BR112019018349B1 - BACTERIAL LACTIC ACID COMPOSITION FOR THE PREPARATION OF FERMENTED FOOD PRODUCTS WITH HIGH NATURAL SWEETNESS AND FLAVOR - Google Patents

Abstract

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

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a composition for producing a fermented dairy product comprising at least one strain of Streptococcus thermophilus (St), wherein the St strain is from galactose fermentation, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on expression of the gene, and (ii) at least one strain of Lactobacillus delbrueckii subsp. bulgaricus (Lb). Such a composition is used for the production of fermented milk products with a high sweetness.

BACKGROUND OF THE INVENTION

[0002] Pure fermented dairy products are recognized by an acidic or sour taste as a result of the conversion of lactose to lactic acid by lactic acid bacteria during fermentation. They are therefore often sweetened by adding fruit, honey, sugar or artificial sweeteners to accommodate customers' desire for a sweeter tasting product.

[0003] The food industry has an increasingly high demand for low-calorie, sweet-tasting food products in order to help overcome the overweight and obesity problems that have become so prevalent over the past 20 years. Sweetness, generally thought of as a pleasant sensation, is produced by the presence of sugars and certain other substances. The perception of sugars is very different. Using sucrose as a 100 reference, the sweetness of lactose is 16, galactose 32 and glucose 74 (Godshall (1988). Food Technology 42(11):71-78). Glucose is thus perceived to be more than 4 times sweeter than lactose, while still having approximately the same level of calories.

[0004] Sugar in fermented food products is more often being replaced by sweeteners such as aspartame, acesulfame K, sucralose and saccharin which can provide sweetness with a lower calorie intake. However, the use of artificial sweeteners can result in an unpleasant taste and several studies indicating that the consumption of artificial sweeteners is connected with inconveniences, such as increased hunger, allergies, cancer, etc., have contributed to consumer preference for fermented milk products that contain only natural sweeteners or, preferably, no added sweeteners.

[0005] Thus, a special challenge remains in the development of fermented dairy products where the natural (internal) sweetness is high.

[0006] The acidity of fermented dairy products largely depends on the lactic acid bacteria present and the process parameters used for preparing the fermented dairy product.

[0007] The fermentation of the disaccharide lactose is very well studied in bacteria because lactic acid is the main source of carbon in milk. In many species, lactose is cleaved by β-galactosidase into glucose and galactose after absorption. Glucose is phosphorylated by glucokinase to glucose-6-phosphate and fermented via the Embden-Meyerhof-Parnas pathway (glycolysis) by most lactic acid bacteria (Figure 1).

[0008] Streptococcus thermophilus is one of the most widely used lactic acid bacteria for commercial thermophilic milk fermentation where the organism is normally used as part of a mixed starter culture, the other component being a Lactobacillus sp. For example. Lactobacillus delbrueckii subsp. bulgaricus for yogurt or Lactobacillus helveticus for Swiss cheese.

[0009] The legal definition of yogurt in many countries requires Streptococcus thermophilus alongside Lactobacillus delbrueckii subsp. bulgaricus. Both species generate desirable amounts of acetaldehyde, an important flavoring component in yogurt.

[0010] Lactose and sucrose are more easily fermented by Streptococcus thermophilus than their component monosaccharides. In the presence of excess galactose only the glucose part of the lactose molecule is fermented and galactose accumulates in fermented milk products when using Streptococcus thermophilus. In yoghurt where high acid concentrations limit fermentation, free galactose remains while free galactose produced in the early stages of Swiss cheese making is later fermented by Lactobacillus helveticus.

[0011] However, galactose fermentation strains of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus have been reported by several researchers (Hutkins et al. (1986) J. Dairy Sci. 69(1):1-8; Vaillancourt et al. (2002) J. Bacteriol. 184(3); 785-793) and WO 2011/026863 (Chr. Hansen) describes a method for obtaining strains of Streptococcus thermophilus which are from galactose fermentation.

[0012] In order to satisfy the requirements of the food industry, it has become relevant to propose new strains, in particular strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus, which provide a more natural sweetness without extra calories directly in the fermented product (internal sweetness) through glucose excretion.

[0013] Pool et al. (2006. Metabolic Engineering 8(5); 456-464) describes the strain of Lactococcus lactis in which glucose metabolism is completely disrupted by deletion of genes encoding glucokinase, EII(man/glc) and glucose-PTS EII (cel) recently discovered. The method of construction is genetic recombination to generate all mutations and the resulting strain is consequently a genetically modified organism (GMO) which at present cannot be used in food products.

[0014] Thompson et al. (1985. J Bacteriol. 162(1); 217-223) studied the metabolism of lactose in Streptococcus lactis (now called Lactococcus lactis). In this work 2-deoxyglucose was used to obtain a mutant in the mannose-PTS system. Subsequently, this mutant was subjected to mutagenesis using UV mutagenesis followed by screening for glucose negative colonies by replica plating. In this way, a double mutant (PTS mannose and glucokinase) was isolated. This double mutant was used to study the mechanisms involved in the regulation of lactose fermentation by "initiator" organisms. These mutants have several disadvantages compared to their parent strain that make them unsuitable for inclusion in a commercial starter culture. The cell yield of the mutants was half that of the parent strain per mole of fermented lactose and the doubling time was significantly increased in the mutants when grown on lactose. Likewise, the yield of lactic acid was half that of the original strain per mole of fermented lactose. The behavior of these strains in milk has not been analysed, but it is anticipated that the acidification rate would be significantly reduced.

[0015] Furthermore, Lactococcus lactis is not generally selected for the production of acetaldehyde and does not contribute to meeting the requirements for the legal definition of yogurt.

[0016] Chervaux et al. (2000. Appl. And Environ. Microbiol., 66, 5306-5311 ) studied the physiology of strains of Lactobacillus delbrueckii subsp. bulgaricus in a new chemically defined medium and isolated 2-deoxyglucose resistant mutants that were defective in glucose fermentation. Several different phenotypes have been observed and strain-specific effects have been reported.

[0017] WO2013/160413 describes strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus with improved properties with regard to the natural sweetening of food products and to decrease the lactose content of fermented milk, and method of screening and isolation of such strains.

[0018] WO2015/193449 describes a method of producing a fermented dairy product with a very low concentration of lactose using a combination of lactase and lactic acid bacteria with a deficiency in glucose metabolism.

[0019] WO2015/193459 describes a method of producing a fermented dairy product with a reduced level of post-acidification, in which one embodiment uses a mixture of four lactic acid bacterial strains with impaired glucose metabolism, and in which another embodiment uses a mixture of lactose-deficient lactic acid bacterial strains.

[0020] The object of the present invention is to provide a composition for producing a fermented milk product comprising at least one strain of glucose-deficient Streptococcus thermophilus and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus, where the composition produces a fermented milk product with an improved taste.

SUMMARY OF THE INVENTION

[0021] This object is achieved with the present invention, which is directed to a composition for the production of a fermented dairy product comprising (i) at least one strain of Streptococcus thermophilus (St), wherein the St strain is of galactose fermentation, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on expression of the gene, and (ii) at least a strain of Lactobacillus delbrueckii subsp. bulgaricus (Lb), in which the Lb strain is lactose deficient and capable of metabolizing a non-lactose carbohydrate.

[0022] It is well known that for conventional starter cultures for the production of fermented milk products comprising a strain of Streptococcus thermophilus and a strain of Lactobacillus delbrueckii subsp. bulgaricus, there is a risk of formation of unwanted off-flavours, such as free amino acids, e.g. glutamic acid. It is still well known that unpleasant flavors are formed by the strain of Lactobacillus delbrueckii subsp. bulgaricus, and that the level of unpleasant flavors formed depends on the growth level of said strain, which varies depending on several factors, such as the level of inoculation of the Lactobacillus delbrueckii subsp. bulgaricus, the type of growth medium and fermentation growth conditions, as well as the strain of Streptococcus thermophilus and the strain of Lactobacillus delbrueckii subsp. bulgaricus used in the starter culture. It has now been found that for starter cultures for the production of a fermented milk product with enhanced sweetness comprising at least one strain of glucose-deficient Streptococcus thermophilus and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus, formation of off-flavors is at a particularly high level. This is consistent with the fact that in such starter cultures the Lactobacillus delbrueckii subsp. bulgaricus grows at a particularly high cell count.

[0023] It has surprisingly been observed that the problem of off-tastes using starter cultures with a glucose-deficient strain of Streptococcus thermophilus can be solved by using a strain of Lactobacillus delbrueckii subsp. bulgaricus deficient in lactose, for example, a strain of Lactobacillus delbrueckii subsp. Bulgaricus glucose-positive lactose deficiency. Likewise, the strain of Lactobacillus delbrueckii subsp. bulgaricus deficient in the lactose used in the composition of the invention does not result in any post-acidification. Finally, it was observed that Lactobacillus delbrueckii subsp. bulgaricus lactose-deficient in combination with the glucose-deficient strains of Streptococcus thermophilus produces a very high cell count compared to the strains of Lactobacillus delbrueckii subsp. conventional bulgaricus, i.e. lactose positive, glucose positive.

