the gene.

[0103] In a particularly preferred embodiment, the mutation results in the replacement of the codon encoding glutamic acid at position 209 of SEQ ID NO.6 of the IIC Man protein of the glucose / mannose phosphotransferase system with a stop codon (not shown). Preferably, the mutation results in the replacement of G at position 625 of SEQ ID NO.5 with T (not shown).

[0104] Preferred Streptococcus thermophilus strains of the present invention

[0105] In a preferred embodiment of the composition of the present invention, the Streptococcus thermophilus strains are selected from the group consisting of: Streptococcus thermophilus CHCC19216 strain deposited under the accession number DSM 32227 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Streptococcus thermophilus CHCC16731 strain deposited under the accession number DSM 28889 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany in June 2014, Streptococcus thermophilus CHCC15757 strain deposited under the accession number DSM 25850 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Streptococcus thermophilus CHCC15887 strain deposited under the accession number DSM 25851 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Streptococcus thermophilus CHCC16404 strain deposited under the accession number DSM 26722 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, and mutant strains derived therefrom, wherein the mutant strains are obtained by using one of the deposited strains as a starting material, and wherein the mutants retain or further improve the textural properties and / or glucose secretion properties of the deposited strains.

[0106] In the context of the present invention, the term "mutant strain" should be understood as a strain that is derived from, or can be derived from, the strain of the present invention (or its parental strain) by, for example, genetic engineering, radiation, and / or chemical treatment. A "strain derived therefrom" can also be a spontaneously occurring mutant. A "strain derived therefrom" is preferably a functionally equivalent mutant, such as a mutant having substantially the same or improved characteristics as its parental strain. In particular, the term "mutant strain" refers to a strain obtained by subjecting the strain of the present invention to any conventionally used mutagenesis treatment, including treatment with chemical mutagens such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ultraviolet treatment, or a spontaneously occurring mutant. The mutant may have been subjected to several mutagenesis treatments (a single treatment is considered one mutagenesis step followed by a screening / selection step), but currently it is preferred to carry out 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 1%, less than 0.1%, less than 0.01%, less than 0.001%, or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced or deleted compared to the parental strain.

[0107] Method for obtaining sweetened Streptococcus thermophilus strains

[0108] In a preferred embodiment of the present invention, the starting strain for developing a sweetened Streptococcus thermophilus strain is a structured Streptococcus thermophilus strain, which can be obtained as described in other parts of this specification.

[0109] In a preferred embodiment, such a structured starting strain is selected from the group consisting of: Streptococcus thermophilus strain CHCC11342 deposited at the German Collection of Microorganisms with the deposit number DSM22932, Streptococcus thermophilus strain CHCC11976 deposited at the German Collection of Microorganisms with the deposit number DSM 22934, Streptococcus thermophilus strain CHCC12339 deposited at the German Collection of Microorganisms with the deposit number DSM 24090, and strains derived therefrom.

[0110] Alternatively, the starting strain for developing a sweetened Streptococcus thermophilus strain is an unstructured Streptococcus thermophilus strain. In this case, the structuring property is then introduced into the resulting sweetened Streptococcus thermophilus strain, i.e., the sweetened strain is used as the starting strain in the method for obtaining a structured strain described elsewhere in this specification.

[0111] The first step of the method for sweetening Streptococcus thermophilus strains is to provide galactose-positive strains, i.e., strains capable of using galactose as a carbohydrate source. Galactose-positive strains can be obtained by a method comprising the following steps: streaking the bacteria to be tested on an agar plate, such as an M17 agar plate containing a certain concentration, for example, 2% galactose (the sole carbohydrate source), and identifying the colonies capable of growing on said plate.

[0112] The determination of the growth pattern of bacteria in M17 medium + 2% galactose compared to that in M17 medium + 2% glucose, along with 2-deoxyglucose, is used to select bacteria having a mutation in the glucokinase (glcK) gene.

[0113] A method for screening and isolating Streptococcus thermophilus strains having a mutated glcK gene, comprising the following steps:

[0114] a) providing a galactose-fermenting Streptococcus thermophilus parent strain;

[0115] b) selecting and isolating a pool of mutant Streptococcus thermophilus strains resistant to 2-deoxyglucose from a pool of mutant Streptococcus thermophilus strains derived from the parent strain; and

[0116] c) if the growth rate of the mutant Streptococcus thermophilus strain in M17 medium + 2% galactose is higher than that in M17 medium + 2% glucose, then selecting and isolating said mutant Streptococcus thermophilus strain from the pool of mutant Streptococcus thermophilus strains resistant to 2-deoxyglucose.

[0117] As used herein, the term "resistant to 2-deoxyglucose" is defined as the ability of a specific mutant bacterial strain to grow into colonies after incubation at 40 °C for 20 hours when streaked on an M17 medium plate containing 2% lactose or 2% galactose and containing 20 mM 2-deoxyglucose. The presence of 2-deoxyglucose in the medium prevents the growth of non-mutant strains, while the growth of mutant strains is not affected or is not significantly affected. Non-mutant strains that can be used as a sensitive reference strain during resistance assessment include strain CHCC11976.

[0118] In an embodiment, the method further comprises step a1): subjecting the parent strain to mutagenesis, such as subjecting the parent strain to chemical and / or physical mutagens.

[0119] In another embodiment, the method further comprises step d): if the growth rate of the Streptococcus thermophilus strain in M17 medium + 2% sucrose is high, but zero or at least 0 - 50% lower compared to the growth rate of the parent strain in M17 medium + 2% glucose, then selecting and isolating said Streptococcus thermophilus strain from the pool of 2-deoxyglucose-resistant Streptococcus thermophilus strains derived from the Streptococcus thermophilus strain selected in step c).

[0120] The Streptococcus thermophilus parent strain capable of galactose fermentation can grow on / in M17 medium + 2% galactose and is defined herein as having the ability to lower the pH in M17 broth containing 2% galactose as the sole carbohydrate to 5.5 or lower when inoculated with 1% of an overnight culture and incubated at 37 °C for 24 hours. Such galactose-positive strains are described in WO2011 / 026863 (Chr. Hansen A / S) and WO2011 / 092300 (Chr. Hansen A / S).

