EA042276B1 - COMPOSITION OF LACTIC BACTERIA FOR OBTAINING FERMENTED FOOD PRODUCTS WITH IMPROVED NATURAL SWEETNESS AND TASTE - Google Patents

Description

Field of invention

The present invention relates to a composition for producing a fermented dairy product containing (1) at least one strain of Streptococcus thermophilus (St), where this St strain is galactose-fermenting, where this strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, where this the mutation inactivates the protein glucokinase or has a negative effect on the expression of this gene, and (2) at least one strain of Lactobacillus delbrueckii sub. bulgaricus (Lb). Such a composition is used to produce fermented dairy products with improved sweet taste.

Prior Art

Pure fermented dairy products are recognized by their tart or sour taste as a result of lactose being converted to lactic acid by lactic acid bacteria during fermentation. They are therefore often sweetened with fruit, honey, sugar or artificial sweeteners to satisfy consumer desire for a sweeter tasting product.

The food industry has an increasing need for low-calorie, sweet-tasting foods to help overcome the overweight and obesity problems that have become so prevalent over the past 20 years. Sweet taste, usually considered as a pleasant sensation, is caused by the presence of sugars and some other substances. The taste perception of sugars is very different. Using sucrose as a reference as 100, the sweetness of lactose is 16, galactose is 32 and glucose is 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 calorie level.

Sugar in fermented foods is more often replaced with sweeteners such as aspartame, acesulfame K, sucralose, and saccharin, which can produce a sweet taste with fewer calories. However, the use of artificial sweeteners can lead to off-flavours, and several studies showing that the consumption of artificial sweeteners is associated with disadvantages such as increased hunger, allergies, cancer, etc. have contributed to consumer preference for fermented dairy products, which contain only natural sweeteners or preferably do not contain added sweetener.

Thus, it is a particular challenge to develop fermented dairy products that have a strong natural (intrinsic) sweet taste.

The acidity of fermented dairy products is highly dependent on the lactic acid bacteria present and the process parameters used to prepare the fermented dairy product.

The fermentation of the disaccharide lactose has been very well studied in lactic acid bacteria, as it is the main source of carbon in milk. In many species, lactose is broken down after consumption by β-galactosidase into glucose and galactose. Glucose is phosphorylated by glucokinase to glucose-6-phosphate and fermented via the Embden-Meyerhof-Parnass pathway (glycolysis) by most lactic acid bacteria (see drawing).

Streptococcus thermophilus is one of the most commonly used lactic acid bacteria for industrial thermophilic fermentation of milk, where the organism is usually used as part of a mixed starter culture, with the other component being Lactobacillus species, for example Lactobacillus delbrueckii subsp. bulgaricus for yogurt or Lactobacillus helveticus for Swiss type cheese.

The standardized definition of yogurt in many countries requires the presence of Streptococcus thermophilus along with Lactobacillus delbrueckii subsp. bulgaricus. Both types generate desirable amounts of acetaldehyde, which is an important flavor component in yogurt.

Lactose and sucrose are more readily fermented by Streptococcus thermophilus than their constituent monosaccharides. In the presence of excess galactose, only the glucose portion of the lactose molecule is fermented, and when Streptococcus thermophilus is used, galactose accumulates in fermented dairy products. In yogurt, where high acid concentrations limit fermentation, free galactose remains, while the free galactose produced in the early stages of Swiss cheese making is later fermented by Lactobacillus helveticus.

However, galactose-fermenting strains of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus have been described by several investigators (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 producing strains of Streptococcus thermophilus that are galactose-fermenting.

In order to meet the requirements of the food industry, it has become suitable to offer new strains, in particular strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus, which give a more natural sweet taste without additional calories directly in the fermented product (own sweet taste) through the release of glucose.

- 1 042276

Pool et al. (2006. Metabolic Engineering 8(5); 456-464) disclose a strain of Lactococcus lactis in which glucose metabolism is completely impaired by the deletion of genes encoding glucokinase, EII(man/glc) and the recently discovered glucose-PTS EII(cel). The design method is genetic recombination to obtain all mutations, and the resulting strain is therefore a genetically modified organism (GMO) that cannot currently be used in food.

Thompson et al. (1985. J Bacteriol. 162(1); 217-223) examined lactose metabolism in Streptococcus lactis (now renamed Lactococcus lactis). In this work, 2deoxyglucose was used to obtain a mutant in the mannose-PTS system. This mutant was then subjected to ultraviolet (UV) mutagenesis followed by fingerprint screening for glucose negative colonies. This method was used to isolate a double mutant (mannose-PTS and glucokinase). This double mutant was used to investigate the mechanisms involved in the regulation of lactose fermentation by starter organisms. These mutants have several disadvantages compared to their parental strain that make them unsuitable for inclusion in a commercially available starter culture. The cell yield of the mutants was half that of the parental strain per mole of fermented lactose, and the mutants had significantly increased doubling time when grown on lactose. Similarly, the yield of lactic acid was half that of the parent strain per mole of fermented lactose. The behavior of these strains in milk has not been analyzed, but it is expected that the rate of acidification would be significantly reduced.

In addition, Lactococcus lactis is generally not selected for acetaldehyde production and does not contribute to the standardized determination of yogurt.

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

WO2013/160413 discloses Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus strains with improved natural food sweetening and fermented milk lactose reduction properties, and a method for screening and isolating such strains.

WO2015/193449 discloses a method for producing a fermented dairy product with a very low concentration of lactose using a combination of lactase and lactic acid bacteria deficient in glucose metabolism.

WO2015/193459 discloses a process for producing a fermented dairy product with a reduced level of post acidification, which in one embodiment uses a mixture of four strains of lactic acid bacteria deficient in glucose metabolism, and which in another embodiment uses a mixture of lactose deficient strains of lactic acid bacteria.

The object of the present invention is to provide a composition for producing a fermented dairy product containing at least one glucose-deficient strain of Streptococcus thermophilus and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus, wherein the composition provides a fermented dairy product with improved taste.

Brief summary of the invention

This object is achieved using the present invention, which is directed to a composition for producing a fermented dairy product containing (1) at least one strain of Streptococcus thermophilus (St), where this St strain is galactose-fermenting, where this strain carries a mutation in the DNA sequence of the glcK gene , encoding a glucokinase protein, where this mutation inactivates the glucokinase protein or has a negative effect on the expression of this gene, and (2) at least one strain of Lactobacillus delbrueckii subsp. bulgaricus (Lb), where this Lb strain is lactose-deficient and is able to metabolize a non-lactose carbohydrate.

It is well known that conventional starter cultures for the production of fermented dairy products containing the strain of Streptococcus thermophilus and the strain of Lactobacillus delbrueckii subsp. bulgaricus run the risk of developing undesirable off-flavours, eg from free amino acids, eg glutamic acid. In addition, it is well known that off-flavors are produced by Lactobacillus delbrueckii subsp. bulgaricus and that the level of off-flavors formed depends on the level of growth of said strain, which varies depending on a number of factors such as the level of inoculation of the Lactobacillus delbrueckii subsp. bulgaricus strain, the type of growth medium, and fermentation growth conditions, as well as a specific strain of Streptococcus thermophilus and a strain of Lactobacillus delbrueckii subsp. bulgaricus used in starter culture. It has now been found that for starter cultures for producing a fermented dairy product with enhanced sweet taste, containing at least one glucose-deficient strain of Streptococcus thermophilus and at least one strain of Lactobacillus delbrueckii subsp. bulgaricus, the formation of undesirable off-flavors is at a particularly high level. This is consistent with the fact that in such starter cultures the Lactobacillus delbrueckii subsp. bulgaricus strain grows to a particularly high cell number.

- 2 042276

Surprisingly, it has been found that the problem of off-flavors when using starter cultures with a glucose-deficient strain of Streptococcus thermophilus can be solved by using a lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, for example a lactose-deficient glucose-positive strain of Lactobacillus delbrueckii subsp. bulgaricus. It has also been found that the lactose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus used in the composition of the invention does not lead to any subsequent acidification. Finally, it was found that lactose-deficient Lactobacillus delbrueckii subsp. bulgaricus in combination with glucose-deficient strains of Streptococcus thermophilus gave a very large number of cells compared to traditional ones, i.e. lactose-positive glucose-positive strains of Lactobacillus delbrueckii subsp. bulgaricus.

It is quite surprising that it is possible to avoid the formation of off-flavours, for example from a lactose-deficient glucose-positive strain of Lactobacillus delbrueckii subsp. bulgaricus, and to avoid subsequent acidification in view of the fact that Lactobacillus delbrueckii subsp. bulgaricus is glucose-positive and is present in a fermented dairy product containing glucose, where in theory this the strain would have the ability to continue growing during storage of the fermented dairy product.

In addition, even more surprising is that it is possible to avoid the formation of off-flavors from Lactobacillus delbrueckii subsp. bulgaricus and to avoid subsequent acidification, while obtaining a large number of cells of Lactobacillus delbrueckii subsp. bulgaricus, since both off-flavors and subsequent acidification occurs due to the growth of a strain of Lactobacillus delbrueckii subsp. bulgaricus.