[0024] It is very surprising that it is possible to prevent the formation of unpleasant flavors, for example, a strain of Lactobacillus delbrueckii subsp. blgaricus deficient in glucose-positive lactose, and to avoid post-acidification in view of the fact that the strain of Lactobacillus delbrueckii subsp. bulgaricus is glucose positive and is present in a fermented dairy product containing glucose, where in theory the strain would be able to continue growth during storage of the fermented dairy product.

[0025] What's more, it is even more surprising that it is possible to prevent the formation of unpleasant tastes from the strain of Lactobacillus delbrueckii subsp. bulgaricus and avoid post-acidification while at the same time achieving high cell counts from the Lactobacillus delbrueckii subsp. bulgaricus, as it was generally expected that both off-flavors and post-acidification would result from the growth of the Lactobacillus delbrueckii subsp. bulgaricus.

[0026] Without being bound by theory, it is believed that the surprising effects of the present invention are due to differences in the metabolism of Lactobacillus delbrueckii subsp. bulgaricus lactose-deficient of the invention compared to Lactobacillus delbrueckii subsp. bulgaricus positive on conventional lactose.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGURE 1 is a schematic representation of lactose catabolism in Streptococcus thermophilus. GlcK, glucokinase; LacS, lactose transporter; LacZ, β-galactosidase; GalM, mutarotase; GalK, galactokinase; GalT, galactose-1-phosphate uridyltransferase; GalE, UDP-glucose 4 epimerase; Gal1P, galactose-1-phosphate.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0028] As used herein, the term "lactic acid bacterium" means a gram-positive, microaerophilic or anaerobic bacterium that ferments sugars with the production of acids including lactic acid as the predominant acid produced, acetic acid and propionic acid. The most industrially useful lactic acid bacteria are found within the order of "Lactobacillales" which includes lactic acid bacteria Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp., including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry as frozen or freeze-dried cultures for bulk starter propagation or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a container or tank of fermentation for the production of a dairy product, such as a fermented milk product. Such cultures are generally referred to as "starter cultures" or "initiators".

[0029] The use of the terms "a" and "an" and "the" and "the" and similar referents in the context of describing the invention (especially in the context of the claims that follow) shall be interpreted to cover both the singular or plural, unless otherwise indicated in this invention or clearly contradicted by the context. The terms "comprising", "having", "including" and "containing" shall be interpreted as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise noted. The recitation of ranges of values in this invention is merely intended to serve as a shorthand method of individually referring to each separate value that falls within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it were individually recited here. All methods described herein may be performed in any suitable order, unless otherwise indicated in this invention or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to further clarify the invention and does not impose a limitation on the scope of the invention, unless claimed otherwise. No language in the specification should be construed as indicating any element not claimed to be essential to the practice of the invention.

[0030] In connection with the present invention, the expression "increased sweetness" means an increase in sweetness compared to the sweetness produced by a parent strain that does not carry a mutation in the DNA sequence of the glck gene encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on gene expression.

[0031] The expression "CFU" means Colony Forming Units.

Strains of Streptococcus thermophilus from the composition of the invention

[0032] In some countries, the legal definition of yogurt requires the presence of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Both species generate desirable amounts of acetaldehyde, an important flavoring component in yogurt.

[0033] Cheese, such as Mozzarella and Pizza cheese as well as Feta, can also be prepared by fermentation using either Streptococcus thermophilus or Lactobacillus delbrueckii subsp. bulgaricus (H0ier et al. (2010) in The Technology of Cheese making, 2nd Ed. Blackwell Publishing, Oxford; 166-192).

[0034] In order to satisfy the requirements of the food industry, it has become desirable to develop new strains, in particular strains of Lactobacillus delbrueckii subsp. bulgaricus and strains of Streptococcus thermophilus, which produce more natural sweetness directly in the fermented product (internal sweetness) without the contribution of extra calories.

[0035] Streptococcus thermophilus is one of the most widely used lactic acid bacteria for commercial milk fermentation where the organism is normally used as part of a mixed starter culture, the other component being a Lactobacillus sp., e.g. Lactobacillus delbrueckii subsp. . bulgaricus for yogurt and Lactobacillus helveticus for Swiss cheese.

[0036] Only the glucose part of the lactose molecule is fermented by Streptococcus thermophilus and galactose accumulates in fermented milk products when Streptococcus thermophilus is used. In yogurt, where high acid concentrations limit fermentation, free galactose remains while free galactose produced in the early stages of Swiss cheese making is later fermented by Lactobacillus helveticus. Lactococcus lactis found in many starter cultures used for cheese production is also capable of consuming the galactose produced by Streptococcus thermophilus.

[0037] In order to ensure Streptococcus thermophilus strains with as optimal a growth performance as possible, the present inventors have exposed the galactose fermentation strains of Streptococcus thermophilus to the selective agent 2-deoxymglucose. Typically, 2-deoxyglucose resistant mutants have mutations in the gene encoding glucokinase and in 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 a mutation in the glucokinase gene, CHCC16731 and CHCC19216 both have a mutation that means that secreted glucose is not transported back into cells again.

[0038] Surprisingly, such mutants alone are still fully capable of acidifying milk, although the acidification time at pH 5 is delayed by 2 to 5 hours. They are therefore, as such, useful in fermented milk applications and they have preserved the ability of the mother strains to acidify milk which is characteristic of yoghurt. Furthermore, the mutants were found to excrete a high level of glucose when 9.5% milk B with 0.05% sucrose was inoculated with the mutants and fermented at 40°C for at least 20 hours. At the same time, very low levels of lactose remain in fermented milk. Therefore, the use of such strains for the production of fermented milk products may be of importance for people with lactose intolerance.

[0039] Consequently, the final fermented milk has an increased internal sweetness index calculated as described by Godshall (1988. Food Technology 42(11):71-78).

[0040] In one aspect of the present invention, the Streptococcus thermophilus strain is a galactose fermentation mutant strain of Streptococcus thermophilus, wherein the mutant strain carries a mutation in the DNA sequence of the glck gene encoding a 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 expression of the glucokinase gene are readily known (Porter et al. (1982) Biochim. Biophys. Acta, 709;178-186) and include enzyme assays with commercially available kits. and transcriptomics or quantitative PCR using materials that are readily available.

[0041] In a preferred embodiment of the invention, the Streptococcus thermophilus strain of the invention is resistant to 2-deoxyglucose.

[0042] A bacterial "strain" as used herein refers to a bacterium that remains genetically unchanged when cultured or multiplied. A multiplicity of identical bacteria is included.

[0043] The term "galactose fermentation Streptococcus thermophilus strains" as used herein refers to those strains of Streptococcus thermophilus which are capable of growth in M17 medium + 2% galactose. Galactose fermentation Streptococcus thermophilus strains are defined herein as strains of Streptococcus thermophilus which reduce the pH of M17 broth containing 2% galactose as the sole carbohydrate to 5.5 or less when inoculated from an overnight culture in 1% and incubated for 24 hours at 37°C.

[0044] The term "protein glucokinase inactivation mutation" as used herein, refers to a mutation that results in an "protein glucokinase inactivation", a protein glucokinase that, if present in a cell, is unable to exert its normal function as well as mutations that prevent the formation of protein glucokinase or result in degradation of protein glucokinase.

[0045] In particular, an inactivated protein glucokinase is a protein that compared to a functional protein glucokinase is not capable of facilitating the phosphorylation of glucose to glucose-6-phosphate or facilitates the phosphorylation of glucose to glucose-6-phosphate in a significantly reduced rate. The gene encoding such a glucokinase protein inactivated compared to the gene encoding a functional glucokinase protein comprises a mutation in the open reading frame (ORF) of the gene, wherein said mutation may include, but is not limited to, a deletion, a frameshift mutation, introduction of a stop codon, or a mutation that results in an amino acid substitution that alters the functional properties of the protein, or a promoter mutation that reduces or abolishes transcription or translation of the gene.

[0046] In preferred embodiments, the mutation reduces the activity (the rate of phosphorylation of glucose to glucose-6-phosphate) of the protein glucokinase by at least 50%, such as at least 60%, such as at least 70%, such as such as at least 80%, such as at least 90%.

[0047] Glucokinase activity can be determined by enzymatic glucokinase assays as described by Pool et al. (2006. Metabolic Engineering 8;456-464).

[0048] The term "functional protein glucokinase" as used herein, refers to a protein glucokinase which, if present in a cell, facilitates the phosphorylation of glucose to glucoses-6-phosphate. In particular, a functional glucokinase protein can be encoded by a gene comprising an ORF having a sequence corresponding to position 1-966 in SEQ ID NO. 1 or a sequence that has 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, with the sequence corresponding to position 1-966 of SEQ ID NO. 1.

[0049] The percent identity of two sequences can be determined using mathematical algorithms, such as the algorithm of Karlin and Altschul (1990. Proc. Natl. Acad. Sci. USA 87;2264), the modified algorithm described in Karlin and Altschul ( 1993. Proc. Natl. Acad. Sci. USA 90;5873-5877); the Myers and Miller algorithm (1988. CABIOS 4;11-17); the algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48;443-453); and the Pearson and Lipman algorithm (1988. Proc. Natl. Acad. Sci. USA 85;2444-2448). Computer software for determining nucleic acid or amino acid sequence identity based on these mathematical algorithms is also available. For example, nucleotide sequence comparison can be performed with the BLASTN program, score = 100, basic information unit size = 12. Amino acid sequence comparison can be performed with the BLASTX program, score = 50, unit size basic information unit = 3. For remaining parameters of BLAST programs, the default parameters can be used.