[0121] In this context, the term "strains derived therefrom" should be understood to mean strains derived from or derivable from a structured or galactose-fermenting Streptococcus thermophilus parent strain by, for example, genetic engineering, radiation and / or chemical treatment. "Strains derived therefrom" can also be spontaneously occurring mutants. Preferably, "strains derived therefrom" are functionally equivalent mutants, such as mutants having substantially the same or improved characteristics (e.g., regarding texture or galactose fermentation) as their parent strain. In particular, the term "strains derived therefrom" refers to strains obtained by subjecting the strains of the present invention to any conventionally used mutagenesis treatment, including treatment with chemical mutagens such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ultraviolet treatment, or to spontaneously occurring mutants. The mutants 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 preferably not more than 20, not more than 10 or not more than 5 treatments (or screening / selection steps). In currently preferred mutants, less than 1%, less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced or deleted compared to the parent strain.

[0122] In the present invention, the expression "glcK gene encoding glucokinase" refers to any DNA sequence of Streptococcus thermophilus encoding a protein having glucokinase activity, including the specific glucokinase encoded by the DNA sequence of SEQ ID NO.:1. Glucokinase catalyzes the reaction of converting glucose into glucose-6-phosphate, see Figure 1 .

[0123] Lactobacillus delbrueckii subsp. bulgaricus strains of the composition of the present invention

[0124] The terms "lactose metabolism defective" and "lactose defective" are used in the context of the present invention to characterize LAB that have partially or completely lost the ability to use lactose as a source for cell growth or maintaining cell viability. Each LAB is capable of metabolizing one or several carbohydrates selected from sucrose, galactose, and / or glucose or another fermentable carbohydrate. Since these carbohydrates are not naturally present in milk in amounts sufficient to support fermentation by lactose-deficient mutants, these carbohydrates must be added to milk. Lactose-deficient and partially defective LAB can be characterized as white colonies on a medium containing lactose and X-Gal.

[0125] For the present invention, the term "X-Gal" refers to 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, which is a chromogenic substrate for β-galactosidase. β-Galactosidase hydrolyzes X-Gal into colorless galactose and 5-bromo-4-chloro-indophenol, which spontaneously dimerize to form a blue pigment.

[0126] In a specific embodiment of the present invention, the lactose-deficient strain is capable of metabolizing a non-lactose carbohydrate selected from the group consisting of sucrose, galactose, and glucose, preferably sucrose. In a specific embodiment of the present invention, the lactose-deficient strain is capable of metabolizing galactose.

[0127] In a specific embodiment of the composition of the present invention, the Lb strain is selected from the group consisting of the strain deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig under the deposit number DSM 28910 on June 12, 2014, and mutant strains derived therefrom, wherein the mutant strains are further characterized by the ability to form white colonies on a medium containing lactose and X-Gal.

[0128] Lactose-deficient strains can be obtained by mutation from a suitable lactose-positive parent strain. After UV-mutagenesis, lactose-deficient strains are selected as white colonies (indicating the lactose-deficient phenotype) on a suitable medium, such as an MRS agar plate with 1% lactose and 200 mg / ml X-Gal. Lactose-positive strains have β-galactosidase activity, and due to the activity of β-galactosidase, the colonies of lactose-positive strains are blue. As the lactose-positive parent strain for generating lactose-deficient strains, the strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig under the deposit number DSM 19252 on April 3, 2007 for WO2011 / 000879 can be used.

[0129] Composition

[0130] The present invention further relates to comprising 104 -10 12 CFU (colony forming units) / g Streptococcus thermophilus strains, such as 10 5 -10 11 CFU / g, such as 10 6 -10 10 CFU / g, or such as 10 7 -10 9 CFU / g of a composition of Streptococcus thermophilus strains.

[0131] In a preferred embodiment, the Streptococcus thermophilus strains of the composition cannot acidify 9.5% B-lactose, defined as when inoculating 9.5% B-lactose with 10 6 -10 7 CFU / ml of Streptococcus thermophilus strains and incubating at 40 °C for 14 hours, resulting in a pH decrease of less than 1.0, and the composition further contains a certain amount of a compound that can trigger the acidification of 9.5% B-lactose by Streptococcus thermophilus strain CHCC16404, which is deposited at the German Collection of Microorganisms with the deposit number DSM 26722, defined as when inoculating 9.5% B-lactose with 10 6 -10 7 CFU / ml of the Streptococcus thermophilus strains and incubating at 40 °C for 14 hours, resulting in a pH decrease of 1.0 or higher.

[0132] Preferably, the compound is sucrose.

[0133] Preferably, the amount of sucrose is 0.000001% - 2%, such as 0.00001% - 0.2%, such as 0.0001% - 0.1%, such as 0.001% - 0.05%.

[0134] In a particularly preferred embodiment, the composition further contains 10 4 -10 12 CFU / g of Lactobacillus delbrueckii subsp. bulgaricus strains, such as 10 5 -10 11 CFU / g, such as 10 6 -10 10 CFU / g, or such as 10 7 -10 9 CFU / g of Lactobacillus delbrueckii subsp. bulgaricus strains.

[0135] In a specific embodiment of the present invention, the composition contains at least two St strains, preferably at least three St strains, more preferably three St strains. In a specific embodiment of the present invention, the composition contains no more than three Lb strains, preferably no more than two Lb strains, more preferably one Lb strain.

[0136] In a specific embodiment of the present invention, the composition contains at least one St strain selected from the group consisting of: Streptococcus thermophilus CHCC16731, which was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, under the deposit number DSM 28889 on June 4, 2014; Streptococcus thermophilus CHCC15757 strain, which was deposited at the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 25850; Streptococcus thermophilus CHCC16404 strain, which was deposited at the German Collection of Microorganisms and Cell Cultures under the deposit number DSM 26722; and an Lb strain, which was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, under the deposit number DSM 28910 on June 12, 2014.

[0137] Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are commonly used as starter cultures for technical purposes in the production of various foods, for example in the dairy industry, for example for fermenting dairy products. Therefore, in another preferred embodiment, the composition is suitable as a starter culture.

[0138] In addition to liquid starter cultures, starter cultures can be provided as frozen or dried starter cultures. Therefore, in yet another preferred embodiment, the composition is in frozen form, freeze-dried form or liquid form.