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

Brief description of graphic materials

The drawing shows 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.

The term lactic acid bacterium as used herein means a gram-positive microaerophilic or anaerobic bacterium that ferments sugars to form acids, including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The most industrially useful lactic acid bacteria are within the order Lactobacillales, which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Lactic acid bacteria, including bacteria of the species Lactobacillus sp. and Streptococcus thermophilus are commonly supplied to the dairy industry either as frozen or freeze-dried cultures for bulk sourdough propagation, or as so-called direct inoculation cultures (DVS) intended for direct inoculation into a fermentation vessel or vat to produce a dairy product such as fermented dairy product. Such cultures are generally referred to as starter cultures or sourdoughs.

The use of terms in the singular in the context of the description of this invention (especially in the context of the following claims) should be construed as embracing the terms in both the singular and the plural, unless otherwise indicated here, or this is clearly contrary to the context. The terms containing, having, including, and containing are to be construed as open terms (i.e., meaning including, but not limited to), unless otherwise noted. The listing of ranges of values here is simply intended to serve as a shorthand way of individually referring to each individual value falling within a given range, unless otherwise noted here, and each individual value is included in this specification as if it were here individually. listed. All methods described here can be performed in any suitable order, unless otherwise indicated here, or otherwise clearly does not contradict the context. The use of any and all examples provided herein, or exemplary language (eg, such as) is merely intended to better illuminate the invention and is not intended to limit the scope of the invention unless otherwise stated. Nothing in the description of the invention should be construed as indicating any unclaimed element essential to the practice of this invention.

In the context of the present invention, the expression "enhancement of sweet taste" means an increase in sweet taste compared to the sweet taste caused by a mother strain that does not carry a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, where this mutation inactivates the glucokinase protein or has a negative effect on the expression of this gene. .

- 3 042276

The expression CFU means colony forming units.

Strains of Streptococcus thermoyhilus compositions according to the invention.

In some countries, the standardized definition of yogurt requires the presence of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Both types generate desirable amounts of acetaldehyde, which is an important flavor component in yogurt.

Cheese such as mozzarella and pizza cheese, as well as feta, can also be fermented using both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (H0ier et al. (2010) in The Technology of Cheese making, ^2nd Ed. Blackwell Publishing, Oxford; 166-192).

In order to meet 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 give a more natural sweet taste directly in the fermented product (own sweet taste) without providing additional calories.

Streptococcus thermophilus is one of the most commonly used lactic acid bacteria for commercial milk fermentation, where the organism is usually used as part of a mixed starter culture, with the other component being a Lactobacillus species, for example, Lactobacillus delbrueckii subsp. bulgaricus for yogurt and Lactobacillus helveticus for Swiss-type cheese.

Only the glucose part of the lactose molecule is fermented by Streptococcus thermophilus, while galactose accumulates in fermented dairy products when using Streptococcus thermophilus. In yogurt, in which high acid concentrations limit fermentation, free galactose is retained, while free galactose, produced in the early stages of Swiss cheese making, is later fermented by Lactobacillus helveticus. Lactobacillus lactis, found in many starter cultures used to make cheese, is also able to consume the galactose produced by Streptococcus thermophilus.

In order to provide Streptococcus thermophilus strains with as optimal growth efficiency as possible, the present inventors exposed galactose-fermenting Streptococcus thermophilus strains to the selective agent 2-deoxyglucose. Typically, 2-deoxyglucose-resistant mutants have mutations in the gene encoding glucokinase and in the genes encoding glucose transport proteins. An isolated CHCC16731 mutant that is 2deoxyglucose resistant has a mutation in its glucokinase (glcK) gene. In addition to the mutation in the glucokinase gene, both CHCC16731 and CHCC19216 have a mutation which means that the secreted glucose is not transported back into the cells.

Surprisingly, such mutants alone are still fully capable of acidifying milk, although the acidification time to pH 5 is delayed by 2-5 hours. Therefore, as such, they are useful in fermented milk applications, and they have retained the ability of the mother strains to acidify milk, which is characteristic of yoghurt. Additionally, the mutants were found to release high levels of glucose when the mutants were inoculated into 9.5% B-milk with 0.05% sucrose and fermented at 40° C. for at least 20 hours. At the same time, very low levels of lactose are retained in fermented milk. Therefore, the use of such strains to produce fermented dairy products may be important for people with lactose intolerance.

Therefore, the final fermented milk has an increased intrinsic sweetness index calculated as described by Godshall (1988. Food Technology 42(11): 71-78).

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

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

A bacterial strain, as used herein, refers to a bacterium that remains genetically unchanged through growth and reproduction. A number of identical bacteria are included.

The term galactose-fermenting strains of Streptococcus thermophilus, as used herein, refers to strains of Streptococcus thermophilus that are capable of growing on/in M17 plus 2% galactose medium. Galactose-fermenting strains of Streptococcus thermophilus are defined here as strains of Streptococcus thermophilus that lower the pH of M17 broth containing 2% galactose as the only carbohydrate to 5.5 or lower when inoculated from an overnight culture at 1% and incubated for 24 hours at 37°C.

The term mutation inactivates the glucokinase protein, as used herein, refers to a mutation that results in an inactivated glucokinase protein, a glucokinase protein that, if present in a cell, is unable to perform its normal function, as well as mutations that prevent the formation of the glucokinase protein or lead to degradation of the glucokinase protein.

In particular, an inactivated glucokinase protein is a protein that, compared to a functional glucokinase protein, is unable to facilitate the phosphorylation of glucose to glucose-6-phosphate or facilitates the phosphorylation of glucose to glucose-6-phosphate at a significantly reduced rate. The gene encoding such an inactivated glucokinase protein, as compared to the gene encoding a functional glucokinase protein, contains a mutation in the open reading frame (ORF) of that gene, where said mutation may include a deletion, a mutation leading to a reading frame shift, the 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, but is not limited to.

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

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

The term functional glucokinase protein, as used herein, refers to a glucokinase protein that, when present in a cell, facilitates the phosphorylation of glucose to glucose-6-phosphate. In particular, a functional glucokinase protein may be encoded by a gene containing an ORF that has a sequence corresponding to position 1-966 in SEQ ID NO: 1, or a sequence that has at least 85% identity, for example at least 90% a specific identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, to the sequence corresponding to position 1-966 in SEQ ID NO: 1.

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), a modified algorithm described by Karlin and Altschul (1993. Proc. Natl. Acad Sci USA 90;58735877); the algorithm of Myers and Miller (1988. CABIOS 4; 11-17); the algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48; 443-453); and the algorithm of Pearson and Lipman (1988. Proc. Natl. Acad. Sci. USA 85; 2444-2448). Also available is a computer program for determining the identity of nucleic acid sequences or amino acid sequences based on these mathematical algorithms. For example, comparison of nucleotide sequences can be carried out using the BLASTN program, score - 100, word length -12. Comparison of amino acid sequences can be carried out using the BLASTX program, score - 50, word length - 3. As for the remaining parameters of the BLAST programs, you can use the default parameters.

In many countries, the use of genetically modified organisms (GMOs) for fermented dairy products is unacceptable. Instead, the present invention provides naturally occurring or induced mutant strains that can provide the desired accumulation of glucose in a fermented dairy product.

Thus, in an even more preferred embodiment of the present invention, the mutant strain is a naturally occurring mutant or an induced mutant.

The terms mutant bacterium or mutant strain, as used herein, refers to a natural (spontaneous, naturally occurring) mutant bacterium or an induced mutant bacterium containing in its genome (DNA) one or more mutations that are not present in wild-type DNA. An induced mutant is a bacterium where 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 a naturally occurring mutant has not been mutagenized by humans. The mutant bacteria here are not GMOs (Genetically Modified Organisms), i.e. they are not modified by recombinant DNA technology.

The term wild-type strain refers to the unmutated form of a bacterium as it occurs in nature.

Terms such as strains with a sweet taste enhancing property, strains that can provide a desirable accumulation of glucose in a fermented dairy product, and strains with improved natural sweet taste enhancing properties in foods are used interchangeably herein to characterize the useful aspect of using the strains of the present invention in fermentation of dairy products.

In a preferred embodiment, the mutant strain of Streptococcus thermophilus according to the invention increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into 9.5% B-milk at a concentration of 106-10 ⁷ CFU/ ml and growing at 40°C for less

- 5 042276 at least 20 hours.

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

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

In more preferred embodiments of the invention, the mutant strain results in an increase in glucose to at least 6 mg/mL, such as at least 7 mg/mL, such as at least 8 mg/mL, such as at least 9 mg/mL, such as at least 10 mg/ml, such as at least 11 mg/ml, such as at least 12 mg/ml, such as at least 13 mg/ml, such as at least 14 mg/ml, such as at least 15 mg /ml, for example at least 20 mg/ml, for example at least 25 mg/ml.