[0050] In many countries the use of genetically modified organisms (GMOs) for fermented dairy products is not accepted. The present invention instead provides naturally occurring or induced mutant strains that can provide a desirable accumulation of glucose in the fermented dairy product.

[0051] Thus, in a very preferred embodiment of the present invention, the mutant strain is a naturally occurring mutant or an induced mutant.

[0052] A "mutant bacterium" or a "mutant strain" as used herein refers to a naturally occurring (spontaneous, naturally occurring) mutant bacterium or an induced mutant bacterium comprising one or more mutations in its genome (DNA) that are absent in wild-type DNA. An "induced mutant" is a bacterium in which the mutation has been induced by human treatment, such as treatment with chemical mutagens, UV or gamma radiation, etc. In contrast, a "spontaneous mutant" or "naturally occurring mutant" has not been subjected to human mutagenesis. The mutated bacteria are in this invention non-GMO (non-genetically modified organism), i.e. not modified by recombinant DNA technology.

[0053] "Wild-type strain" refers to the unmutated form of a bacterium as found in nature.

[0054] Terms such as "strains with a sweetening property", "strains that can provide a desirable accumulation of glucose in the fermented dairy product" and "strains with enhanced properties for the natural sweetening of food products" are used interchangeably herein to characterize an advantageous aspect of using the strains of the present invention in the fermentation of dairy products.

[0055] In a preferred embodiment, the mutant strain of Streptococcus thermophilus according to the invention increases the amount of glucose in 9.5% B milk by at least 5 mg/ml when inoculated into 9.5% B milk in a concentration of 106 to 107 CFU/ml and cultured at 40°C for at least 20 hours.

[0056] In another preferred embodiment, the mutant strain of Streptococcus thermophilus according to the invention increases the amount of glucose in milk B to 9.5% with sucrose 0.05% by at least 5 mg/ml when inoculated into milk B to 9.5% with 0.05% sucrose at a concentration of 106 to 107 CFU/ml and cultured at 40°C for at least 20 hours.

[0057] In the present context, 9.5% B milk is boiled milk prepared with low-fat skimmed milk powder reconstituted at a dry matter level of 9.5% and pasteurized at 99°C for 30 minutes , followed by cooling to 40°C.

[0058] In the most preferred embodiments of the invention, the mutant strain leads to an increase in the amount of glucose by at least 6 mg/ml, such as at least 7 mg/ml, such as at least 8 mg/ml, such as at least 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.

[0059] In another embodiment of the invention, the mutant strain of Streptococcus thermophilus is resistant to 2-deoxyglucose.

[0060] The term "2-deoxyglucose resistant" is defined by the fact that a bacterial strain subjected to the particular mutation has the ability to grow into a colony when placed on a plate of M17 medium containing 20 mM 2-deoxyglucose after incubation at 40°C for 20 hours. The presence of 2-deoxyglucose in the culture medium will impede the growth of the non-mutated strains whereas the growth of the mutated strains is unaffected or not significantly affected. Unmutated strains that can be used as sensitive reference strains in assessing resistance preferably include strains CHCC14994 AND CHCC11976.

[0061] The mutant Streptococcus thermophilus strains of the invention excrete glucose in milk when 9.5% B milk is inoculated with 106 to 107 CFU/ml of a Streptococcus thermophilus strain according to the invention and fermented with the strains of Streptococcus thermophilus according to the invention at 40°C for at least 20 hours. Preferably, such mutant strains alone will excrete at least 5 mg/ml of glucose in B-milk when the 9.5% B-milk is inoculated with 106 to 107 CFU/ml of a Streptococcus thermophilus strain according to the invention and fermented with Streptococcus thermophilus strains at 40°C for at least 20 hours. The strains are still fully capable of acidifying milk, although the acidification time at pH 5 is delayed by 2 to 5 hours. The final fermented milk contains less than 15 mg/ml of lactose in fermented milk. The final fermented milk consequently has an internal sweetness index of approximately 2 times or more.

[0062] In yet another embodiment, the mutant strain according to the invention can be characterized by its growth pattern. This is illustrated by the finding that the growth rate of the mutant strain is greater in M17 medium + 2% galactose than in M17 medium + 2% glucose. The growth rate is measured as the development in the optical density of the culture exponentially increasing at 600 nanometers (OD600) with time.

[0063] In a preferred embodiment, the growth rate is at least 5% greater, such as at least 10% greater, such as at least 15% greater, such as at least 20% greater, in M17 medium + galactose at 2 % than in M17 medium + 2% glucose. Mutation in the glcK gene

[0064] In a preferred embodiment, the mutation results in the replacement of the codon encoding glycine with the codon encoding arginine at position 249 in SEQ ID NO. 2. Preferably, the mutation in the glcK gene results in the replacement of a G with an A at position 745 in SEQ ID NO. 1. The CHCC16731 strain has such a mutation.

[0065] In a preferred embodiment, the mutation results in the replacement of the codon encoding serine with the codon encoding proline at position 72 in SEQ ID NO. 2 (not shown). Preferably, mutation in the glcK gene results in the replacement of a T with a C at position 214 in SEQ ID NO. 1 (not shown).

[0066] In another preferred embodiment, the mutation results in the replacement of the codon encoding threonine with the codon encoding isoleucine at position 141 in SEQ ID NO. 2 (not shown).

[0067] Preferably, mutating the NO glck gene results in the replacement of a C with a T at position 422 in SEQ ID NO. 1 (not shown).

[0068] It should be emphasized that the glcK gene of a Streptococcus thermophilus can be inactivated by other types of mutations at other sites of the glcK gene.

Mutation that reduces the transport of glucose into the cell

[0069] In a preferred embodiment, the Streptococcus thermophilus strain carries a mutation that reduces glucose transport into the cell.

[0070] The term "a mutation that reduces glucose transport into the cell" as used herein, refers to a mutation in a gene encoding a protein involved in glucose transport that results in an accumulation of glucose in the environment of the cell.

[0071] The glucose level in the culture medium of a Streptococcus thermophilus strain can be measured by methods known to the person skilled in the art, also when the culture medium is a milk substrate.

[0072] In preferred embodiments, 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 %.

[0073] The transport of glucose into the cell can be determined by the glucose uptake assay as described in Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423-5432).

[0074] Preferably, the strain of Streptococcus thermophilus carries a mutation in a gene encoding a component of a glucose transporter, wherein the mutation inactivates the glucose transporter protein or has a negative effect on gene expression.

[0075] The component can be any component of a glucose transporter protein that is critical for glucose transport. It is, for example, contemplated that inactivation of any component of the glucose/mannose PTS in Streptococcus thermophilus will result in inactivation of the glucose transporter function.

[0076] The term "mutation inactivates the glucose transporter" as used herein, refers to a mutation that results in an "inactivated glucose transporter", a glucose transporter protein that, if present in a cell, is not capable of exercising its normal function as well as mutations that prevent the formation of the glucose transporter protein or result in the degradation of the glucose transporter protein.

[0077] In particular, an inactivated glucose transporter protein is a protein that compared to a functional glucose transporter protein is not capable of facilitating the transport of glucose across a plasma membrane or facilitates the transport of glucose across a plasma membrane in a significantly reduced rate. The gene encoding such a glucose transporter protein inactivated compared to the gene encoding a functional glucose transporter protein comprises a mutation in the open reading frame (ORF) of the gene, wherein said mutation may include, but is not limited to these, a deletion, a frameshift mutation, introduction of a stop codon, or a mutation that results in an amino acid substitution that alters the functional properties of the protein, or a promoter mutation that reduces or abolishes transcription or translation of the gene.

[0078] In preferred embodiments, the mutation reduces the activity (the rate of glucose transport) of the glucose transport 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%.

[0079] Glucose transporter activity can be determined by the glucose uptake assay as described by Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423-5432).

[0080] The term "functional glucose transporter protein" as used herein, refers to a glucose transporter protein which, if present in a cell, facilitates the transport of glucose over a plasma membrane.

[0081] In a preferred embodiment of the invention, the Streptococcus thermophilus strain of the invention carries a mutation in the DNA sequence of the manN gene encoding the IIDMan protein of the glucose/mannose phosphotransferase system, wherein the mutation inactivates the IIDMan protein or has a negative effect on gene expression.

[0082] CHCC16731 has a threonine to proline change at position 79 in the manN gene encoding the IIDMan Protein of the glucose/mannose phosphotransferase system. Preferably, the mutation in the ManN gene results in the replacement of an A with a C at position 235 of SEQ ID NO. 3.

[0083] Thus, in a preferred embodiment, the Streptococcus thermophilus strain of the invention carries a mutation in the DNA sequence of the manN gene encoding the IIDMan protein of the glucose/mannose phosphotransferase system, wherein the mutation results in the substitution of threonine for proline at position 79 of SEQ ID No. 4.