[0139] As disclosed in WO 2005 / 003327, it is beneficial to add certain cryoprotectants to the starter culture. Therefore, the starter culture composition of the present invention may contain one or more cryoprotectants selected from the following: inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5'-monophosphate (GMP), uridine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, inosine, orotidine, thymidine, inosine and derivatives of any such compounds.

[0140] Method for producing fermented dairy products

[0141] The present invention also relates to a method for producing fermented dairy products, including inoculating and fermenting a milk substrate with the composition of the present invention.

[0142] The term "milk" should be understood as the milk secretion obtained by milking any mammal such as cows, sheep, goats, buffaloes or camels. In a preferred embodiment, the milk is cow's milk.

[0143] The term "milk substrate" can be any raw milk material and / or processed milk material that can be fermented according to the method of the present invention. Thus, useful milk substrates include, but are not limited to, solutions or suspensions of any milk or milk-like product containing proteins, such as whole milk or low-fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, dried milk, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream. Obviously, the milk substrate can be derived from any mammal, for example, substantially pure mammalian milk or reconstituted milk powder.

[0144] Preferably, at least part of the proteins in the milk substrate are proteins naturally present in milk, such as casein or whey protein. However, part of the proteins can be proteins not naturally present in milk.

[0145] Before fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.

[0146] As used herein, "homogenization" means intense mixing to obtain a soluble suspension or emulsion. If homogenization is carried out before fermentation, then homogenization can be carried out so as to break the milk fat into smaller sizes so that the milk fat no longer separates from the milk. This can be done by passing the milk through small holes under high pressure.

[0147] As used herein, "pasteurization" means treating the milk substrate to reduce or eliminate the presence of live organisms such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specific time. The specified temperature is usually obtained by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. Subsequently, a rapid cooling step can be carried out.

[0148] In the method of the present invention, "fermentation" means the conversion of carbohydrates into alcohols or acids by the action of microorganisms. Preferably, fermentation in the method of the present invention includes the conversion of lactose into lactic acid.

[0149] Fermentation processes for producing fermented dairy products are well known, and those skilled in the art will know how to select suitable process conditions, such as temperature, oxygen, the amount and characteristics of microorganisms, and process time. Obviously, the fermentation conditions are selected to support the implementation of the present invention, that is, to obtain a dairy product (fermented dairy product) in solid or liquid form.

[0150] The term "fermented dairy product" as used herein refers to a food or feed product, wherein the preparation of the food or feed product includes fermenting a milk substrate with lactic acid bacteria. "Fermented dairy products" as used herein include, but are not limited to, thermophilic fermented dairy products such as yogurt, mesophilic fermented dairy products such as sour cream and buttermilk, and products such as fermented whey.

[0151] The term "thermophile" as used herein refers to microorganisms that grow best at temperatures above 35°C. The most industrially useful thermophilic bacteria include species of the genus Streptococcus and species of the genus Lactobacillus. The term "thermophilic fermentation" as used herein refers to fermentation at a temperature above about 35°C, such as at about 35°C to about 45°C. The term "thermophilic fermented dairy product" refers to a fermented dairy product prepared by thermophilic fermentation with a thermophilic starter culture, and includes fermented dairy products such as set yogurt, stirred yogurt, and drinking yogurt (such as Yakult).

[0152] The term "mesophile" as used herein refers to microorganisms that grow best at moderate temperatures (15°C - 35°C). The most industrially useful mesophilic bacteria include species of the genus Lactococcus and species of the genus Leuconostoc. The term "mesophilic fermentation" as used herein refers to fermentation at a temperature of about 22°C to about 35°C. The term "mesophilic fermented dairy product" refers to a fermented dairy product prepared by mesophilic fermentation with a mesophilic starter culture, including fermented dairy products such as buttermilk, acid milk, fermented milk, smetana, sour cream, kefir, and fresh cheese, such as quark, tvarog, and cream cheese.

[0153] In a preferred embodiment, the concentration of inoculated Streptococcus thermophilus cells is 10 4 -10 9 CFU of Streptococcus thermophilus cells per ml of milk substrate, such as 10 4 CFU to 10 8 CFU of Streptococcus thermophilus cells.

[0154] In another preferred embodiment of the method of the present invention, the Streptococcus thermophilus strain cannot acidify 9.5% B - milk, defined as resulting in a pH decrease of less than 1.0 when inoculating 9.5% B - milk with 10 6 -10 7 CFU / ml of the Streptococcus thermophilus strain and incubating at 40°C for 14 hours, and a certain amount of a compound is added to the milk substrate that effectively triggers the Streptococcus thermophilus to acidify 9.5% B - milk, defined as resulting in a pH decrease of 1.0 or more when inoculating 9.5% B - milk with 10 6 -10 7 CFU / ml of the Streptococcus thermophilus strain and incubating at 40°C for 14 hours.

[0155] Preferably, the compound is sucrose.

[0156] Preferably, the amount of sucrose is 0.000001% - 2%, such as 0.00001% - 0.2%, such as 0.0001% - 0.1%, such as 0.001% - 0.05%.

[0157] In another preferred embodiment, the fermented dairy product is yogurt or cheese.

[0158] Examples of cheeses prepared by fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus include mozzarella cheese and pizza cheese ( et al. (2010), in The Technology of Cheesemaking, 2 nd Ed. Blackwell Publishing, Oxford; 166 - 192).

[0159] Preferably, the fermented dairy product is yogurt.

[0160] In the context of the present invention, a yogurt starter culture is a bacterial culture comprising at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus. Accordingly, the term "yogurt" refers to a fermented dairy product obtained by inoculating and fermenting milk with a composition comprising a strain of Lactobacillus delbrueckii subsp. bulgaricus and a strain of Streptococcus thermophilus.

[0161] Fermented dairy products

[0162] The present invention also relates to a fermented dairy product comprising the composition of the present invention.

[0163] In another preferred embodiment, the fermented dairy product can be yogurt, cheese, sour cream and buttermilk, and fermented whey. Preferably, the fermented dairy product is yogurt.

[0164] Use of the composition of the present invention

[0165] Another aspect of the present invention relates to the use of the composition of the present invention for the preparation of a fermented dairy product.

[0166] The following describes embodiments of the present invention by way of non - limiting examples.

[0167] Sequence Listing

[0168] SEQ ID NO.1 shows the DNA sequence of the mutant glucokinase gene of strain CHCC16731.

[0169] SEQ ID NO.2 shows the amino acid sequence encoded by SEQ ID NO.1.