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

The term 2-deoxyglucose resistant is defined herein in that a particular mutant bacterial strain has the ability to grow to a colony when streaked onto an M17 medium plate containing 20 mM 2-deoxyglucose after incubation at 40° C. for 20 hours. The presence of 2deoxyglucose in the culture medium will prevent the growth of non-mutated strains, while the growth of mutated strains is unaffected or not significantly affected. Non-mutated strains that can be used as sensitive control strains in the evaluation of resistance preferably include strains CHCC14994 and CHCC11976.

The mutant strains of Streptococcus thermophilus according to the invention release glucose into milk upon inoculation of 9.5% B-milk with 10 ⁶ -10 ⁷ CFU/ml of the strain of Streptococcus thermophilus according to the invention and fermentation with strains of Streptococcus thermophilus according to the invention at 40°C for at least 20 hours. Preferably, such mutant strains alone will release at least 5 mg/ml glucose into B-milk when 9.5% B-milk is inoculated with 10 ⁶ -10 ⁷ CFU/ml of the Streptococcus thermophilus strain of the invention and fermented with Streptococcus thermophilus strains at 40°C for at least 20 hours. These strains are still fully capable of acidifying milk, although the acidification time to pH 5 is delayed by 2-5 hours. The final fermented milk contains less than 15 mg/ml of lactose in the fermented milk. The final fermented milk therefore has a higher intrinsic sweet taste index increased by about 2 times or more.

In another embodiment, the mutant strain according to the invention may differ in its growth pattern. This is illustrated by the finding that the growth rate of the mutant strain is faster in M17 plus 2% galactose than in M17 plus 2% glucose. Growth rate is measured as the development of the optical density of an exponentially growing culture at 600 nanometers (OD ₆₀₀ ) over time.

In a preferred embodiment, the growth rate is at least 5% faster, such as at least 10% faster, such as at least 15% faster, such as at least 20% faster in M17 plus 2% galactose medium than in M17 plus 2% glucose.

Mutation in the glcK gene.

In one 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 G with A at position 745 in SEQ ID NO: 1. The CHCC16731 strain has this mutation.

In a preferred embodiment, this 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, a mutation in the glcK gene results in a T being replaced by a C at position 214 in SEQ ID NO: 1 (not shown).

In another preferred embodiment, this 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).

Preferably, a mutation in the glcK gene results in a change from C to T at position 422 in SEQ ID NO: 1 (not shown).

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

A mutation that reduces the transport of glucose into the cell.

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

The term mutation that reduces the transport of glucose into the cell as used here

- 6 042276 refers to a mutation in the gene encoding a protein involved in the transport of glucose, which leads to the accumulation of glucose in the cell environment.

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

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%.

Transport of glucose into a cell can be determined by analyzing glucose uptake as described by Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423-5432).

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

This component can be any component of the glucose transporter protein that is critical for glucose transport. For example, it is contemplated that inactivation of any glucose/mannose PTS component in Streptococcus thermophilus will result in inactivation of glucose transporter function.

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 unable to perform its normal function, as well as mutations that prevent the formation of the glucose transporter protein or lead to degradation of the glucose transporter protein.

In particular, an inactivated glucose transporter protein is a protein that, compared to a functional glucose transporter protein, is unable to facilitate the transport of glucose across the plasma membrane or facilitates the transport of glucose across the plasma membrane at a significantly reduced rate. The gene encoding such an inactivated glucose transporter protein, compared to the gene encoding a functional glucose transporter protein, contains a mutation in the open reading frame (ORF) of this gene, where the specified mutation may include a deletion, a mutation leading to a reading frame shift, the introduction of a stop a codon or mutation that results in an amino acid substitution that changes the functional properties of the protein, or a promoter mutation that reduces or abolishes transcription or translation of a gene, but is not limited to.

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

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

The term functional glucose transport protein, as used herein, refers to a glucose transport protein that, when present in a cell, facilitates the transport of glucose across the plasma membrane.

In a preferred embodiment of the invention, the strain of Streptococcus thermophilus of the invention carries a mutation in the DNA sequence of the manN gene encoding the protein IID ^Man of the glucose/mannose phosphotransferase system, where this mutation inactivates the IIDMan protein or has a negative effect on the expression of this gene.

CHCC16731 has a threonine-to-proline substitution at position 79 in the manN gene encoding the IIDMan protein of the glucose/mannose phosphotransferase system. Preferably, this mutation in the manN gene results in a change from A to C at position 235 in SEQ ID NO:3.

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 protein IIDMan of the glucose/mannose phosphotransferase system, where this mutation results in the replacement of threonine by proline at position 79 of SEQ ID NO: 4.

In another preferred embodiment of the invention, the Streptococcus thermophilus strain of the invention carries a mutation in the DNA sequence of the tamM gene encoding ^{the Man} IIC protein of the glucose/mannose phosphotransferase system, where the mutation inactivates ^{the Man} IIC protein or has a negative effect on the expression of this gene.

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

Preferred strains of Streptococcus thermophilus according to the invention.

In a preferred embodiment of the composition according to the invention, the Streptococcus thermophilus strain

- 7 042276 is selected from the group consisting of Streptococcus thermophilus strain CHCC19216 which has been deposited with the German Collection of Microorganisms and Cell Cultures under Accession No. DSM 32227, Streptococcus thermophilus strain CHCC16731 which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, June 4, 2014, Accession No. DSM 28889, Streptococcus thermophilus strain CHCC15757, which has been deposited with the German Collection of Microorganisms and Cell Cultures, Accession No. DSM 25850, Streptococcus thermophilus strain CHCC15887, which has been deposited with German Collection of Microorganisms and Cell Cultures, Accession No. DSM 25851, Streptococcus thermophilus strain CHCC16404, which was deposited with the German Collection of Microorganisms and Cell Cultures, Accession No. DSM 26722, and a mutant strain derived therefrom, where this mutant strain is obtained using one of deposited strains as starting material, and where this mutant has retained or further improved texturing ability and the ability of the specified deposited strain to secrete glucose.

In the present context, the term mutant strain should be understood as strains obtained, or strains that can be obtained from the strain (or their parent strain) according to the invention by, for example, genetic engineering, irradiation treatment and/or chemical treatment. Strains derived from them can also be spontaneously occurring mutants. It is preferred that the strains derived therefrom are functionally equivalent mutants, for example mutants that have substantially the same or improved properties with respect to their parent strain. The term mutant strains particularly refers to strains obtained by subjecting a strain of the invention to any conventionally used mutagenic treatment, including treatment with a chemical mutagen such as ethanemethanesulfonate (EMS) or K-methyl-K'-nitro-K-nitroguanidine (NTG), UV light, or to a spontaneously emerging mutant. The mutant may have been subjected to several mutagenization treatments (one treatment should be understood as one mutagenesis 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 be carried out ( or screening/selection steps). In the preferred mutant of the present invention, 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 with a different nucleotide or deleted compared to the parent strain.

A method for producing a strain of Streptococcus thermophilus that enhances sweet taste.

In a preferred embodiment of the invention, the parent strain used to develop the sweet taste enhancing strain of Streptococcus thermophilus is a texturizing strain of Streptococcus thermophilus, which can be produced as described herein.

In a preferred embodiment, such texturizing strain is selected from the group consisting of Streptococcus thermophilus strain CHCC11342 which has been deposited with the German Collection of Microorganisms and Cell Cultures under accession no. DSM 22932, Streptococcus thermophilus strain CHCC11976 which has been deposited with the German collection of microorganisms and cell cultures under accession no. accession DSM 22934, Streptococcus thermophilus strain CHCC12339, which was deposited with the German Collection of Microorganisms and Cell Cultures under Accession No. DSM 24090, and strains derived therefrom.

Alternatively, the starting strain used to develop the sweet taste-enhancing Streptococcus thermophilus strain is a non-texturing Streptococcus thermophilus strain, in which case the texturizing ability is introduced into the sweet taste-enhancing Streptococcus thermophilus strain as soon as it is produced, i. the sweet taste enhancing strain is used as the starting strain in the method for producing the texturizing strain described in this specification.

The first step in the process for producing a sweet taste enhancing strain of Streptococcus thermophilus is to provide a galactose positive strain, i. a strain that is able to use galactose as a carbohydrate source. Galactose-positive strains can be obtained by a method comprising the step of streaking the bacteria to be tested on agar plates, such as M17 agar plates, containing a certain concentration, for example 2%, of galactose (the only source of carbohydrates), and identifying colonies capable of growing on these cups.

For the selection of bacteria having a mutation in the glucokinase (glcK) gene, 2deoxyglucose is used and the growth pattern of bacteria in M17 plus 2% galactose medium compared to M17 plus 2% glucose medium is used.

One method for screening and isolating a strain of Streptococcus thermophilus with a mutated glcK gene includes the following steps:

a) providing a galactose-fermenting parent strain of Streptococcus thermophilus;

b) selection and isolation from the pool of mutant strains of Streptococcus thermophilus obtained from the parent strain, the pool of mutant strains of Streptococcus thermophilus that are resistant to 2-deoxyglucose; And

- 8 042276

c) selection and isolation from the pool of mutant strains of Streptococcus thermophilus that are resistant to 2-deoxyglucose, the mutant strain of Streptococcus thermophilus, if the growth rate of this mutant strain of Streptococcus thermophilus is higher in the medium M17 plus 2% galactose than in the medium M17 plus 2% glucose .