[0084] In another preferred embodiment of the invention, the Streptococcus thermophilus strain of the invention carries a mutation in the DNA sequence of the manM gene that encodes the IICMan protein of the glucose/mannose phosphotransferase system, in which the mutation inactivates the IICMan protein or has a negative effect on gene expression.

[0085] In a specific preferred embodiment the mutation results in the replacement of the codon encoding glutamic acid with a stop codon at position 209 of SEQ ID NO. 6 of the IICMan protein of the glucose/mannose phosphotransferase system (not shown). Preferably, the mutation results in the replacement of a G with a T at position 625 of SEQ ID NO. 5 (not shown).

Preferred Streptococcus thermophilus strains of the invention

[0086] In a preferred embodiment of the composition of the invention, the strain of Streptococcus thermophilus is selected from the group consisting of the strain of Streptococcus thermophilus CHCC19216 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 32227, of the strain of Streptococcus thermophilus CHCC16731 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on June 4, 2014 under Accession No. DSM 28889, of the Streptococcus thermophilus strain CHCC15757 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 25850, from a strain of Streptococcus thermophilus CHCC15887 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession No. and a mutant strain 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 texturing property and the glucose secretion property of said deposited strain.

[0087] In the present context, the term "mutant strain" should be understood as derived strains, or strains that can be derived from a strain (or its parent strain) of the invention by means of, for example, genetic engineering, radiation and/ or chemical treatment. "Strains derived therefrom" may also be spontaneously occurring mutants. It is preferred that "strains derived therefrom" are functionally equivalent mutants, e.g., mutants that have substantially the same or improved properties as their parent strain. Specifically, the term "mutant strains" refers to strains obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as methane ethane sulfonate (EMS) or N-methyl-N' -nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment is to be understood as a mutagenization step followed by a screening/selection step), but it is currently preferred that no more than 20, or no more than 10 , or no more than 5, treatments (or screening/selection steps) are performed. In a currently preferred mutant 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 by another nucleotide, or deleted, compared to the parent strain.

Method for obtaining a strain of Streptococcus thermophilus sweetener

[0088] In a preferred embodiment of the invention, the starting strain used for developing a sweetener strain of Streptococcus thermophilus is a texturing strain of Streptococcus thermophilus, which can be obtained as described elsewhere in this specification.

[0089] In a preferred embodiment, such a texturizing starter strain is selected from the group consisting of Streptococcus thermophilus strain CHCC11342 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 22932, Streptococcus strain thermophilus CHCC11976 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 22934, the strain of Streptococcus thermophilus CHCC12339 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. .

[0090] Alternatively, the starting strain used to develop a sweetening Streptococcus thermophilus strain is a non-textured Streptococcus thermophilus strain, in which case the texturing property is introduced into the sweetening Streptococcus thermophilus strain as soon as it is produced, i.e. the sweetening strain is used as a starting strain in the method of obtaining a texturizing strain described elsewhere in this specification.

[0091] The first step in the method of sweetening a strain of Streptococcus thermophilus is to provide a galactose-positive strain, ie, a strain that is capable of utilizing galactose as a source of carbohydrate. Galactose-positive strains can be obtained by a method that comprises the step of placing the bacteria to be tested on agar plates, such as M17 agar plates containing a certain concentration, such as 2%, of galactose (the only source of carbohydrate ) and identify the colonies capable of developing on the plates.

[0092] 2-deoxyglucose and a determination of the growth pattern of bacteria in M17 medium + 2% galactose compared to M17 medium + 2% glucose are used for the selection of bacteria having a mutation in the glucokinase gene ( glcK).

[0093] A method for screening and isolating a strain of Streptococcus thermophilus with a mutated glcK gene comprises the following steps:

[0094] a) provide a parent strain of Streptococcus thermophilus from galactose fermentation;

[0095] b) selecting and isolating from a group of mutant Streptococcus thermophilus strains derived from the parent strain from a group of mutant Streptococcus thermophilus strains that are resistant to 2-deoxyglucose; It is

[0096] c) selecting and isolating from the group of mutant Streptococcus thermophilus strains that are resistant to 2-deoxyglucose a mutant Streptococcus thermophilus strain if the growth rate of the mutant Streptococcus thermophilus strain is higher in M17 medium + 2% galactose than in M17 medium + 2% glucose.

[0097] The term "2-deoxyglucose resistant" in this invention is defined by the fact that a bacterial strain subjected to the particular mutation has the ability to develop into a colony when placed on a plate of M17 medium containing 2% lactose or 2% galactose containing 20 mM 2-deoxyglucose after incubation at 40°C for 20 hours. The presence of 2-deoxyglucose in the culture medium will impede the growth of non-mutated strains, whereas the growth of the mutated strains is not affected or not significantly affected. Unmutated strains that can be used as sensitive reference strains in assessing resistance include strain CHCC11976.

[0098] In one embodiment, the method further comprises step a1) which subjects the parent strain to mutagenization, such as subjecting the parent strain to a chemical and/or physical mutagenic agent.

[0099] In another embodiment, the method further comprises a step d) selecting and isolating from a group of strains of Streptococcus thermophilus resistant to 2-deoxyglucose derived from the strain of Streptococcus thermophilus selected in step c) a strain of Streptococcus thermophilus if the rate growth rate of the Streptococcus thermophilus strain is increased in M17 medium + 2% sucrose, but zero or at least 0 to 50% reduced compared to the growth rate of the parent strain in M17 medium + 2% glucose.

[00100] The mother strains of Streptococcus thermophilus from galactose fermentation are able to develop in M17 medium + 2% galactose and are defined here by the fact that they have the ability to lower the pH in M17 broth containing 2% galactose as a single carbohydrate to 5.5 or lower when inoculated from a 1% overnight culture and incubated for 24 hours at 37°C. Such galactose positive strains have been described in WO2011/026863 (Chr. Hansen A/S) and WO11/092300 (Chr. Hansen A/S).

[00101] In the present context, the term "strains derived from these" shall be understood as derived strains, or strains that can be derived from texturing or strains of Streptococcus thermophilus from galactose fermentation by means of, for example, genetic engineering, radiation and/or chemical treatment. "Strains derived therefrom" may also be spontaneously occurring mutants. It is preferred that "strains derived therefrom" are functionally equivalent mutants, e.g., mutants that have substantially the same or improved properties (e.g. with respect to texture or galactose fermentation) as their parent strain. In particular, the term "strains derived therefrom" refers to strains obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as methane ethane sulfonate (EMS) or N-methyl-N '-nitro-N-nitroguanidine (NTG), UV light, or to a spontaneously occurring mutant. A mutant may have been subjected to several mutagenization treatments (a single treatment shall be understood to comprise a mutagenization step followed by a screening/selection step) but it is currently preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening/selection steps) are performed. In a currently preferred mutant, 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 by another nucleotide, or deleted, compared to the parent strain.

[00102] In the present invention, the expression "glcK gene encoding a glucokinase" means any DNA sequence from a Streptococcus thermophilus that encodes a protein having glucokinase activity, including the specific glucokinase encoded by the DNA sequence of SEQ ID NO: 1 A glucokinase catalyzes the reaction that converts glucose to glucose-6-phosphate, cfe. to Figure 1.

Strains of Lactobacillus delbrueckii subsp. bulgaricus from the composition of the invention

[00103] The terms "lactose metabolism deficiency" and "lactose deficiency" are used in the context of the present invention to characterize LAB that partially or completely lose the ability to use lactose as a source for cell growth or to maintain cell viability. The respective LAB are capable of metabolizing one or several carbohydrates selected from sucrose, galactose and/or glucose or other fermentable carbohydrate. Since these carbohydrates are not naturally present in milk in sufficient amounts to support fermentation by lactose-deficient mutants, it is necessary to add these carbohydrates to milk. Lactose-deficient and partially lactose-deficient LABs can be characterized as white colonies on a medium containing lactose and X-Gal.

[00104] In connection with the present invention, the term "X-Gal" means 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside, which is a chromogenic substrate for beta-galactosidase, which hydrolyzes X-Gal to colorless galactose and 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to form a blue pigment.

[00105] In a particular embodiment of the 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 particular embodiment of the invention, the lactose-deficient strain is capable of metabolizing galactose.

[00106] In a particular embodiment of the composition of the invention, the Lb strain is selected from the group consisting of the strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under Accession No. DSM 28910, and a mutant strain derived from DSM 28910, wherein the mutant strain is further characterized as having the ability to generate white colonies on a medium containing lactose and X-Gal.

[00107] Lactose-deficient strains can be obtained from a suitable lactose-positive parent strain by mutation. Lactose-deficient strains were selected after UV mutagenesis as white colonies (indicating a lactose-deficient phenotype) on a suitable medium, eg MRS agar plates with 1% lactose and 200 mg/ml X-Gal. Lactose-positive strains have β-galactosidase activity, and lactose-positive strain colonies appear blue due to β-galactosidase activity. As the lactose-positive parent strain for the production of a lactose-deficient strain, the Lactobacillus delbrueckii ssp. bulgaricus CHCC10019 filed as WO2011/000879 with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 03.04.2007 under DSM Accession No. 19252, can be used.