[0170] SEQ ID NO.3 shows the DNA sequence of the mutant ManN gene of strain CHCC16731.

[0171] SEQ ID NO.4 shows the amino acid sequence encoded by EQ ID NO.3.

[0172] SEQ ID NO.5 shows the DNA sequence of the ManM gene (unmutated) of strain CHCC16731.

[0173] SEQ ID NO.6 shows the amino acid sequence encoded by SEQ ID NO.5.

[0174] Examples

[0175] Materials and Methods

[0176] Culture medium:

[0177] For Streptococcus thermophilus, the culture medium used is M17 medium known to those skilled in the art.

[0178] M17 agar medium per liter of H2O has the following composition:

[0179] Agar: 12.75 g

[0180] Ascorbic acid: 0.5 g

[0181] Casein peptone (tryptic): 2.5 g

[0182] Sodium β-glycerophosphate pentahydrate: 19 g

[0183] Magnesium sulfate hydrate: 0.25 g

[0184] Meat extract: 5 g

[0185] Meat peptone (pepsin): 2.5 g

[0186] Soy peptone (papain): 5 g

[0187] Yeast extract: 2.5 g

[0188] Final pH 7.1 ± 0.2 (25 °C)

[0189] M17 broth per liter of H2O has the following composition:

[0190] Ascorbic acid: 0.5 g

[0191] Magnesium sulfate: 0.25 g

[0192] Meat extract: 5 g

[0193] Meat peptone (pepsin): 2.5 g

[0194] Sodium glycerophosphate: 19 g

[0195] Soy peptone (papain): 5 g

[0196] Tryptone: 2.5 g

[0197] Yeast extract: 2.5 g

[0198] Final pH 7.0 ± 0.2 (25 °C)

[0199] The added carbon source is sterile lactose 20 g / l, glucose 20 g / l or galactose 20 g / l.

[0200] As is known to those skilled in the art, M17 medium is considered a medium suitable for the growth of Streptococcus thermophilus. In addition, as understood by those skilled in the art, in the context of the present invention, M17 concentrate can be supplied by different suppliers, and for the cells of interest relevant herein, one will obtain the same 2-deoxyglucose resistance results relevant herein (within the standard measurement uncertainty) independently of the specific supplier.

[0201] The medium for culturing Lactobacillus delbrueckii subsp. bulgaricus is MRS-IM medium. MRS-IM is used in the form of agar plates or broths.

[0202] The MRS-IM agar medium per liter of H2O has the following composition:

[0203]

[0204] Adjust the pH to 6.9 ± 0.1 at 25 °C after autoclaving.

[0205] The MRS-IM broth used for the liquid cultures in the examples below per liter of H2O has the following composition:

[0206]

[0207] Adjust the pH to 6.9 ± 0.1 at 25 °C after autoclaving. First, the carbon source, namely lactose 20 g / l or glucose 20 g / l, is aseptically filtered and then added to the autoclaved broth.

[0208] The above MRS-IM medium can be modified to a certain extent without affecting the ability of the medium to support the growth of Lactobacillus delbrueckii subsp. bulgaricus. In addition, as will be understood by those skilled in the art, MRS-IM concentrate or the various components described above can be obtained from different suppliers and used to prepare MRS-IM medium. These media will equally be used in the examples below, particularly for the 2-deoxyglucose resistance selection assay.

[0209] Mother strain

[0210] Streptococcus thermophilus CHCC11976 (a galactose-fermenting strain with a mutation in the GalK gene as described in WO2011 / 026863).

[0211] Streptococcus thermophilus CHCC12339 (phage resistance and texturization as described in WO2011 / 092300).

[0212] Streptococcus thermophilus CHCC18948 (a galactose-fermenting mutant of CGCC12339 with a mutation in the GalK gene)

[0213] 2-Deoxy-glucose resistant strain

[0214] Streptococcus thermophilus CHCC16165 (a 2-deoxyglucose-resistant mutant of CHCC11976).

[0215] Streptococcus thermophilus CHCC16731 (a sweetening and texturizing mutant of CHCC16165).

[0216] Streptococcus thermophilus CHCC19216 (a sweetening and texturizing mutant of CHCC18948).

[0217] Example 1: Isolation of glucokinase mutants of Streptococcus thermophilus CHCC11976 with enhanced glucose secretion using 2-deoxyglucose.

[0218] To isolate mutants of the Streptococcus thermophilus strain CHCC11976, cells derived from single colony growth were inoculated into 10 ml of M17 broth containing 2% lactose and grown overnight at 40 °C.

[0219] The next day, the strain was serially diluted and plated on M17 agar plates containing 2% galactose and 2-deoxyglucose at a concentration of 20 mM (CHCC11976) and incubated at 40 °C for 20 hours. The resistant colonies were first re-streaked on agar plates of the same type as when they were selected. The survivors were used to inoculate fresh M17 broth containing 2% lactose, 2% galactose, or 2% glucose, and growth was measured.

[0220] Thereby, as outlined in Example 2, many mutants that were able to grow faster on galactose than on glucose were identified. One such mutant is CHCC16165.

[0221] Example 2: Growth pattern of 2-deoxyglucose resistant mutants

[0222] To ensure the selection of 2-deoxyglucose-resistant mutants that can grow on galactose, two strains selected from a collection of galactose-fermenting strains were used. Although these galactose-fermenting strains still grew at least 10% faster in the exponential phase in M17 broth + 2% glucose than in M17 broth + 2% galactose, on the other hand, the 2-deoxyglucose-resistant mutant derivative of CHCC11976, namely CHCC16165, was characterized by growing faster in the exponential phase in M17 culture medium + 2% galactose than in M17 culture medium + 2% glucose.

[0223] Growth in the exponential phase was measured herein as the development of the optical density over time at 600 nanometers (OD 600 ) of a culture growing exponentially at 40 °C.

[0224] As is known to those skilled in the art, when a culture is in exponential growth, it can vary with the species. Those skilled in the art will know how to determine growth in the exponential phase, for example, between OD 600 0.1 - 1.0.

[0225] The optical density (OD) of the culture was measured in a spectrophotometer.

[0226] Conclusion

[0227] Based on the 2-deoxyglucose-resistant mutant growth pattern of this Example 2 - for a particular strain of interest (e.g., a strain from a related commercial product) - those skilled in the art can routinely test whether this particular strain of interest has the relevant growth pattern herein for the selected mutant characteristics.