The term 2-deoxyglucose-resistant is defined herein in that a particular mutated bacterial strain has the ability to grow to a colony when streaked on a plate of M17 medium containing 2% lactose or 2% galactose and containing 20 mM 2-deoxyglucose after incubation at 40°C. C within 20 hours. The presence of 2-deoxyglucose in the culture medium will prevent the growth of non-mutated strains, while the growth of mutated strains is unaffected or not significantly affected. Non-mutated strains that can be used as sensitive control strains in the evaluation of resistance include strain CHCC11976.

In one embodiment, the method further comprises the step al) of subjecting the mother strain to mutagenesis, for example, exposing the mother strain to a chemical and/or physical mutagen.

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

Streptococcus thermophilus galactose-fermenting parent strains are capable of growing on/in M17 plus 2% galactose medium and are defined herein in that they have the ability to lower the pH in M17 broth containing 2% galactose as the sole carbohydrate to 5.5 or lower when inoculated from culture grown overnight at 1% and incubated for 24 hours at 37°C. Such galactose positive strains have been described in WO2011/026863 (Chr. Hansen A/S) and WO2011/092300 (Chr. Hansen A/S).

In the context of the present invention, the term strains derived from is to be understood as strains derived from, or strains that can be obtained from texturizing or galactose fermenting parent strains of Streptococcus thermophilus, through, for example, genetic engineering, irradiation treatment and/or chemical treatment. Strains derived from them may also be spontaneously occurring mutants. It is preferred that strains derived therefrom are functionally equivalent mutants, eg mutants that have substantially the same or improved properties (eg texture or galactose fermentation) as their parent strain. In particular, the term strains derived therefrom refers to strains obtained by subjecting the strain of the invention to any conventionally used mutagenic treatment, including treatment with a chemical mutagen such as ethanemethanesulfonate (EMS) or K-methyl-K'-nitro-K-nitroguanidine ( NTG), UV light, or to a spontaneously occurring mutant. The mutant may have been subjected to multiple mutagenization treatments (one treatment should be understood as one mutagenesis step, followed by a screening/selection step), but in the present invention it is preferred that no more than 20, or no more than 10, or no more than 5 treatments (or steps screening/selection). In the present invention, in the 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 with a different nucleotide or deleted compared to the parent strain.

In the present invention, the expression glcK gene encoding glucokinase means any DNA sequence of Streptococcus thermophilus encoding a protein having glucokinase activity, including the specific glucokinase encoded by the DNA sequence of SEQ ID NO: 1. Glucokinase catalyzes the reaction that converts glucose to glucose-6-phosphate, see .drawing.

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

The terms lactose deficient and lactose deficient are used in the context of the present invention to characterize LABs that either partially or completely lose their ability to use lactose as a source for cell growth or cell viability. The respective LABs are capable of metabolizing one or more carbohydrates selected from sucrose, galactose and/or glucose, or another fermentable carbohydrate. Since these carbohydrates are not naturally present in milk in sufficient quantities to support lactose-deficient mutant fermentation, these carbohydrates must be added to milk. Lactose-deficient and partially deficient LABs can be characterized as white colonies on medium containing lactose and X-Gal.

In the context of the present invention, the term X-Gal means 5-bromo-4-chloro-3-indolyl-beta-Ogalactopyranoside, which is a chromogenic substrate for beta-galactosidase, which

- 9 042276 hydrolyzes X-Gal to colorless galactose and 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to form a blue pigment.

In a specific 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 specific embodiment of the invention, the lactose-deficient strain is capable of metabolizing galactose.

In a specific embodiment of the composition of the invention, the Lb strain is selected from the group consisting of a strain deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 06/12/2014 under accession number DSM 28910, and a mutant strain derived from DSM 28910, where this mutant strain is additionally characterized by the ability to form white colonies on a medium containing lactose and X-Gal.

Lactose-deficient strains can be obtained from a suitable lactose-positive maternal strain by mutation. Lactose-deficient strains were selected after UV mutagenesis as white colonies (indicative of a lactose-deficient phenotype) on a suitable medium, eg MRS agar plates with 1% lactose and 200 mg/ml X-Gal. The lactose-positive strains have β-galactosidase activity, and the colonies of the lactose-positive strain appear blue due to β-galactosidase activity. Lactobacillus delbrueckii subsp. bulgaricus strain CHCC10019 deposited for WO2011/000879 at DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig 04/03/2007 under DSM Accession No. 19252.

Composition.

The present invention further relates to a composition containing from 10 ⁴ to 10 ¹² CFU (colony forming unit)/g of Streptococcus thermophilus strain, for example from 105 to 10 ¹¹ CFU/g, for example from 106 to 10 ¹⁰ CFU/g, or for example from 10 ⁷ up to 109 CFU/g of Streptococcus thermophilus strain.

In a preferred embodiment, the Streptococcus thermophilus strain of this composition is unable to acidify 9.5% B-milk, defined as resulting in a pH decrease of less than 1.0 when 9.5% B-milk is inoculated 106-10 ⁷ CFU/ ml of Streptococcus thermophilus strain and incubation for 14 hours at 40°C, and this composition additionally contains an amount of a compound that can start the acidification of 9.5% B-milk with Streptococcus thermophilus strain CHCC16404, which was deposited in the German Collection of Microorganisms and Cellular of cultures under accession number DSM 26722, defined as resulting in a decrease in pH of 1.0 or more when inoculated with 9.5% B-milk with 106-10 ⁷ cfu/ml of a strain of Streptococcus thermophilus and incubated for 14 hours at 40° WITH.

Preferably, this compound is sucrose.

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

In a particularly preferred embodiment, the composition further comprises from 10 ⁴ to 10 ¹² CFU/g of Lactobacillus delbrueckii subsp. bulgaricus, for example from 105 to 10 ¹¹ CFU/g, for example from 106 to 10 ¹⁰ CFU/g, or for example from 10 ⁷ to 109 CFU/g strain Lactobacillus delbrueckii subsp. bulgaricus.

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

In a specific embodiment of the present invention, the composition comprises at least one St strain selected from the group consisting of the Streptococcus thermophilus strain CHCC16731, which has been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, June 4, 2014, Accession No. DSM 28889, Streptococcus thermophilus strain CHCC15757, which was deposited with the German Collection of Microorganisms and Cell Cultures, Accession No. DSM 25850, and Streptococcus thermophilus strain CHCC16404, which was deposited to the German Collection of Microorganisms and Cell Cultures under Accession No. DSM 26722, and the Lb strain deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 06/12/2014, DSM Accession No. 28910. Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are commonly used as starter cultures for technological purposes in the production of various food products, for example in the dairy industry. , for example for fermented dairy products. Thus, in another preferred embodiment, this composition is suitable as a starter culture.

Starter cultures may be provided as frozen or dried starter cultures, in addition to liquid starter cultures. Thus, in another preferred

- 10 042276 embodiment, the composition is in frozen, lyophilized or liquid form.

As disclosed in WO 2005/003327, it is useful to add certain cryoprotectants to the starter culture. Thus, the starter culture composition of the present invention may contain one or more cryoprotectants 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 derivative of any such compound.

Method for obtaining a fermented dairy product.

This invention is further directed to a process for producing a fermented dairy product, comprising inoculating and fermenting a dairy substrate with a composition according to the present invention.

The term milk should be understood as the secret of the mammary glands obtained by milking any animal such as cow, sheep, goat, buffalo or camel. In a preferred embodiment, the milk is cow's milk.

The term milk substrate can refer to any raw and/or processed milk material that can be fermented according to the process of the invention. Thus, useful dairy substrates include solutions/suspensions of any milk or milk-like products containing protein, such as whole or reduced fat milk, skim milk, buttermilk, reconstituted milk powder, condensed milk, milk powder, whey, whey permeate, lactose, lactose crystallization mother liquor, whey protein concentrate or cream, but are not limited to. Obviously, the milk substrate can be derived from any mammal, for example, be essentially pure mammalian milk or diluted milk powder.

Preferably, at least a portion of the protein in the milk substrate is a protein naturally occurring in milk, such as casein or whey protein. However, some of the protein may be proteins that do not occur naturally in milk.

Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.

The term homogenization, as used here, means intensive mixing to obtain a soluble suspension or emulsion. When homogenization is carried out before fermentation, it can be carried out in such a way as to break up the milk fat to smaller sizes, so that it no longer separates from the milk. This can be done by passing milk through small holes at high pressure.

The term pasteurization as used herein means the treatment of a milk substrate to reduce or eliminate the presence of living organisms such as microorganisms. Preferably, pasteurization is achieved by maintaining a specific temperature for a specific period of time. This particular temperature is usually reached by heating. The temperature and length of time may be chosen to kill or inactivate certain bacteria, such as harmful bacteria. This may be followed by a rapid cooling step.