Composition

[00108] The present invention also relates to a composition comprising 104 to 1012 CFU (colony forming units)/g of the Streptococcus thermophilus strain, such as 105 to 1010 CFU/g, such as 106 to 1010 CFU/g, or such as 107 to 109 CFU/g of the Streptococcus thermophilus strain.

[00109] In a preferred embodiment, the Streptococcus thermophilus strain of the composition is unable to acidify B milk at 9.5%, defined as resulting in a decrease in pH to less than 1.0 when B milk at 9.5 % is inoculated with 106 to 107 CFU/ml of the Streptococcus thermophilus strain and incubated for 14 hours at 40°C, and the composition further comprises an amount of a compound which can cause the strain to acidify B milk to 9.5% of Streptococcus thermophilus CHCC16404 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 26722, defined as resulting in a decrease in pH of 1.0 or more when 9.5% B milk is inoculated with 106 a 107 CFU/ml of Streptococcus thermophilus strain and incubated for 14 hours at 40°C.

[00110] Preferably, the compound is sucrose.

[00111] Preferably, the amount of sucrose is from 0.000001% to 2%, such as from 0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001 % to 0.05%.

[00112] In an especially preferred embodiment, the composition further comprises from 104 to 1012 CFU/g of the strain of Lactobacillus delbrueckii subsp. bulgaricus, such as from 10 5 to 10 11 CFU/g, such as from 10 6 to 10 10 CFU/g, or such as from 10 7 to 10 9 CFU/g from the Lactobacillus delbrueckii subsp. bulgaricus.

[00113] In a particular embodiment of the invention, the composition contains at least two strains of St, preferably at least three strains of St, and more preferably three strains of St. In a particular embodiment of the invention, the composition contains not more than three strains of Lb, preferably no more than two strains of Lb, and most preferably one strain of Lb.

[00114] In a particular embodiment of the invention, the composition contains at least one strain of St selected from the group consisting of the strain Streptococcus thermophilus CHCC16731 which has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on June 4, 2014 under DSM Accession No. 28889, the Streptococcus thermophilus strain CHCC15757 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under DSM Accession No. 25850, and the strain Streptococcus thermophilus CHCC16404 which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under Accession No. DSM 26722, and the Lb strain deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under no. access code DSM 28910.

[00115] Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are commonly used as starter cultures that serve a technological purpose in the production of various foods, such as in the dairy industry, such as for fermented milk products. Thus, in another preferred embodiment, the compound is suitable as a starter culture.

[00116] Starter cultures can be provided as frozen or dry starter cultures in addition to liquid starter cultures. Thus, in yet another preferred embodiment, the composition is in frozen, freeze-dried or liquid form.

[00117] As described in WO 2005/003327, it is beneficial to add certain cryoprotective agents to a starter culture. Thus, a starter culture composition according to the present invention may comprise one or more cryoprotectant agents selected from the group consisting of inosine-5'-monophosphate (IMP), adenosine-5'-monophosphate (AMP), guanosine-5' -monophosphate (GMP), uranosine-5'-monophosphate (UMP), cytidine-5'-monophosphate (CMP), adenine, guanine, uracil, cytosine, adenosine, guanosine, uridine, cytidine, hypoxanthine, xanthine, hypoxanthine, orotidine, thymidine, inosine and a derivative of any such compound.

Method of producing a fermented dairy product

[00118] The invention is further directed to a method of producing a fermented milk product which comprises inoculating and fermenting a milk substrate with the composition according to the present invention.

[00119] The term "milk" should be understood as the milky secretion obtained by milking any mammal, such as a cow, a sheep, a goat, a buffalo or a camel. In a preferred embodiment, the milk is cow's milk.

[00120] The term "milk substrate" can be any natural and/or processed milk material that can be subjected to fermentation according to the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions/suspensions of any milk or milk-like products comprising protein, such as whole or low-fat milk, skim milk, cream, milk in reconstituted powder, condensed milk, powdered milk, whey, whey permeate, lactose, lactose crystallization mother liquor, whey protein concentrate or cream. Obviously, the milk substrate can originate from any mammal, for example, substantially pure mammalian milk, or reconstituted milk powder.

[00121] Preferably, at least part of the protein in the milk substrate are naturally occurring proteins in milk, such as casein or whey protein. However, some of the protein may be proteins that are not naturally occurring in milk.

[00122] Before fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.

[00123] "Homogenization" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed before fermentation, it can be performed in order to break the milk fat into smaller sizes so that it no longer separates from the milk. This can be accomplished by forcing milk under high pressure through small holes.

[00124] "Pasteurization" as used herein means treating the milk substrate to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. The specified temperature is usually achieved by heating. Temperature and duration can be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A quick cool-down step may follow.

[00125] "Fermentation" in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism. Preferably, fermentation in the methods of the invention comprises the conversion of lactose to lactic acid.

[00126] Fermentation processes to be used in the production of fermented dairy products are well known and the person of skill in the art will know how to select the appropriate process conditions, such as temperature, oxygen, quantity and characteristics of microorganisms and process time. Obviously, the fermentation conditions are selected so as to support carrying out the present invention, i.e. to obtain a dairy product in solid or liquid form (fermented dairy product).

[00127] The term "fermented dairy product" as used herein refers to a food or feed product, wherein the preparation of the food or feed product involves fermenting a milk substrate with a lactic acid bacteria. "Fermented dairy product" as used in this invention includes, but is not limited to, products such as thermophilic fermented dairy products, for example, yogurt, mesophilic fermented dairy products, for example, sour cream, as well as fermented whey.

[00128] The term "thermophile" refers here to microorganisms that develop better at temperatures above 35°C. The most industrially useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term "thermophilic fermentation" herein refers to fermentation at a temperature above about 35°C, such as between about 35°C to about 45°C. The term "thermophilic fermented dairy product" refers to fermented dairy products prepared by thermophilic fermentation of a thermophilic starter culture and includes such fermented dairy products as conventional yoghurt, stirred yoghurt and drinkable yoghurt, e.g. Yakult.

[00129] The term "mesophile" refers here to microorganisms that develop better at moderate temperatures (15°C to 35°C). The most industrially useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term "mesophilic fermentation" in this invention refers to fermentation at a temperature between about 22°C and about 35°C. The term "mesophilic fermented dairy product" refers to fermented dairy products prepared by mesophilic fermentation of a mesophilic starter culture and includes such fermented dairy products as cream, curd, cultured milk, cream, sour cream , kefir and fresh cheese, such as ricotta, tvarog and creamy cheese.

[00130] In a preferred embodiment, the concentration of inoculated Streptococcus thermophilus cells is 104 to 109 CFU of Streptococcus thermophilus cells per ml of milk substrate, such as 104 CFU to 108 CFU of Streptococcus thermophilus cells per ml of milk substrate.

[00131] In another preferred embodiment of the method of the invention, the Streptococcus thermophilus strain is unable to acidify B milk at 9.5%, defined as resulting in a pH decrease of less than 1.0 when B milk at 9 .5% is inoculated with 106 to 107 CFU/ml of Streptococcus thermophilus strain and incubated for 14 hours at 40°C, and the milk substrate is added in an amount of a compound, effective to trigger the acidification of milk B to 9.5% by the Streptococcus thermophilus strain, defined as resulting in a pH decrease of 1.0 or more when 9.5% B milk is inoculated with 106 to 107 CFU/ml of the Streptococcus thermophilus strain and incubated for 14 hours at 40°C.

[00132] Preferably, the compound is sucrose.

[00133] Preferably, the amount of sucrose is from 0.000001% to 2%, such as from 0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001 % to 0.05%.

[00134] In another preferred embodiment, the fermented milk product is a yogurt or a cheese.

[00135] Examples of cheeses that are prepared through fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus include mozzarella and pizza cheese (H0ier et al. (2010) in The Technology of Cheesemaking, 2nd Ed. Blackwell Publishing, Oxford; 166-192).

[00136] Preferably the fermented milk product is a yogurt.

[00137] In the present context, 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. As used herein, the term "yoghurt" refers to a fermented milk product obtainable by inoculating and fermenting milk with a composition comprising a strain of Lactobacillus delbrueckii subsp. bulgaricus and a strain of Streptococcus thermophilus.

fermented dairy product

[00138] The present invention further relates to a fermented milk product comprising the composition according to the invention.

[00139] In another preferred embodiment, the fermented milk product can be a yogurt, a cheese, sour cream and cream as well as fermented whey. Preferably, the fermented milk product is a yoghurt.

Use of the composition of the invention

[00140] A further aspect of the invention relates to the use of the composition according to the invention for the preparation of a fermented milk product.

[00141] Embodiments of the present invention are described below, by way of non-limiting examples. Sequence listing SEQ ID NO. 1 shows the DNA sequence of the mutated glucokinase gene from strain CHCC16731. SEQ ID NO. 2 shows the amino acid sequence encoded by SEQ ID NO. 1 SEQ ID NO. 3 shows the DNA sequence of the mutated ManN gene from strain CHC16731. SEQ ID NO. 4 shows the amino acid sequence encoded by SEQ ID NO. 3. SEQ ID NO. 5 shows the DNA sequence of the ManM gene (unmutated) from strain CHCC16731. SEQ ID NO. 6 shows the amino acid sequence encoded by SEQ ID NO. 5.