[0228] Example 3: Selection of high-lactose-fermenting and glucose-secreting mutants of Streptococcus thermophilus CHCC16165

[0229] To isolate high-lactose-fermenting and glucose-secreting mutants of the Streptococcus thermophilus strain CHCC16165, cells derived from the growth of a single colony were inoculated into 10 ml of M17 broth containing 2% galactose and grown overnight at 40 °C.

[0230] The next day, the strain was serially diluted and plated onto M17 agar plates containing 2% sucrose and 2-deoxyglucose at a concentration of 40 mM and incubated at 40 °C for 20 hours. Ten random colonies were picked from the plates and used to inoculate fresh M17 broth containing 2% sucrose and incubated overnight at 40 °C. The CHCC16165 mutants were transferred to fresh M17 medium containing 2% sucrose.

[0231] Example 4. Carbohydrate analysis of fermented milk.

[0232] The mutants obtained in Example 3 and the strain CHCC16165 were grown in 9.6% B - milk containing 0.01% sucrose. After acidification, the lactose concentration in the milk acidified with CHCC16165 was 14.9, the galactose concentration was 8.4, and the glucose concentration was 5.7. In contrast, for the milk acidified with the best - performing mutant of CHCC16165, the lactose concentration was 9.3, the galactose concentration was 10.5, and the glucose concentration was 9.9. The best mutant of CHCC16165 was named CHCC16731 and was further tested in milk fermentation when used in combination with Lactobacillus delbrueckii subsp. bulgaricus strains CHCC16159 and CHCC16160 (described in WO2013 / 160413).

[0233] Overnight cultures of CHCC16165 and CHCC16731 were combined with each of CHCC16159 and CHCC16160 and added to 200 ml B - milk samples and acidified at 40°C.

[0234] The results are presented in Table 1:

[0235]

[0236] From the results in Table 1, it can be seen that CHCC16731 was able to remove almost all of the lactose from the milk. In addition, CHCC16731 produced and secreted high levels of galactose and glucose into the milk. Considering that sucrose is 100 as a reference, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the sweetness of the fermented dairy product produced with CHCC16731 increased significantly.

[0237] Example 5. Generation of galactose - positive mutants of Streptococcus thermophilus CHCC12339

[0238] Streptococcus thermophilus CHCC12339 was grown overnight at 40 °C in M17 containing 2% lactose. The overnight culture was plated (100 μl) on M17 plates containing 2% galactose and incubated at 40 °C under anaerobic conditions. Twenty-two colonies appeared on the M17 plates containing 2% galactose. Sixteen of these colonies were re-streaked on plates of the same type and eight of the colonies were transferred to liquid M17 containing 2% galactose, all of which resulted in growth and four of these cultures were frozen. Two of the frozen cultures were plated on M17 plates containing 2% galactose and 40 mM 2-deoxyglucose and for each culture, eight of the resulting colonies were selected. The 16 2-deoxyglucose mutants were transferred to liquid M17 containing 2% lactose and 2% galactose and grown overnight at 40 °C and then the mutants were grown for three nights in liquid M17 containing 2% galactose, the production of galactose and glucose was measured and their growth was followed. Based on the production of galactose and glucose, seven cultures were selected for lactic acidification tests by adding 2 ml of the culture to 200 ml of 9.5% B-lact containing 0.05% sucrose. Three mutants had acidification characteristics similar to the parental strain CHCC12339 and the three mutants were streaked to single colonies and incubated for 48 hours. Subsequently, the colonies were transferred to liquid M17 containing 2% galactose and the growth of the three mutants was tested relative to CH12339. The fastest growing mutant was selected and purified again by streaking three times to single colonies to produce a purified galactose-positive strain named CHCC18948 (galactose-positive variant of CHCC12339).

[0239] Example 6. Generation of 2-deoxyglucose mutants of Streptococcus thermophilus CHCC18948 with enhanced glucose secretion

[0240] 100 μl of the CHCC18949 culture was spread on M17 plates containing 2% galactose and 20 mM or 40 mM 2 - deoxyglucose and incubated overnight at 40°C. Eight colonies were selected from the 20 mM plate and eight colonies were selected from the 40 mM plate and grown in liquid M17 containing 2% galactose and 20 mM 2 - deoxyglucose. The resulting cultures were frozen and re - streaked, and 12 of the resulting colonies were started in M17 medium and used to acidify β - milk. A 1% overnight culture of the 2 - deoxyglucose mutants was inoculated into 200 ml of 9.5% β - milk containing 0.05% sucrose and acidified at 40°C. All 12 2 - deoxyglucose mutants of CHCC18948 produced fermented dairy products with low lactose concentration and high galactose and glucose concentrations. Seven mutants were selected for individual retesting and tested together with Lactobacillus delbrueckii subsp. bulgaricus strain CHCC16159 (described in WO2013 / 160413). Again, a 1% overnight culture of the 2 - deoxyglucose mutants was inoculated into 200 ml of 9.5% β - milk containing 0.05% sucrose and acidified at 40°C.

[0241] The results are shown in Table 2:

[0242]

[0243] The three best mutants were mutants 2, 3, and 12 and were retested individually and in combination with Lactobacillus delbrueckii subsp. bulgaricus strains CHCC16159, CHCC16160, and CHCC16161 (described in WO2013 / 160413) in β - milk containing 0.05% sucrose.

[0244] The results are shown in Table 3:

[0245]

[0246] Based on the above data, CHCH18948 mutant 3 was selected as the best mutant and named CHCC19216. As can be seen from the above data, CHCC19216 can remove almost all lactose from milk. In addition, CHCC19216 produces and secretes high levels of galactose and glucose into milk. Considering that sucrose is 100 as a reference, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the sweetness of the fermented dairy product produced with CHCC19216 is significantly increased.