Fermentation in the methods of the present invention means the conversion of carbohydrates into alcohols or acids through the action of a microorganism.

Preferably, the fermentation in the methods of the invention includes the conversion of lactose to lactic acid.

Fermentation methods for use in making fermented dairy products are well known and one skilled in the art knows how to select suitable process conditions such as temperature, oxygen content, amount and characteristics of the microorganism(s), and process time. Obviously, the fermentation conditions are chosen in such a way as to ensure the implementation of the present invention, i. to obtain a dairy product in solid or liquid form (fermented dairy product).

The term fermented dairy product as used herein refers to a food or feed product, where the preparation of the food or feed product involves the fermentation of a milk substrate with a lactic acid bacterium. The term fermented dairy product as used herein includes, but is not limited to, products such as thermophilically fermented dairy products such as yogurt, mesophilically fermented dairy products such as sour cream and buttermilk, and fermented whey.

The term thermophile here refers to microorganisms that live best at temperatures above 35°C. The most industrially useful thermophilic bacteria include Streptococcus spp. and Lactobacillus spp. The term thermophilic fermentation here refers to fermentation at a temperature higher than about 35°C, for example from about 35 to about 45°C. The term thermophilically fermented dairy product refers to fermented dairy products.

- 11 042276 products obtained by thermophilic fermentation of thermophilic starter culture, and it includes fermented dairy products such as oven-produced yogurt, curd-disrupted yogurt, and drinking yogurt, such as Yakult Japanese bio-yogurt.

The term mesophile here refers to microorganisms that live best at moderate temperatures (15-35°C). The most commercially useful mesophilic bacteria include Lactococcus spp. and Leuconostoc spp. The term mesophilic fermentation here refers to fermentation at a temperature of from about 22 to about 35°C. The term mesophilically fermented dairy product refers to fermented dairy products obtained by the mesophilic fermentation of a mesophilic starter culture and includes fermented dairy products such as buttermilk, curdled milk, sour milk, sour cream, kefir, and young cheese such as cottage cheese and cream cheese.

In a preferred embodiment, the concentration of inoculated Streptococcus thermophilus cells is from 10 ⁴ to 10 ⁹ CFU of Streptococcus thermophilus cells per ml of milk substrate, for example from 10 ⁴ to 10 ⁸ CFU of Streptococcus thermophilus cells per ml of milk substrate.

In another preferred embodiment of the method according to the invention, the strain of Streptococcus thermophilus is not able to acidify 9.5% B-milk, which is defined as resulting in a decrease in pH of less than 1.0 upon inoculation of 9.5% B-milk 10 ⁶ - 10 ⁷ cfu/ml of Streptococcus thermophilus strain and incubation for 14 hours at 40° C. and this milk substrate adds an effective amount of the compound to trigger acidification of 9.5% B-milk by Streptococcus thermophilus strain, defined as resulting in a decrease in pH by 1.0 or more when inoculated with 9.5% B-milk 10 ⁶ -10 ⁷ CFU/ml of Streptococcus thermophilus strain and incubated for 14 hours at 40°C.

Preferably, this compound is sucrose.

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

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

Examples of cheeses that are fermented using Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus include mozzarella and pizza cheese (Hoier et al. (2010) in The Technology of Cheesemaking, ^2nd Ed. Blackwell Publishing, Oxford; 166-192).

Preferably, the fermented dairy product is yogurt.

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

Fermented dairy product.

The present invention further relates to a fermented dairy product containing a composition according to the present invention.

In another preferred embodiment, the fermented dairy product may be yogurt, cheese, sour cream and buttermilk, as well as fermented whey. Preferably, the fermented dairy product is yogurt.

The use of the composition according to the invention

Another aspect of this invention relates to the use of the composition according to the invention for the preparation of a fermented dairy product.

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 of 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 of strain CHCC 16731.

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) strain CHCC16731.

SEQ ID NO: 6 shows the amino acid sequence encoded by SEQ ID NO: 5.

- 12 042276

Examples

Materials and methods.

Wednesday.

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

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 meat extract - 5 g meat peptone (peptic) - 2.5 g soy peptone (papaic) - 5 g yeast extract - 2.5 g final pH 7.1 plus/minus 0.2 (25°C);

and broth Ml7 has the following composition per liter of H ₂ O:

ascorbic acid - 0.5 g magnesium sulfate - 0.25 g meat extract - 5 g meat peptone (peptic) - 2.5 g sodium glycerophosphate - 19 g soy peptone (papaic) - 5 g tryptone - 2.5 g yeast extract - 2.5 g final pH 7.0 plus/minus 0.2 (25°C).

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

As known to the person skilled in the art, M17 medium is a medium that is considered suitable for the growth of Streptococcus thermophilus. In addition, as one skilled in the art will understand, in the context of the present invention, the M17 concentrate can be supplied from different suppliers and, regardless of the particular supplier (within the standard range of measurements), the same relevant 2-deoxyglucose resistance result will be obtained here for the relevant cell of interest here.

The medium used for culturing Lactobacillus delbrueckii subsp. bulgaricus was MRS-IM medium. MRS-IM was used either as agar plates or broth.

The MRS-IM agar medium had the following composition per liter H ₂ O:

trypton Oxoid L 42 10.0 g yeast extract Oxoid L 21 5.0 g Tween 80 Merck, no. 8.22187 1.0 g K ₂ NRO ₄ Merck, No. 105104 2.6 g Na-acetate Merck, No. 106267 5.0 g Diammonium hydrocitrate Merck, No. 101154 2.0 g MgSO ₄ 7 H2O Merck, No. 105882 0.2 g MnSO ₄ H2O Merck, No. 105941 0.05 g agar SO-BI-GEL 13.0 g

After autoclaving, the pH was adjusted to 6.9 plus/minus 0.1 at 25°C.

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

- 13 042276

trypton Oxoid L 42 10.0 g yeast extract Oxoid L 21 5.0 g Tween 80 Merck, no. 8.22187 1.0 g K ₂ NRO ₄ Merck, No. 105104 2.6 g Na-acetate Merck, No. 106267 5.0 g Diammonium hydrocitrate Merck, No. 101154 2.0 g MgSO ₄ 7 H2O Merck, No. 105882 0.2 g MnSO ₄ H2O Merck, No. 105941 0.05 g

After autoclaving, the pH is adjusted to 6.9 plus/minus 0.1 at 25°C. Carbon sources, 20 g/l lactose or 20 g/l glucose, were first subjected to sterilizing filtration and then added to the autoclaved broth.

The above MRS-IM media can be modified to some extent without affecting the ability of these media to support the growth of Lactobacillus delbrueckii subsp. bulgaricus. In addition, as will be appreciated by those skilled in the art, the MRS-IM concentrate or the various components described above may be purchased from various vendors and used to prepare the MRS-IM medium. These media will likewise be used in the examples below, in particular in the 2-deoxyglucose resistance selection assay.

mother strains.

Streptococcus thermophilus CHCC11976 (galactose fermenting strain with a mutation in the GalK gene as described in WO 2011/026863).

Streptococcus thermophilus CHCC12339 (phage resistant and texturizing as described in WO2011/092300).

Streptococcus thermophilus CHCC18948 (galactose-fermenting mutant CGCC12339 with a mutation in the GalK gene).

Strains resistant to 2-deoxyglucose.

Streptococcus thermophilus CHCC16165 (CHCC11976 2-deoxyglucose resistant mutant).

Streptococcus thermophilus CHCC16731 (sweet-enhancing and texturing mutant CHCC16165).

Streptococcus thermophilus CHCC19216 (sweet-enhancing and texturing mutant CHCC18948).

Example 1 Use of 2-deoxyglucose to isolate a glucokinase mutant from Streptococcus thermophilus CHCC11976 with enhanced glucose release.

In order to isolate mutants of Streptococcus thermophilus strain CHCC11976, single colony cells were inoculated into 10 ml of M17 broth containing 2% lactose and grown overnight at 40°C.

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

Based on this, a number of mutants were identified that could grow faster on galactose than on glucose, as described in Example 2. One such mutant was CHCC16165.

Example 2 Growth pattern of the 2-deoxyglucose resistant mutant.

To ensure the selection of 2-deoxyglucose resistant mutants that can grow on galactose, two strains were used that were selected from a set of galactose fermenting strains. While these galactose fermenting strains still grow at least 10% faster in the exponential phase in M17 broth plus 2% glucose than in M17 broth plus 2% galactose, the 2-deoxyglucose resistant mutant is a derivative of CHCC11976, i.e. . CHCC16165, on the other hand, grows faster in the exponential phase in M17 broth plus 2% galactose than in M17 broth plus 2% glucose.

Growth in the exponential phase is measured here as the development of the optical density of an exponentially growing culture at 600 nanometers (OP ₆₀₀ ) over time at 40°C.

As one skilled in the art would know, it can vary from species to species when the culture is in exponential growth. One skilled in the art would know how to determine growth in an exponential phase, such as in the OD ₆₀₀ range of 0.1-1.0.