EXAMPLES Materials and methodsMedium:

[00142] For Streptococcus thermophilus, the medium used is the M17 medium known to persons skilled in the art.

[00143] The M17 agar medium has the following composition per liter of H2O: agar, 12.75 g ascorbic acid, 0.5 g casein peptone (tryptic), 2.5 g disodium β-glycerophosphate pentahydrate, 19 g magnesium sulfate hydrate, 0.25 g beef extract, 5 g beef peptone (peptic), 2.5 g soy peptone (papain), 5 g yeast extract, 2.5 g final pH 7.1 ± 0.2 (25°C) and the M17 broth has the following composition per liter of H2O: ascorbic acid, 0.5 g magnesium sulfate, 0.25 g meat extract, 5 g meat peptone (peptide), 2.5 g sodium glycerophosphate, 19 g soy peptone (papain), 5 g tryptone, 2.5 g yeast extract, 2.5 g final pH 7.0 ± 0.2 (25°C)

[00144] Added carbon sources are sterile lactose 20 g/l, glucose 20 g/l or galactose 20 g/l.

[00145] As known to the person skilled in the art, the M17 medium is a medium which is considered to be suitable for the growth of Streptococcus thermophilus. Furthermore, as understood by the person skilled in the art, in the present context, an M17 concentrate can be supplied from different suppliers and irrespective of the specific supplier (within standard measurement uncertainty) the same relevant result of resistance to 2- deoxyglucose to a cell of relevant interest in this invention.

[00146] The medium used for the cultivation of Lactobacillus delbrueckii subsp. bulgaricus was the MRS-IM medium. MRS-IM was used in the form of agar plates or broth.

[00147] The MRS-IM agar medium had the following composition per liter of H2O. Tryptone Oxoid L 42 10.0 g Yeast Extract Oxoid L 21 5.0 g Tween 80 Merck nr 8.22187 1.0 g K2HPO4 Merck nr 105104 2.6 g Na-acetate Merck nr 106267 5.0 g Diammonium Hydrogen Citrate Merck nr 101154 2.0 g MgSO4, 7 H2O Merck nr 105882 0.2 g MnSO4, H2O Merck nr 105941 0.05 g SO-BI-GEL Agar 13.0 g

[00148] The pH was adjusted after autoclaving to 6.9 ± 0.1 at 25°C.

[00149] The MRS-IM broth used in the examples below for liquid cultures had the following composition per liter of H2O: Tryptone Oxoid L 42 10.0 g Yeast extract Oxoid L 21 5.0 g Tween 80 Merck nr 8.22187 1, 0 g K2HPO4 Merck nr 105104 2.6 g Na-acetate Merck nr 106267 5.0 g Diammonium Hydrogen Citrate Merck nr 101154 2.0 g MgSO4, 7 H2O Merck nr 105882 0.2 g MnSO4, H2O Merck nr 105941 0 .05 g

[00150] The pH is adjusted after autoclaving to 6.9 ± 0.1 at 25°C. The carbon sources, lactose 20 g/l or glucose 20 g/l, were first sterile filtered and then added to the autoclaved broth.

[00151] The above MRS-IM medium can be varied to some extent without affecting the ability of the medium to support the growth of Lactobacillus delbrueckii subsp. bulgaricus. Furthermore, as will be understood by the person skilled in the art, an MRS-IM concentrate or the various components described above can be obtained from different suppliers and used for the preparation of an MRS-IM medium. These media will also be used in the examples below, in particular in the 2-deoxyglucose resistance selection assay.

mother strains

[00152] Streptococcus thermophilus CHCC11976 (galactose fermentation strain with a mutation in the GalK gene as described in WO 2011/026863),

[00153] Streptococcus thermophilus CHCC12339 (phage resistant and texturing as described in WO2011/092300).

[00154] Streptococcus thermophilus CHCC18948 (galactose fermentation mutant of CGCC12339 with a mutation in the GalK gene)

2-deoxyglucose resistant strains

[00155] Streptococcus thermophilus CHCC16165 (2-deoxyglucose resistant mutant of CHC11976).

[00156] Streptococcus thermophilus CHCC16731 (sweetening and texturing mutant of CHCC16165).

[00157] Streptococcus thermophilus CHCC19216 (sweetening and texturing mutant of CHCC18948) EXAMPLE 1: Use of 2-deoxyglucose to isolate Streptococcus thermophilus glucose kinase mutant CHCC11976 with enhanced glucose excretion.

[00158] In order to isolate mutants of the strain De Streptococcus thermophilus CHCC11976, cells derived from the growth of a single colony were inoculated into 10 ml of M17 broth containing 2% lactose A and cultured overnight at 40°C.

[00159] The following day, the strains were plated in serial dilutions onto M17 agar plates containing 2% galactose and a concentration of 20 mM 2-deoxyglucose (CHCC11976) and incubated for 20 hours at 40°C. Resistant colonies were first replaced on the same type of agar plates as they were selected. Survivors were used to inoculate fresh M17 broth containing 2% lactose, 2% galactose or 2% glucose and growth was measured.

[00160] From this, a number of mutants that were able to grow faster on galactose than on glucose were identified as outlined in Example 2. One such mutant was CHC16165. EXAMPLE 2: 2-deoxyglucose resistance mutant growth pattern

[00161] To ensure the selection of 2-deoxyglucose resistant mutants that can grow on galactose, two strains that were selected from a collection of galactose fermentation strains were used. Although these galactose fermentation strains still grow at least 10% faster in the exponential phase in the M17 broth + 2% glucose than in the M17 broth + 2% galactose, the 2-deoxyglucose resistant mutant derived from CHCC11976, in the absence of CHCC16165, on the other hand, are characterized by faster growth in exponential phase in M17 broth + 2% galactose than in M17 broth + 2% glucose.

[00162] Exponential phase growth is measured here as the development in optical density of the exponentially growing culture at 600 nanometers (OD600) with time at 40°C.

[00163] As known by the knowledgeable person, it may vary from species to species when the culture is in exponential growth. The knowledgeable person will know how to determine the growth in the exponential phase, for example between OD600 0.1 to 1.0.

[00164] The optical density (OD) of the culture is measured in a spectrophotometer.

Conclusion:

[00165] Based on the 2-deoxyglucose resistance mutant growth pattern of this Example 2 - for a specific strain of interest (e.g., that of a relevant commercial product) - the skilled person can routinely test whether this specific strain of interest has the relevant growth pattern in this invention which is a property of the selected mutants. EXAMPLE 3: Selection of a hyperlactose fermentation and glucose secretion mutant of Streptococcus thermophilus CHCC16165

[00166] In order to isolate a hyperlactose fermentation and glucose secretion mutant 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 cultured for overnight at 40°C.

[00167] The next day, the strain was plated in serial dilutions on M17 agar plates containing 2% sucrose and a concentration of 2-deoxyglucose of 40 mM and incubated for 20 hours at 40°C. 10 random colonies were selected from the plate and used to inoculate fresh M17 broth containing 2% sucrose and incubated overnight at 40°C. CHCC16165 mutants were transferred to fresh M17 media containing 2% sucrose. Example 4. Carbohydrate analysis of fermented milk

[00168] The mutants obtained in Example 3 and the CHCC16165 strain were grown in 9.6% B milk containing 0.01% sucrose. After acidification, milk acidified with CHCC16165 had a lactose concentration of 14.9, a galactose concentration of 8.4 and a glucose concentration of 5.7. In comparison, milk acidified with the best performing mutant of CHCC16165 had a lactose concentration of 9.3, a galactose concentration of 10.5, and a glucose concentration of 9.9. Said best CHCC16165 mutant was designated CHCC16731 and was subjected to further testing in milk fermentation when used in combination with strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 and CHCC16160 (described in WO2013/160413).

[00169] Overnight cultures of CHCC16165 and CHCC16731 in combination with each of CHCC16159 and CHCC16160 were added into 200 ml milk samples B and acidified at 40°C.