[0247] Example 7. Texturization properties of CHCC16731 and CHCC19216

[0248] The texturization properties of strains CHCC16731 and CHCC19216 were tested by measuring shear stress using the following determinations:

[0249] After 7 days of incubation, the fermented milk was brought to 13 °C and gently stirred with a rod equipped with a perforated disc until the sample was homogeneous. The rheological properties of the samples were evaluated on a rheometer (Anton Paar Physica ASC / DSR301 rheometer (autosampler), Anton GmbH, Austria) using the following settings:

[0250] Waiting time (to re-establish an initial structure)

[0251] 5 minutes without oscillation or rotation

[0252] Oscillation (to measure G' and G”, for calculating G*)

[0253] y = 0.3%, frequency (f) = [0.5…8] Hz

[0254] 6 measurement points within 60 s (one every 10 s)

[0255] Rotation (to measure shear stress at 300 1 / s etc.)

[0256] and

[0257] 21 measurement points within 210 s (one every 10 s), rising to 300 1 / s,

[0258] and

[0259] 21 measurement points within 210 s (one every 10 s), falling to 0.2707 1 / s

[0260] For further analysis, the shear stress at 300 1 / s was selected.

[0261] Using strains CHCC16731 and CHCC19216 in the mixtures listed in Table 4, a 0.024% inoculum was used to acidify 200 ml of 9.5% B milk sample until the pH reached 4.55.

[0262] The results are shown in Table 4:

[0263]

[0264]

[0265] Lb: Lactobacillus delbrueckii subsp. bulgaricus strain CHCC16159

[0266] St: Streptococcus thermophilus

[0267] As can be seen from the results, strains CHCC16731 and CHCC19216 produce fermented dairy products with shear stresses of 41.3 Pa and 51.5 Pa respectively when used as the sole Streptococcus thermophilus in the culture mixture. When used in combination with strains CHCC16731 and CHCC19216, a fermented dairy product with a shear stress of 43.3 Pa is produced.

[0268] This level of shear stress is high compared to traditional sweetening strains disclosed in, for example, WO2013 / 160413.

[0269] Example 8. Flavor and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) in the culture composition for the culture of the present invention compared to a culture with lactose-positive, glucose-positive Lb

[0270] Experimental plan

[0271] Table 5: Composition of the cultures tested

[0272]

[0273] The culture of the present invention consists of three glucose-deficient Streptococcus thermophilus (St) and one lactose-deficient Lb. Culture 2 (comparative culture) consists of three glucose-deficient St and one lactose-positive, glucose-positive (traditional) Lb.

[0274] Prepare two cultures with the composition shown in Table 5 and use them as starter cultures in fermentation to produce yogurt at 43 °C until the target pH of 4.55 is reached. Compare the flavor, Lb growth, post-acidification, carbohydrates, and rheology of the two cultures.

[0275] Milk matrix

[0276] The milk matrix contains 4.5% protein, 1.0% fat, and 0.1% sucrose.

[0277] Mix 0.5% fat milk and 1.5% fat milk so that the fat content in the final milk matrix is 1%, and add 0.1% sucrose. Adjust the protein level to 4.5% using skim milk powder (SMP). Hydration is carried out at 6 °C for 2 hours. Thereafter, the milk is homogenized at 65 °C at 200 / 50 bar and pasteurized at 95 °C for 5 minutes.

[0278] Yogurt production parameters

[0279] Measure the pH during fermentation online using Intap. When the yogurt reaches pH 4.55, break the gel using a rod with a drilled disc. Then process the gel in a post-treatment unit (PTU) at 2 bar and 25 °C, fill it into 120 mL yogurt cups, and store at 6 °C for 28 days.

[0280] Measurement of shear stress

[0281] Use the method described in Example 7, except that the sample is not stirred with a rod equipped with a drilled rod because the gel has broken during PTU treatment to obtain sample uniformity.

[0282] Measurement of colony-forming units

[0283] M17 is used as a selective medium for St. MRSlac (MRS + 8% lactose) is used as a selective medium for Lb. Make a 10× dilution series from the yogurt. Plate each dilution separately on M17 and MRSlac agar plates and incubate anaerobically at 37 °C for 2 days. Thereafter, count the colony-forming units.

[0284] Acidification

[0285] Culture 1 took 10.8 hours to reach a pH of 4.55. Culture 2 took 12.1 hours to reach a pH of 4.55.

[0286] Post-acidification

[0287] Table 6: Post-acidification

[0288] pH Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Culture 1 4.55 4.53 4.51 4.54 4.50 4.54 Culture 2 4.55 4.51 4.50 4.51 4.50 4.54

[0289] Growth of Lb

[0290] Table 7: Culture growth at the end of fermentation (colony-forming units (CFU))

[0291] Lb (CFU) St (CFU) Culture 1 3E+07 9E+08 Culture 2 9E+05 8E+08

[0292] As can be seen from Table 7, the Lb cell count of the culture of the present invention is much higher than that of the comparative culture of traditional Lb, i.e., lactose-positive, glucose-positive Lb. Therefore, the combination of St with a grape defect and Lb with a lactose defect produces a higher level of Lb at the end of fermentation.

[0293] Rheology

[0294] The shear stress of Culture 1 at 300 1 / s at the end of fermentation was 41.1 Pa.

[0295] The shear stress of Culture 2 at 300 1 / s at the end of fermentation was 27.3 Pa.

[0296] Carbohydrates

[0297] Table 8: Carbohydrate levels at the end of fermentation

[0298]

[0299] Flavor

[0300] The flavor was evaluated by a tasting panel. Culture 1 had no off - flavors.

[0301] Example 9. Flavor and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) in the culture composition for the cultures of the present invention compared to cultures with glucose - defective Lb

[0302] Experimental plan

[0303] Table 9: Composition of the cultures tested

[0304]

[0305] The cultures of the present invention consist of three glucose - defective Sts and one lactose - defective Lb. Culture 2 (comparative culture) consists of three glucose - defective Sts and one lactose - positive, glucose - defective Lb.

[0306] Two cultures with the composition shown in Table 9 were prepared and used as starter cultures in fermentation to produce yogurt at 43 °C until the target pH of 4.55 was reached. The flavors (amino acid levels), volatile organic components, carbohydrates, and rheology of the two cultures were compared.

[0307] Milk matrix

[0308] Two milk matrices were adjusted using skim milk powder (SMP) or whey protein (WP) and contained 4.5% protein, 1% fat, and 0.1% sucrose.

[0309] 0.5% fat milk and 1.5% fat milk were mixed to give a final fat content of 1% in the milk matrix, and 0.1% sucrose was added. The protein level was adjusted to 4.5% using SMP or WP. Hydration was carried out at 6 °C for 2 hours. Thereafter, the milk was homogenized at 65 °C at 200 / 50 bar and pasteurized at 95 °C for 5 minutes.