The optical density (OD) of the culture is measured on a spectrophotometer.

- 14 042276

Conclusion.

Based on the growth pattern of the 2-deoxyglucose resistant mutant of this Example 2 - for a particular strain of interest (e.g., a strain from a relevant commercial product) - one skilled in the art can test, according to established practice, whether that particular strain of interest has a suitable growth pattern, which is a property of the selected mutants.

Example 3 Selection of a lactose hyperfermenting and glucose secreting Streptococcus thermophilus CHCC16165 mutant.

In order to isolate the lactose hyperfermenting and glucose secreting mutant of Streptococcus thermophilus strain CHCC16165, single colony cells were inoculated into 10 ml of M17 broth containing 2% galactose and grown overnight at 40°C.

The next day, this strain was seeded in serial dilutions on plates with M17 agar containing 2% sucrose and 40 mm concentration of 2-deoxyglucose, and incubated for 20 hours at 40°C. 10 random colonies were selected from the dish and used to inoculate fresh M17 broth containing 2% sucrose and incubated overnight at 40°C. The CHCC16165 mutants were transferred to fresh M17 media containing 2% sucrose.

Example 4 Carbohydrate analysis of fermented milk.

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

Overnight grown cultures of CHCC16165 and CHCC16731 in combination with each of CHCC16159 and CHCC16160 were added to 200 ml B-milk samples and sour at 40°C.

The results are shown in table. 1.

Table 1

Blend of Strains Lactose mg/ml Galactose mg/ml Glucose mg/ml SNSS16165 + SNSS16159 14.0 10.9 6.5 SNSS16165 + SNSS16160 13.5 and about 6.9 SNSS 16731 + SNSS16159 1.0 22.0 16.2 SNSS 16731 + SNSS16160 1.0 22.5 16.0 B-milk 48.9 Not detected Not detected

As can be seen from the results of Table. 1, CHCC16731 is able to remove almost all lactose from milk. CHCC16731 also produces and secretes high levels of both galactose and glucose into milk. Considering that sucrose is a control of 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the fermented dairy product obtained using CHCC16731 has a significantly enhanced sweet taste.

Example 5 Obtaining a galactose-positive mutant of Streptococcus thermophilus CHCC 12339.

Streptococcus thermophilus CHCC12339 was grown overnight in M17 containing 2% lactose at 40°C. The overnight culture was seeded (100 μl) onto an M17 plate containing 2% galactose and incubated anaerobically at 40°C. On plates with M17 containing 2% galactose, 22 colonies appeared. 16 of these were re-streaked on the same type of plates and 8 of them were transferred to liquid M17 containing 2% galactose resulting in overall growth and 4 of these cultures were frozen. 2 of the frozen cultures were spread on M17 plates containing 2% galactose and 40 mM 2 deoxyglucose, and 8 colonies formed were selected for each culture. The 16 mutants selected with 2-deoxyglucose were transferred into liquid M17 containing 2% lactose and 2% galactose and grown overnight at 40°C, after which these mutants were grown in liquid M17 containing 2% galactose for 3 nights, and the production of galactose and glucose was measured and monitored throughout growth. Based on the production of galactose and glucose, 7 cultures were selected to test milk acidification by adding 2 ml of the culture to 200 ml of 9.5% B-milk containing 0.05% sucrose. The 3 mutants had acidification profiles similar to those of the parent strain CHCC12339, and these 3 mutants were streaked to form single colonies and incubated for 48 hours. Then these colonies were transferred into liquid M17 containing

- 15 042276 containing 2% galactose, and the growth of these 3 mutants was tested against CH12339. The mutant having the strongest growth was selected and purified again by streaking a single colony 3 times to obtain a purified galactose-positive strain named CHCC18948 (galactose-positive variant CHCC12339).

Example 6 Preparation of 2-deoxyglucose resistant mutant of Streptococcus thermophilus CHCC18948 with increased glucose release.

100 μl of CHCC18949 culture was distributed on plates with M17 containing 2% galactose and 20 mm or 40 mm 2-deoxyglucose, and incubated overnight at 40°C. 8 colonies were selected from 20 mM plates and 8 colonies from 40 mM plates and grown in liquid M17 containing 2% galactose and 20 mM 2-hydroxyglucose. The resulting cultures were frozen and streaked again, and 12 of the formed colonies were included in M17 medium and used to acidify B-milk. A 1% overnight culture of the 2-deoxyglucose resistant mutant was inoculated into 200 ml of 9.5% B-milk containing 0.05% sucrose and sour at 40°C. All 12 2-deoxyglucose-resistant CHCC18948 mutants produced fermented dairy products with low lactose concentrations and high galactose and glucose concentrations. The 7 mutants were selected for retesting individually and for testing together with Lactobacillus delbrueckii subsp. bulgaricus strains CHCC16159 (described in WO2013/160413). Again, a 1% overnight culture of the 2-deoxyglucose resistant mutant was inoculated into 200 ml of 9.5% B-milk containing 0.05% sucrose and sour at 40°C.

The results are shown in table. 2.

table 2

Strain / mixture of strains Lactose mg/ml Galactose mg/ml Glucose mg/ml CHCH18948-Mutant1 Not detected 17.9 20.8 CHSN18948- Mutant2 Not detected 18.9 23.8 CHCH18948- Mutant3 9.5 13.6 14.5 CHSN18948- Mutant? Not detected 19.1 24.2 CHCH18948-Mutant 10 1.7 16.0 21.1 CHCH18948-Mutant 11 Not detected 17.5 22.9 CHCH18948- Mutant12 3.6 16.9 18.7 CHCH18948- Mutant 1+ SNSS16159 7.9 16.7 9.6 CHCH18948- Mutant2+ SNSS16159 9.1 18.4 10.3 CHCH18948- Mutant3+ SNSS16159 3.5 18.6 I,9 CHSN18948- Mutant? + SNSS16159 10.7 15.1 7.7 SNSN18948- MutantYu + SNSS16159 10.2 16.2 8.5 CHSN18948- MutantI + SNSS16159 10.5 15.9 8.4 CHCH18948- Mutant12+ SNSS16159 3.3 19.0 10.9 B-milk 47.1 Not detected Not detected

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

The results are shown in table. 3.

- 16 042276

Table 3

Strain / mixture of strains Lactose mg/ml Galactose mg/ml Glucose mg/ml CHSN16159 9.9 14.8 8.7 CHSN16160 1.6 18.5 17.5 CHSN16161 12.8 13.7 8.6 CHCH18948-Mutant2+ CHSN16159 6.8 19.4 12.0 CHCH18948-Mutant2+ CHSN16160 Not detected 21.3 17.6 CHCH18948-Mutant2+ CHSN16161 Not detected 20.5 17.1 CHCH18948-Mutant 12+ CHSN16159 5.0 19.3 12.1 SNSN 18948-Mutant 12+ SNSS16160 1.4 21.8 16.5 SNSN 18948-Mutant 12+ SNSS16161 4.2 20.2 14.6 CHSN18948-Mutant3+ SNSS16159 1.6 19.8 13.2 CHSN18948-Mutant3+ SNSS16160 Not detected 20.2 16.5 CHSN18948-Mutant3+ SNSS16161 h, o 19.5 16.5 CHSN18948-Mutant2 Not detected 19.7 27.3 CHSN18948-Mutant3 Not detected 19.9 27.1 SNSN 18948-Mutant 12 4.7 15.1 17.0 B-milk 42.8 Not detected Not detected

Based on the above data, CHCH18948-Mutant3 was selected as the best mutant and was designated CHCC19216. As can be seen from the above data, CHCC19216 is able to remove almost all lactose from milk. CHCC19216 also produces and secretes high levels of both galactose and glucose into milk. Considering that sucrose is a control of 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, the fermented dairy product obtained using CHCC19216 has a significantly enhanced sweet taste.

Example 7: Texturizing ability of CHCC16731 and CHCC19216.

The texturing ability of strains CHCC16731 and CHCC19216 was tested by measuring shear stress using the following assay.

Seven days after incubation, the fermented milk was brought to 13°C and gently stirred with a stick equipped with a disc with holes until the sample was homogeneous. The rheological properties of the sample were evaluated on a rheometer (Anton Paar Physica ASC/DSR301 rheometer (autosampler), Anton Paar® GmbH, Austria) using the following setups.

Waiting time (to restore to some extent the original structure):

minutes without oscillation or rotation, oscillation (for measuring G' and G for calculating G*), y is 0.3%, frequency (f) is [0.5... 8] Hz, measurement points in 60 s (one measurement every 10 s), rotation (to measure shear stress at 300 1/s, etc.), y = [0.2707-300] 1/s, and y = [275-0.2707] 1/s a 210 s measurement point (one every 10 s) going up to 300 1/s, and a 210 s measurement point (one every 10 s) going up to 0.2707 1/s.

- 17 042276

The shear stress at 300 1/s was chosen for further analysis.