[00170] The results were as listed in Table 1:

[00171] As will appear from the results in Table 1, CHCC16731 is able to remove almost all of the lactose from milk. Likewise, CHCC16731 produces and secretes elevated levels of both galactose and glucose in milk. Taking into account that sucrose is a reference 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the fermented milk product produced with CHCC16731 has a highly high sweetness. EXAMPLE 5 - Production of a galactose positive mutant of Streptococcus thermophilus CHCC12339

[00172] Streptococcus thermophilus CHCC12339 was grown overnight in M17 containing 2% lactose at 40°C. An overnight culture was plated (100 µl) onto an M17 plate containing 2% galactose and incubated at 40°C anaerobically. 22 colonies appear on 2% galactose plates containing M17. 16 of these are replaced on the same type of plates, and 8 of these are transferred to liquid M17 containing 2% galactose, all of which result in growth, and 4 of these cultures are frozen. 2 of the frozen cultures are spread onto M17 plates containing 2% galactose and 40 mM 2-deoxyglucose, and for each culture 8 of the resulting colonies are selected. The 16 2-deoxyglucose mutants were transferred into M17 liquid containing 2% lactose and 2% galactose and cultured overnight at 40°C, after which the mutants were cultured in M17 liquid containing 2% galactose for 3 nights and galactose and glucose production was measured and followed throughout the growth. On the basis of glucose and glucose production, 7 cultures were selected for the milk acidification test by adding 2 ml of culture to 200 ml of 9.5% B milk containing 0.05% sucrose. 3 mutants have acidification profiles that resemble that of the CHCC12339 parent strain, and these 3 mutants are placed in isolated colonies and incubated for 48 hours. Subsequently, colonies are transferred to M17 liquid containing 2% galactose and growth for the 3 mutants is tested against CH12339. The mutant having the highest growth is selected and purified once more by placing in a single colony 3 times to produce a purified galactose positive strain called CHCC18948 (galactose positive variant of CHCC12339). EXAMPLE 6. Production of a Streptococcus thermophilus CHCC18948 2-deoxyglucose mutant with enhanced glucose excretion

[00173] 100 µl of the CHCC18948 culture is spread onto M17 plates containing 2% galactose and 20 mM or 40 mM 2-deoxyglucose and incubated overnight at 40°C. 8 colonies from the 20 mM plates and 8 colonies from the 40 mM plates are selected and grown in M17 liquid containing 2% galactose and 20 mM 2-oxyglucose. The resulting cultures are frozen and replaced, and 12 of the resulting colonies are started in M17 medium and used to acidify the B milk. 1% of an overnight culture of the 2-deoxyglucose mutant is inoculated into 200 ml of B milk at 9 .5% containing 0.05% sucrose and acidified at 40°C. All 12 CHCC18948 2-deoxyglucose mutants produced fermented mill products with low concentrations of lactose and high concentrations of galactose and glucose. 7 Mutants were selected for retesting alone and for testing together with strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 (described in WO2013/160413). Again 1% of an overnight culture of the 2-deoxyglucose mutant is inoculated into 200 ml of 9.5% milk B containing 0.05% sucrose and acidified at 40°C.

[00174] The results are given in Table 2:

[00175] The three best mutants are mutants 2, 3 and 12, and are again tested in milk B containing 0.05% sucrose alone and in combination with strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, CHCC16160 and CHCC16161 (described in WO2013/160413).

[00176] The results are given in Table 3:

[00177] Based on the above data C HCH18948-Mutant3 was selected as the best mutant and was designated CHCC19216. As will emerge from the above data, CHCC19216 is capable of removing almost all of the lactose from milk. Likewise, CHCC19216 produces and secretes elevated levels of both galactose and glucose in milk. Taking into account that sucrose is a reference 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the fermented milk product produced with CHCC19216 has a highly high sweetness. EXAMPLE 7. Texturing property of CHCC16731 and CHCC19216

[00178] The texturing property of strains CHCC16731 and CHCC19216 were tested by measuring the shear stress using the following test:

[00179] Seven days after incubation, the fermented milk was brought to 13°C and gently stirred using a stick fitted with a perforated disc until sample homogeneity. The rheological properties of the sample were evaluated in a rheometer (Anton Paar Physica ASC/DSR301 Rheometer (Autosampler), Anton Paar® GmbH, Austria) using the following settings: Waiting time (to reconstruct a somewhat original structure) 5 minutes without oscillation or rotation Oscillation (measure G' and G" to calculate G*) Y = 0.3%, frequency (f) = [0.5...8] Hz 6 measuring points during 60 s (one every 10 s) Rotation (measure shear stress at 300 1/s, etc.) y = [0.2707-300] 1/s and y = [275-0.2707] 1/s 21 measuring points during 210 s (one every 10 s) up to 300 1/s and 21 measurement points over 210 s (one every 10 s) down to 0.2707 1/s

[00180] For further analysis, the shear stress at 300 1/s was selected.

[00181] The CHCC16731 and CHCC19216 strains in the mixtures listed in Table 4 were used to acidify 200 ml samples of 9.5% B milk using a 0.024% inoculation material until the pH reached 4.55.

[00182] The results are given in Table 4: Lb: L. delbruecki spp. Bulgaricus strain CHCC16159. St: Streptococcus thermophilus.

[00183] As will emerge from the results, strains CHCC16731 and CHCC19216 when used as the only S. thermophilus in the culture mixture, produce a fermented dairy product with a shear stress of 41.3 Pa and 51.5 Pa, respectively . When used in combination CHCC16731 and CHCC19216 strains produce a fermented dairy product with a shear stress of 43.3 Pa.

[00184] This level of shear stress is high compared to conventional sweetening strains as described, for example, in WO2013/160413. EXAMPLE 8. Taste and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for the culture composition of the culture of the invention compared to the lactose positive, glucose positive Lb culture Experimental plan Table 5: Composition of tested cultures

[00185] The culture of the invention is composed of three Staphylococcus thermophilus deficient in glucose (St) and one Lb deficient in lactose. Culture 2 (comparative culture) is composed of three glucose-deficient Sts and one lactose-positive, glucose-positive Lb (conventional).

[00186] The two cultures with the composition indicated in Table 5 were prepared and used as a starter culture in a fermentation to produce yogurt at 43°C until a target pH of 4.55 is reached. The two cultures were compared with respect to taste, Lb growth, post-acidification, carbohydrates and rheology.

milk base

[00187] The milk base contained 4.5% protein, 1.0% fat and 0.1% sucrose.

[00188] Milk with a fat content of 0.5% and milk with a fat content of 1.5% were mixed in order to obtain a fat content of 1% in the final milk base and 0.1% sucrose has been added. Protein levels were adjusted to 4.5% using skimmed milk powder (SMP). Hydration took place at 6°C for 2 hours. After that, the milk was homogenized at 200/50 bar at 65°C and pasteurized at 95°C for 5 minutes.

Yoghurt production parameters

[00189] The pH during fermentation was measured in real time using Intap. When the yoghurts reached pH 4.55, the gel was broken using a stick with a perforated disc. The gel was then treated in a post-treatment unit (PTU) at 2 bar at 25°C, loaded into 120 ml yoghurt cups and stored at 6°C for 28 days.

Shear stress measurement

[00190] The method described in Example 7 was used with the exception that the samples were not stirred with a stick fitted with a perforated stick, since the gel had already been broken during the PTU treatment to obtain homogeneity of the samples.

Measurement of Colony Forming Units

[00191] M17 was used as selective medium for St. MRSlac (MRS + 8% lactose) was used as selective medium for Lb. A series of 10X dilutions was performed with the yoghurts. Each dilution was plated on agar plates with M17 and MRSlac, respectively, and incubated anaerobically at 37°C for 2 days. After that, colony-forming units were counted.

acidification

[00192] 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. Post-acidification Table 6: Post-acidification Lb Cultivation Table 7: Culture growth (Colony Forming Units (CFU)) at the end of fermentation

[00193] As will appear from Table 7, the Lb cell count of the culture of the invention was much higher than the Lb count of the comparative culture with a conventional Lb, i.e., a lactose positive Lb, a glucose. Thus, the combination of glucose-deficient St and lactose-deficient Lb produces a higher level of Lb at the end of the fermentation.

rheology

[00194] Culture 1 has a shear stress at the end of fermentation at 300 1/s of 41.1 Pa.

[00195] Culture 2 has a shear stress at the end of fermentation at 300 1/s of 27.3 Pa. Carbohydrates Table 8: Level of carbohydrates at the end of fermentation

Flavor

[00196] The taste was evaluated through the evaluation by a tasting panel. Culture 1 did not have any off-flavors. EXAMPLE 9. Taste and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for the culture composition of the culture of the invention compared to the culture with glucose-deficient Lb Experimental plan Table 9: Composition of tested cultures

[00197] The culture of the invention is composed of three glucose-deficient St and one lactose-deficient Lb. Culture 2 (comparative culture) is composed of three glucose-deficient Sts and one glucose-deficient lactose-positive Lb.

[00198] The two cultures with the composition indicated in Table 9 were prepared and used as a starter culture in a fermentation to produce yogurt at 43°C until a target pH of 4.55 was reached. The two cultures were compared with respect to flavor (level of amino acids), volatile organic components, carbohydrates and rheology.

milk base

[00199] Two milk bases were used, adjusted with Skimmed Milk Powder (SMP) or whey protein (WP) and contained 4.5% protein, 1% fat and 0.1% sucrose. Milk with 0.5% fat content and milk with 1.5% fat content were mixed to obtain a fat content of 1% in the final milk base and 0.1% sucrose was added. Protein levels were adjusted to 4.5% using SMP or WP. Hydration took place at 6°C for 2 hours. After that, the milk was homogenized at 200/50 bar at 65°C and pasteurized at 95°C for 5 minutes. Yoghurt production parameters

[00200] The pH during fermentation was measured in real time in bottles using CINAC. When the yoghurts reached pH 4.55, the gel was broken and homogenized using a stick with a perforated disc. After that, the yogurt was stored in baby bottles.

Shear stress measurement

[00201] The method described in Example 7 was used. rheology

[00202] Culture 1 in SMP milk base has a shear stress at the end of fermentation at 300 1/s of 46.2 Pa.

[00203] Culture 1 in WP milk base has a shear stress at the end of fermentation at 300 1/s of 39.6 Pa.

[00204] Culture 2 in SMP milk base has a shear stress at the end of fermentation at 300 1/s of 42.0 Pa.