[0310] Yogurt production parameters

[0311] The pH during fermentation was measured online using CINAC in a baby bottle. When the yogurt reached pH 4.55, the gel was broken and homogenized using a rod with a drilled disc. Thereafter, the yogurt was stored in the baby bottle.

[0312] Measurement of shear stress

[0313] Use the method described in Example 7.

[0314] Rheology

[0315] The shear stress of Culture 1 in the SMP milk matrix at the end of fermentation was 46.2 Pa at 300 l / s.

[0316] The shear stress of Culture 1 in the WP milk matrix at the end of fermentation was 39.6 Pa at 300 l / s.

[0317] The shear stress of Culture 2 in the SMP milk matrix at the end of fermentation was 42.0 Pa at 300 l / s.

[0318] The shear stress of Culture 2 in the WP milk matrix at the end of fermentation was 38.4 Pa at 300 l / s.

[0319] It can be seen from the results that Culture 1 had a higher shear stress compared to Culture 2.

[0320] Post - acidification

[0321] Table 10: Post - acidification

[0322]

[0323] Growth of Lb

[0324] Table 11: Culture growth at the end of fermentation (colony - forming units (CFU))

[0325]

[0326] It can be seen from Table 11 that the Lb cell count of the cultures of the present invention was lower than that of the comparative cultures with lactose - positive, glucose - defective Lb.

[0327] Carbohydrates

[0328] Table 12: Carbohydrate levels at the end of fermentation

[0329] Glucose (mg / g) Galactose (mg / g) Lactose (mg / g) Culture 1 in SMP milk matrix 14.4 13.2 15.4 Culture 1 in WP milk matrix 12.0 10.2 9.2 Culture 2 in SMP milk matrix 16.4 16.3 11.7 Culture 2 in WP milk matrix 14.6 14.1 6.1

[0330] Amino acids

[0331] Table 13: Amino acid levels

[0332]

[0333] The sample containing glucose - negative Lb (Culture 2) had a higher amount of TFFA, which indicates higher proteolytic activity and, in turn, can lead to an unpleasant taste.

[0334] In addition, yogurt containing glucose-negative Lb contains a higher amount of glutamic acid, and its production is known to produce an off-flavor described as "broth".

[0335] Valine is a bitter amino acid and is also present in a relatively high amount in yogurt containing glucose-negative Lb.

[0336] Volatile organic compounds (VOCs)

[0337] VOC curves can indicate the flavor characteristics of a sample.

[0338] Fifty-one different volatile organic compounds were measured. For culture 1 in the WP milk matrix, compared with culture 2 in the WP milk matrix, 44 (86%) of the 51 compounds had lower amounts of volatile organic compounds. For culture 1 in the WP milk matrix, compared with culture 2 in the WP milk matrix, 34 (67%) of the 51 compounds had lower amounts of volatile organic compounds. In summary, the content of volatile organic compounds (observed as a group of compounds) in the cultures of the present invention was significantly lower compared to the comparative cultures.

[0339] Example 10. Growth and post-acidification of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for the culture composition of the present invention.

[0340] Experimental plan

[0341] Table 14: Composition of the cultures tested

[0342]

[0343] Cultures with the composition shown in Table 14 were prepared and used as starter cultures in fermentation to produce yogurt at 43 °C until the target pH of 4.55 was reached. The cultures were inoculated at levels of 0.01%, 0.02% and 0.03%. The resulting yogurt was stored at 5 °C, 13 °C and 25 °C. The growth of St and Lb and post-acidification of the cultures were tested.

[0344] Milk matrix

[0345] Table 15: Composition of milk matrices 1 and 2

[0346] Amount Protein Carbohydrate Fat Skim milk 884.2g 1.5% milk 1767.1g Promilk 802FB 10.9g SMP 73.9g Sucrose 2.8g Water 261.0g Milk matrix 3000g 4.57％ 5.61％ 0.92％

[0347] Table 16: Composition of milk matrix 3

[0348] Amount Protein Carbohydrate Fat Skim milk 884.2g 1.5% milk 1767.1g Promilk 802FB 10.9g SMP 73.9g Sucrose 6.0g Water 257.8g Milk matrix 3000g 4.57％ 5.71％ 0.92％

[0349] Post-acidification

[0350] Table 17: Post-acidification

[0351]

[0352]

[0353] As can be seen from Table 17, for all inoculation levels and storage temperatures tested, the level of post-acidification was very low, including the higher storage temperatures of 13 °C and 25 °C, where the growth level of Lb was high for most traditional starter cultures and thus the post-acidification was high.

[0354] Growth of Lb and St

[0355] Table 18: Growth of Lb and St

[0356] Milk matrix 1 1 1 2 2 2 Inoculation 0.01％ 0.02％ 0.03％ 0.01％ 0.02％ 0.03％ Lb CFU / g 5.8E05 6.5E05 7.2E05 3.7E05 4.7E05 8.8E05 St CFU / g 4.2E07 6.5E07 7.4E07 3.8E07 5.6E07 7.0E07

[0357] As shown in Table 18, for all inoculation levels, the Lb cell count was at a high level at the end of fermentation.

[0358] Example 11. Growth, post-acidification and carbohydrate analysis of Lactobacillus delbrueckii subsp. bulgaricus (Lb) in the culture composition for use in the present invention compared to traditional starter cultures

[0359] Experimental plan

[0360] Table 19: Composition of the cultures tested

[0361]

[0362]

[0363] Cultures with the composition shown in Table 19 were prepared and used as starter cultures in fermentation to produce yogurt at 43 °C until the target pH of 4.55 was reached. The cultures were inoculated at a level of 0.02%. The yogurt produced was stored at 5 °C and 13 °C. The cultures were tested for St and Lb growth, post-acidification and carbohydrate analysis.

[0364] YoFlex Premium 1.0 and Yoflex YF-L901 are commercial traditional cultures containing lactose-positive and glucose-positive St and Lb.