Strains SNCC16731 and SNCC19216 in the mixtures listed in Table. 4 was used to acidify the samples with 200 ml of 9.5% B-milk using a 0.024% inoculum until a pH of 4.55 was reached.

The results are shown in table. 4.

Table 4

No. mixtures Content St by invention Content St SNSS 16404 Content St SNSS 15757 Lb content SNSS-16159 Shear stress, Pa 1 72.73% SNSS16731 13.64% 4.54% 9.09% 41.3 2 72.73% SNSS 19216 13.64% 4.54% 9.09% 51.5 3 36.36% SNSS 16731 +- 33.36% SNSS 19216 13.64% 4.54% 9.09% 43.3

Lb: strain CHCC16159 L. delbruecki subsp. bulgaricus, St: Streptococcus thermophilus.

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

This level of shear stress is high compared to conventional sweet taste enhancing strains as disclosed for example in WO2013/160413.

Example 8 Taste and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for a culture composition according to the invention compared to a lactose-positive glucose-positive Lb culture.

Experiment plan.

Table 5. Composition of tested cultures

SNSS 16404 (St)g SNSS 16731 (St) H SNSS 15757 (St)g SNSS 18944 (Lb) g SNSS 12813 (Lb) g 1: culture according to the invention 10.5 10.5 6 1.5 0 2: culture with traditional Lb 10.5 10.5 6 0 1.5

The culture according to the invention consists of three glucose-deficient Streptococcus thermophilus (St) and one lactose-deficient Lb. Culture 2 (comparative culture) consists of three glucose-deficient St and one lactose-positive glucose-positive (traditional) Lb.

Two cultures with the composition indicated in the table. 5 was prepared and used as a starter culture in a yogurt fermentation at 43°C until the target pH of 4.55 was reached. The two cultures were compared in terms of taste, Lb growth, subsequent acidification, carbohydrates and rheology.

Milk base.

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

0.5% fat milk and 1.5% fat milk were mixed to achieve a fat content of 1% in the final milk base, and 0.1% sucrose was added. Protein levels were adjusted to 4.5% using skimmed milk powder (SMP). Hydration took place at 6°C for 2 hours. The milk was then homogenized at 20000/5000 kPa at 65°C and pasteurized at 95°C for 5 minutes.

Options for obtaining yogurt.

pH during fermentation was measured online using Intap. When the yoghurts reached a pH of 4.55, the gel was broken using a stick with a holed disc. The gel was then treated in a post-treatment unit (PTU) at 200 kPa at 25° C., filled into 120 ml yoghurt dishes and stored at 6° C. for 28 days.

- 18 042276

Shear stress measurement.

The method described in Example 7 was used, except that the samples were not agitated with a stick equipped with a perforated disk, since the gel had already been broken down during PTU treatment to obtain sample homogeneity.

Measurement of colony forming units.

M17 was used as a selection medium for St. MRSIac (MRS plus 8% lactose) was used as selective medium for Lb. Did 10x series of dilution of yoghurts. Each dilution was plated onto M17 and MRSIac agar plates, respectively, and anaerobically incubated at 37° C. for 2 days. The colony forming units were then counted.

acidification.

It took 10.8 hours for culture 1 to reach pH 4.55. Culture 2 required 12.1 hours to reach pH 4.55.

Subsequent acidification.

Table 6. Subsequent Acidification

pH Day 0 Day 1 Day 7 Day 14 Day 21 Day 28 Culture 1 4.55 4.53 4.51 4.54 4.50 4.54 culture 2 4.55 4.51 4.50 4.51 4.50 4.54

Lb growth.

Table 7. Culture Growth (Colony Forming Units (CFU)) at the End of Fermentation

Lb (CFU) St (CFU) Culture 1 WE+07 9E+08 culture 2 9E+05 8E+08

As can be seen from Table. 7, the number of Lb cells of the culture according to the invention was significantly higher than the number of Lb cells of a comparative culture with traditional Lb, i.e. lactose-positive glucose-positive Lb. Thus, the combination of glucose-deficient St and lactose-deficient Lb results in a higher level of Lb at the end of fermentation.

Rheology.

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

Culture 2 has a shear stress of 27.3 Pa at the end of fermentation at 300 1/s.

Carbohydrates.

Table 8. Carbohydrate levels at the end of fermentation

Glucose (mg/g) Galactose (mg/g) Lactose (mg/g) Culture 1 15.3 13.8 31.1 Culture 2 15.3 13.6 13.3

Taste.

Taste was analyzed by evaluation by a panel of tasters. Culture 1 did not have any off-flavours.

Example 9 Taste and growth of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for a culture composition according to the invention compared to a glucose-deficient Lb culture.

Experiment plan.

Table 9. Composition of tested cultures

SNSS1640 4(St)g SNSS1673 1 (St) H SNSS1575 7 (St)g SNSS18944 (Lb) g SNSS1615 9 (Lb) g 1: culture according to the invention 10.5 10.5 6 1.5 0 2: culture with glucose- deficient Lb 10.5 10.5 6 0 1.5

The culture according to the invention consists of three glucose-deficient St and one lactose-deficient Lb. Culture 2 (comparative culture) consists of three glucose-deficient St and one lactose-positive glucose-deficient Lb.

Two cultures with the composition indicated in the table. 9, received and used as a starter

- 19 042276 yogurt fermentation cultures at 43° C. until the target pH of 4.55 is reached. The two cultures were compared in terms of taste (amino acid levels), VOCs, carbohydrates and rheology.

Milk base.

Two milk bases were used, adjusted with either skimmed milk powder (SMP) or whey protein (WP), and they contained 4.5% protein, 1% fat and 0.1% sucrose.

0.5% fat milk and 1.5% fat milk were mixed to achieve a 1% fat content 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. The milk was then homogenized at 20000/5000 kPa at 65°C and pasteurized at 95°C for 5 minutes.

Options for obtaining yogurt.

The pH during fermentation was measured online in mini culture flasks using CINAC. When the yoghurts reached a pH of 4.55, the gel was broken and homogenized using a stick with a holed disk. The yogurt was then stored in miniculture flasks.

Shear stress measurement.

The method described in example 7 was used.

Rheology.

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

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

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

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

As can be seen from the results, culture 1 has a higher shear stress compared to culture 2.

Subsequent acidification.

Table 10 Post acidification

pH Day 0 Day 7 Day 14 Day 21 Day 28 Culture 1 in SMP milk base 4.55 4.51 4.56 4.39 4.49 Culture 1 in WP milk base 4.55 4.52 4.55 4.39 4.48 Culture 2 in SMP milk base 4.55 4.45 4.50 4.32 4.43 Culture 2 in WP milk base 4.55 4.42 4.47 4.30 4.40

Lb growth.

Table 11. Culture Growth (Colony Forming Units (CFU)) at the End of Fermentation

LB (CFU) ST (CFU) Culture 1 in SMP milk base 1E+07 2E+08 Culture 1 in WP milk base 2E+07 5E+07 Culture 2 in SMP milk base 2E+08 9E+07 Culture 2 in WP milk base 2E+08 1E+08

As can be seen from Table. 11, the number of Lb cells of the culture according to the invention was less than the number of Lb cells of the comparative culture with lactose-positive glucose-deficient Lb. Table carbohydrates. 12: Carbohydrate level at the end of fermentation.

- 20 042276

Table 12

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

Amino acids.

Table 13. Amino acid levels

Glutamine acid ( ^ppm ) Valine ( ^ppm ) Average value of total free amino acids (mM) Culture 1 in SMP milk base 77 9 5.5 Culture 1 in WP milk base 62 23 4.4 Culture 2 in SMP milk base 137 22 7.1 Culture 2 in WP milk base 85 45 5.4

Samples containing glucose-negative Lb (cultures 2) have higher amounts of TFFA (total free amino acids), indicating higher proteolytic activity, which in turn can lead to off-flavours.

In addition, yoghurts containing glucose-negative Lb contain higher amounts of glutamic acid, which is known for imparting off-flavors described as bouillon.

Valine, which is a bitter amino acid, is also present in higher amounts in yoghurts containing glucose-negative Lb.

Volatile organic compounds (VOC).

The VOC profile can serve as an indication of the flavor profile of the sample.

51 different volatile organic compounds were measured. For culture 1 in WP milk base, VOCs were lower for 44 out of 51 compounds (86%) compared to culture 2 in WP milk base. For culture 1 in WP milk base, VOCs were lower for 34 out of 51 compounds (67%) compared to culture 2 in WP milk base. In conclusion, the cultures according to the invention had a significantly lower content of volatile organic compounds (considered as a group of compounds) compared to comparative cultures.

Example 10 Growth and subsequent acidification of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for a culture composition according to the invention.

Experiment plan.

Table 14. Composition of tested culture

SNSS16404 (St)% SNSS 16731 (St)% SNSS15757 (St)% SNSS 18944 (Lb) % 1: culture according to the invention 36.8 36.8 21.1 5.2

Culture with the composition specified in the table. 14 was prepared and used as a starter culture in a yogurt fermentation at 43°C until the target pH of 4.55 was reached. This culture was inoculated at 0.01, 0.02 and 0.03%. The resulting yoghurt was stored at 5, 13 and 25°C. The culture was tested for St and Lb growth and subsequent acidification.