[00205] Culture 2 in WP milk base has a shear stress at the end of fermentation at 300 1/s of 38.4 Pa.

[00206] As will appear from the results, culture 1 has a higher shear stress compared to culture 2. Post-acidification Table 10: Post-acidification Lb Development Table 11: Culture Development (Colony Forming Units (CFU)) at the end of fermentation

[00207] As will be shown from Table 11, the Lb cell count of the culture of the invention was lower than the Lb count of the comparative culture with a glucose-deficient glucose-positive Lb. Carbohydrates Table 12: Carbohydrate level at the end of fermentation Amino acids Table 13: Level of amino acids

[00208] Samples containing the negative Lb to glucose (Cultures 2) have a higher amount of TFFA, which points to higher proteolytic activity, which in turn can lead to unpleasant aromas.

[00209] Furthermore, yogurts containing the negative Lb to glucose contain a higher amount of glutamic acid which is known to impart unpleasant tastes described as brothy.

[00210] Valine, a bitter amino acid, is also present in higher amounts in yogurts containing negative Lb glucose.

Volatile Organic Compounds (VOC)

[00211] A VOC profile can give indications for the flavor profile of a sample. 51 different volatile organic compounds were measured. For Culture 1 in WP milk base, the amount of volatile organic compound was lower for 44 of 51 compounds (86%) compared to Culture 2 in WP milk base. For Culture 1 in WP milk base, the amount of volatile organic compound was lower for 34 of 51 compounds (67%) compared to Culture 2 in WP milk base. In conclusion, the cultures of the invention had a significantly lower content of volatile organic compounds (viewed as a group of compounds) compared to the comparative cultures. EXAMPLE 10. Growth and post-acidification of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for the culture composition of the invention Experimental plan Table 14: Composition of the tested culture

[00212] The culture with the composition indicated in Table 14 was prepared and used as a starter culture in a fermentation to produce yogurt at 43°C until a target pH of 4.55 was reached. The culture was inoculated at a level of 0.01%, 0.02% and 0.03%. The yogurt produced was stored at 5°C, 13°C and 25°C. the culture was tested for St and Lb growth and post-acidification. Milk bases Table 15: Composition of milk bases 1 and 2 Table 16: Composition of milk base 3 Post-acidification Table 17: Post-acidification

[00213] As will be shown from 1 table 17, the level of post-acidification is very low for all inoculation levels and storage temperatures tested, including the highest storage temperatures of 13°C and 25°C, where the level of Lb growth and consequently post-acidification is high for most conventional starter cultures. Lb and St Development Table 18: Lb and St Development

[00214] As will appear from Table 18, for all inoculation levels, the Lb cell count was at an elevated level at the end of the fermentation. EXAMPLE 11. Growth, post-acidification and carbohydrate analysis of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for the cultivation compositions of the invention compared to conventional starter cultures. Experimental plan Table 19: Composition of tested cultures

[00215] Cultures with the composition indicated in Table 19 were prepared and used as a starter culture in a fermentation to produce yogurt at 43°C until a target pH of 4.55 was achieved. The culture was inoculated at a level of 0.02%. The yogurt produced was stored at 5°C and 13°C. Cultures were tested for St and Lb growth, post-acidification and carbohydrate analysis.

[00216] YoFlex Premium 1.0 and Yoflex YF-L901 are conventional commercial cultures comprising lactose positive and glucose positive St and Lb. Milk base Table 20: Composition of milk base 1 Post-acidification Table 21: Post-acidification

[00217] As will appear from Table 21, the cultures of the invention have a low level of post-acidification, which is at the same level as conventional starter cultures. Lb and St Development Table 22: Lb and St Development

[00218] As will be shown from Table 22, the Lb cell count for the cultures of the invention was at a higher level at the end of the fermentation than the conventional cultures. Carbohydrate Analysis Table 23: Carbohydrate Analysis LO D: Limit of Detection

[00219] All carbohydrate determinations were done in duplicate, and Table 23 lists the mean value of the two determinations. The determinations were performed after 7 days of yogurt storage.

[00220] As will appear from Table 23, the cultures of the invention produced glucose at an elevated level, and after 7 days of storage, glucose was present at a level of approx. 8mg/g.

DEPOSITS AND SPECIALIZED SOLUTIONS

[00221] The applicant requests that a sample of the deposited microorganisms mentioned below may only be made available to a specialist, until the date on which the patent is granted.

[00222] The strain Streptococcus thermophilus CHCC11342 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on September 8, 2009 under DSM Accession No. 22932.

[00223] The strain Streptococcus thermophilus CHCC12339 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on October 14, 2010 under DSM Accession No. 24090.

[00224] The strain Streptococcus thermophilus CHCC11976 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on September 8, 2009 under DSM Accession No. 22934.

[00225] The strain Streptococcus thermophilus CHCC16731 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany on June 4, 2014 under DSM Accession No. 28889.

[00226] The strain Streptococcus thermophilus CHCC19216 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on December 8, 2015 under DSM Accession No. 32227.

[00227] The strain Streptococcus thermophilus CHCC15757 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03.04.2012 under DSM Accession No. 25850.

[00228] The strain Streptococcus thermophilus CHCC15887 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 03.04.2012 under DSM Accession No. 25851.

[00229] The strain Streptococcus thermophilus CHCC16404 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 12.12.2012 under DSM Accession No. 26722.

[00230] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC18944 deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.06.2014 under no. access code DSM 28910.

[00231] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 03.04.2007 under DSM Accession No. 19252.

[00232] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 06.09.2012 under DSM Accession No. 26420.

[00233] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 06.09.2012 under DSM Accession No. 26421.

[00234] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC12813 deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig on 29.09.2010 under DSM Accession No. 24074.

[00235] The deposits were made in accordance with the Budapest treaty on the international recognition of the deposit of microorganisms for the purposes of the patent procedure. REFERENCES WO2011/026863 WO2011/092300 WO2013/160413 Pool et al. (2006) Metabolic Engineering 8(5); 456-464 Thompson et al. (1985) J. Bacteriol. 162(1); 217–223 Chervaux et al. (2000). Appl and Environ Microbiol, 66, 5306-5311 Cochu et al. (2003). Appl and Environ Microbiol, 69(9), 5423-5432 Hoier et al. (2010) in A Technology of Cheesemaking, 2nd Ed. Blackwell Publishing, Oxford; 166-192.

Claims (10)

1. Composition for the production of a fermented milk product, characterized in that it comprises: (i) at least one strain of Streptococcus thermophilus (St), wherein the St strain is from galactose fermentation, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein or has a negative effect on expression of the gene, wherein the ability to ferment galactose is determined as disclosed in the specification, and wherein the mutation reduces the protein glucokinase activity by at least 50%, and wherein the glucokinase activity is determined as indicated in the specification, and (ii) at least one strain of Lactobacillus delbrueckii subsp. bulgaricus (Lb), where the Lb strain is lactose deficient and capable of metabolizing glucose, where 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-beta-D-galacto-pyranoside (X-GaI). 2. Composition according to claim 1, characterized by the fact that the St strain is resistant to 2-deoxyglucose. 3. Composition according to claim 1 or 2, characterized in that the St strain carries a mutation that reduces glucose transport into the cell. 4. Composition according to any one of claims 1 to 3, characterized in that the St strain increases the amount of glucose in B milk at 9.5% by at least 5 mg/ml when inoculated into B milk at 9, 5% at a concentration of 106 to 107 CFU/ml and cultured at 40°C for 20 hours. 5. Composition according to any one of claims 1 to 4, characterized in that the St strain increases the amount of glucose in milk B to 9.5% with sucrose at 0.05% by at least 5 mg/ kg ml when inoculated into 9.5% milk B with 0.05% sucrose at a concentration of 106 to 107 CFU/ml and cultured at 40°C for 20 hours. 6. Composition according to any one of claims 1 to 5, characterized in that the Lb strain is selected from the group consisting of the strain deposited at DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, on 12.6.2014 under accession number DSM 28910, and a functionally equivalent Z mutant strain derived from DSM 28910, in which less than 1% of the nucleotides in the bacterial genome have been replaced by another nucleotide, or deleted , compared to the parent strain. 7. Composition according to any one of claims 1 to 6, characterized in that the strain of St is selected from the group consisting of the strain of Streptococcus thermophilus CHCC19216 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen, under the number of accession DSM 32227, the Streptococcus thermophilus strain CHCC16731 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on 4.6.2014 under accession number DSM 28889, the Streptococcus thermophilus strain CHCC15757 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 25850, a strain of Streptococcus thermophilus CHCC15887 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 25851, the Streptococcus thermophilus strain CHCC16404 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26722, and a mutant strain derived therefrom , in which the mutant strain Z is a functionally equivalent mutant of the deposited strains and in which less than 1% of the nucleotides in the bacterial genome have been replaced by another nucleotide, or deleted, compared to the parent strain. 8. Method for producing a fermented dairy product, characterized in that it comprises the inoculation and fermentation of a milk substrate with the composition, as defined in any one of claims 1 to 7. 9. Fermented dairy product, characterized in that it comprises the composition as defined in any one of claims 1 to 7. 10. Use of the composition, as defined in any one of claims 1 to 7, characterized in that it is for the preparation of a fermented dairy product.