[0365] Milk matrix

[0366] Table 20: Composition of milk matrix 1

[0367] Amount Protein Carbohydrate Fat 1.5% milk 3000g SMP 85g Milk matrix 3085g 4.20％ 6.06％ 1.49％

[0368] Post-acidification

[0369] Table 21: Post-acidification

[0370] Sample 1 2 3 4 5 6 Day 1 at 6°C 4.49 4.48 4.54 4.54 4.53 4.54 Day 7 at 6°C 4.45 4.43 4.52 4.52 4.52 4.53 Day 21 at 6°C 4.44 4.37 4.47 4.47 4.46 4.46

[0371] As shown in Table 21, the cultures of the present invention have a low level of post-acidification, at the same level as traditional starter cultures.

[0372] Growth of Lb and St

[0373] Table 22: Growth of Lb and St

[0374]

[0375]

[0376] As shown in Table 22, the Lb cell count of the cultures of the present invention is higher than that of traditional cultures at the end of fermentation.

[0377] Carbohydrate analysis

[0378] Table 23: Carbohydrate analysis

[0379]

[0380] LOD: Limit of detection

[0381] All determinations of carbohydrates were performed in duplicate, and Table 23 lists the average of the two determinations. The determinations were made after 7 days of yogurt storage.

[0382] As can be seen from Table 23, the cultures of the present invention produce a high level of glucose, and after 7 days of storage, glucose is present at a level of approximately 8 mg / g.

[0383] Deposits and expert solutions

[0384] The applicant requests that the deposited microbial samples described below be available only to experts before the date of patent grant.

[0385] The strain Streptococcus thermophilus CHCC11342 was deposited on September 8, 2009, at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, under the deposit number DSM 22932.

[0386] The strain Streptococcus thermophilus CHCC12339 was deposited on October 14, 2010, at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, under the deposit number DSM 24090.

[0387] The strain Streptococcus thermophilus CHCC11976 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on September 8, 2009, with the deposit number DSM 22934.

[0388] The strain Streptococcus thermophilus CHCC16731 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on June 4, 2014, with the deposit number DSM 28889.

[0389] The strain Streptococcus thermophilus CHCC19216 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on December 8, 2015, with the deposit number DSM 32227.

[0390] The strain Streptococcus thermophilus CHCC15757 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 3, 2012, with the deposit number DSM 25850.

[0391] The strain Streptococcus thermophilus CHCC15887 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 3, 2012, with the deposit number DSM 25851.

[0392] The strain Streptococcus thermophilus CHCC16404 was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany on December 12, 2012, with the deposit number DSM 26722.

[0393] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC18944 was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on June 12, 2014, with the deposit number DSM 28910.

[0394] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on April 3, 2007, with the deposit number DSM 19252.

[0395] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on September 6, 2012, with the deposit number DSM 26420.

[0396] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D - 38124 Braunschweig, on September 6, 2012, under the accession number DSM 26421.

[0397] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC12813 was deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D - 38124 Braunschweig, on September 29, 2010, under the accession number DSM 24074.

[0398] These deposits were made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0399] References

[0400] WO2011 / 026863

[0401] WO2011 / 092300

[0402] WO2013 / 160413

[0403] Pool et al. (2006) Metabolic Engineering 8(5); 456 - 464

[0404] Thompson et al. (1985) J. Bacteriol. 162(1); 217–223

[0405] Chervaux et al. (2000). Appl and Environ Microbiol, 66, 5306 - 5311

[0406] Cochu et al. (2003). Appl and Environ Microbiol, 69(9), 5423 - 5432

[0407] et al. (2010) in The Technology of Cheesemaking, 2 nd Ed. Black - well Publishing, Oxford; 166 - 192.

Claims

1. A composition for producing fermented dairy products, comprising (i) at least one Streptococcus thermophilus (St) strain, wherein the St strain ferments galactose, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on the expression of the gene, and wherein the mutation reduces the activity of the glucokinase protein by at least 50%, and (ii) at least one Lactobacillus delbrueckii subsp. bulgaricus (Lb) strain, wherein the Lb strain is lactose-deficient and capable of metabolizing glucose, wherein the lactose-deficiency is determined by the ability of the Lb strain to form white colonies on a medium containing lactose and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal).

2. The composition according to claim 1, wherein the St strain is resistant to 2-deoxyglucose.

3. The composition according to claim 1 or 2, wherein the St strain carries a mutation that reduces the transport of glucose into the cell.

4. The composition according to claim 1 or 2, wherein when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B-milk and grown at 40°C for 20 hours, the St strain increases the amount of glucose in the 9.5% B-milk to at least 5 mg / ml.

5. The composition according to claim 1 or 2, wherein when inoculated at a concentration of 10 6 -10 7 CFU / ml into 9.5% B-lactose containing 0.05% sucrose and grown at 40 °C for 20 hours, the St strain increases the amount of glucose in 9.5% B-lactose containing 0.05% sucrose to at least 5 mg / ml.

6. The composition according to claim 1 or 2, wherein the Lb strain is selected from: the strain deposited at the DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, under the accession number DSM 28910 on June 12, 2014, and functionally equivalent mutant strains derived from DSM 28910, wherein less than 1% of the nucleotides in the bacterial genome have been replaced or deleted with another nucleotide as compared to the parental strain.

7. The composition according to claim 1 or 2, wherein the St strain is selected from: Streptococcus thermophilus strain CHCC19216 deposited at the German Collection of Microorganisms with the deposit number DSM 32227; Streptococcus thermophilus strain CHCC16731 which has been deposited at the German Collection of Microorganisms (DSMZ), Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, with the deposit number DSM28889 on June 4, 2014; Streptococcus thermophilus strain CHCC15757 deposited at the German Collection of Microorganisms with the deposit number DSM 25850; Streptococcus thermophilus strain CHCC15887 deposited at the German Collection of Microorganisms with the deposit number DSM 25851; Streptococcus thermophilus strain CHCC16404 deposited at the German Collection of Microorganisms with the deposit number DSM 26722; and mutant strains derived therefrom, wherein, The mutant strain is a functionally equivalent variant of the deposited strain, and wherein less than 1% of the nucleotides in the bacterial genome have been replaced or deleted with another nucleotide as compared to the parental strain.

8. A method for producing fermented dairy products, comprising inoculating and fermenting a milk matrix with the composition according to any one of claims 1 - 7.

9. A fermented dairy product, comprising the composition according to any one of claims 1 - 7.

10. Use of the composition according to any one of claims 1 - 7 for the preparation of a fermented dairy product.

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