- 21 042276

Dairy bases.

Table 15. Composition of milk bases 1 and 2

Quantity Protein Carbohydrates Fats Skimmed milk 884.2 g 1.5% milk 1767.1 g Promoloko 802FB 10.9 g SMP 73.9 g sucrose 2.8 g Water 261.0 g milk base 3000 g 4.57% 5.61% 0.92%

Table 16. Composition of milk base 3 Quantity Protein Carbohydrates Fats Skimmed milk 884.2 g 1.5% milk 1767.1 g Promoloko 802FB 10.9 g SMP 73.9 g sucrose 6.0 g Water 257.8 g milk base 3000 g 4.57% 5.71% 0.92%

Subsequent acidification.

Table 17. Post Acidification Dairy the basis 1 1 1 3 3 3 Inoculation 0.01% 0.02% 0.03% 0.01% 0.02% 0.03% 5°C Day 1 4.54 4.54 4.53 4.53 4.53 4.52 5°C Day 7 4.50 4.50 4.49 4.51 4.51 4.51 5°C Day 21 4.48 4.48 4.49 4.50 4.50 4.50 5°C Day 42 4.43 4.42 4.45 4.43 4.45 4.42 13°C Day 1 4.50 4.49 4.50 4.51 4.50 4.50 13°C Day 7 4.46 4.46 4.45 4.50 4.49 4.50 13°C Day 21 4.38 4.38 4.38 4.42 4.45 4.42 25°C Day 1 4.43 4.43 4.43 4.45 4.46 4.45 25°C Day 7 4.40 4.40 4.40 4.43 4.44 4.43

As can be seen from Table. 17, the post-acidification rate is very low for all inoculation levels and storage temperatures tested, including the higher storage temperatures of 13 and 25°C, where the level of Lb growth and hence post-acidification is high for most conventional starter cultures.

Growth Lb and St.

Table 18. Growth of Lb and St

milk base 1 1 1 2 2 2 Inoculation 0.01% 0.02% 0.03% 0.01% 0.02% 0.03% Lb cfu/g 5.8E05 6.5E05 7.2E05 3.7E05 4.7E05 8.8E05 St cfu/g 4.2E07 6.5E07 7.4E07 3.8E07 5.6E07 7.0E07

As can be seen from Table. 18, for all levels of inoculation, the number of Lb cells at the end of fermentation was at a high level.

- 22 042276

Example 11 Growth, post-acidification and analysis of carbohydrates of Lactobacillus delbrueckii subsp. bulgaricus (Lb) for a culture composition according to the invention compared to traditional starter cultures.

Experiment plan.

Table 19. Composition of tested cultures

Sample SNOS 16404 (St)% SNOS 16731 (St)% SNOS 15757 (St)% SNOS 18944 (Lb) % Adding sucrose after fermentation 1 YoFlex Premium 1.0 6.0% 2 Yoflex YF- L901 6.0% 3 Culture according to the invention 36.8 36.8 21.1 5.2 5.0% 4 Culture according to the invention 36.8 36.8 21.1 5.2 4.5% 5 Culture according to the invention 36.8 36.8 21.1 5.2 5.5% 6 Culture according to the invention 36.8 36.8 21.1 5.2 4.8%

Cultures with the composition specified in the table. 19 was prepared and used as a starter culture in a yogurt fermentation at 43°C until the target pH of 4.55 was reached. This culture was inoculated at a level of 0.02%. The resulting yoghurt was stored at 5 and 13°C. Cultures were tested for St and Lb growth, subsequent acidification and carbohydrate analysis.

YoFlex Premium 1.0 and Yoflex YF-L901 are commercially available traditional cultures containing lactose positive and glucose positive St and Lb.

Milk base.

Table 20 Milk Base Composition 1

Quantity Protein Carbohydrates Fats 1.5% milk 3000 g SMP 85 g milk base 3085 g 4.20% 6.06% 1.49%

Subsequent acidification.

Table 21. Subsequent acidification

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

As can be seen from Table. 21, the cultures of the invention have a low post-acidification level that is on the same level as traditional starter cultures.

Growth Lb and St.

Table 22. Growth of Lb and St

Sample 1 2 3 4 5 6 Lb cfu/g 6DE04 1.7E05 3.1E06 2.8E06 ZDE06 2.8E06 St cfu/g 3.0E08 4DE08 3.2E08 ZDE08 3.0E08 ZDE08

As can be seen from Table. 22, the number of Lb cells for cultures according to the invention was at a higher level at the end of fermentation than conventional cultures. Analysis of carbohydrates table. 23: analysis of carbohydrates.

- 23 042276

Table 23

Sample G alactose mg/g Glucose mg/g Lactose mg/g Sucrose mg/g 1 YoFlex Premium 1.0 6.35 <LOD 34.35 46.55 2 Yoflex YF-L901 8.05 1.00 33.05 46.60 3 Culture according to the invention 8.95 8.70 19.90 41.55 4 Culture io invention 8.90 8.75 19.70 37.50 5 Culture io invention 8.60 8.40 19.15 43.10 6 Culture io invention 8.80 8.65 19.40 39.65

LOD: limit of detection.

All determinations of carbohydrates were made in duplicate, and in table. 23 lists the averages of the two determinations. Determinations were carried out after 7 days of storage of yogurt.

As can be seen from Table. 23, the cultures of the invention produced high levels of glucose, and after 7 days of storage, glucose was present at approximately 8 mg/g.

Deposits and expert solutions.

The Applicant requests that a sample of the deposited microorganisms indicated below be made available only to the examiner before the date of grant of the patent.

Streptococcus thermophilus CHCC11342 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, September 8, 2009, accession DSM 22932.

Streptococcus thermophilus CHCC12339 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, October 14, 2010, accession DSM 24090.

Streptococcus thermophilus CHCC11976 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, September 8, 2009, accession DSM 22934.

Streptococcus thermophilus CHCC16731 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, June 4, 2014, accession DSM 28889.

Streptococcus thermophilus CHCC19216 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, December 8, 2015, accession DSM 32227.

Streptococcus thermophilus CHCC15757 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, 04/03/2012, Accession No. DSM 25850.

Streptococcus thermophilus CHCC15887 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, 04/03/2012, Accession No. DSM 25851.

Streptococcus thermophilus CHCC16404 was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, 12/12/2012, Accession No. DSM 26722.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC18944 was deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 6/12/2014 under DSM Accession No. 28910.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 was deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 04/03/2007 under DSM Accession No. 19252.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 was deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 09/06/2012 under DSM Accession No. 26420.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 was deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 09/06/2012 under accession number DSM 26421.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC12813 was deposited with the DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 09/29/2010, accession DSM 24074.

The deposit data was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

-

Claims (10)

Links 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 The Technology of Cheesemaking, ^2nd Ed. Blackwell Publishing, Oxford; 166-192. CLAIM 1. A composition for producing a fermented dairy product, containing: (1) at least one strain of Streptococcus thermophilus (St), where this St strain is galactose-fermenting, where this strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, where this mutation inactivates the glucokinase protein or has a negative effect on the expression of this gene, where the galactose-fermenting activity is determined as indicated in the description of the invention, and where this mutation reduces the activity of the glucokinase protein by at least 50%, and where the glucokinase activity is determined as indicated in the description of the invention, and ( 2) at least one strain of Lactobacillus delbrueckii sub. bulgaricus (Lb), where this Lb strain is lactose-deficient and capable of metabolizing glucose, where 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-betaD-galactopyranoside (X- Gal). 2. The composition of claim 1, wherein the St strain is 2-deoxyglucose resistant. 3. A composition according to claim 1 or 2, wherein the St strain carries a mutation that reduces the transport of glucose into the cell. 4. The composition according to any one of claims 1 to 3, wherein the St strain increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into 9.5% B-milk at a concentration of 106- 10 ⁷ colony forming units (CFU)/ml and grown at 40°C for 20 hours. 5. Composition according to any one of claims 1 to 4, wherein the St strain increases the amount of glucose in 9.5% B milk with 0.05% sucrose to at least 5 mg/ml when inoculated into 9.5% B - milk with 0.05% sucrose at a concentration of 106-10 ⁷ CFU/ml and grown at 40°C for 20 hours. 6. The composition according to any one of claims 1 to 5, where the Lb strain is selected from the group consisting of a strain deposited in the German collection of microorganisms and cell cultures DSMZ, GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, 06/12/2014, accession number DSM 28910, and a functionally equivalent mutant strain derived from DSM 28910, in which less than 1% of the nucleotides in the bacterial genome were replaced by another nucleotide or subjected to a deletion compared to the parent strain . 7. Composition according to any one of claims 1 to 6, wherein the St strain is selected from the group consisting of the Streptococcus thermophilus strain CHCC19216, which has been deposited with the German Collection of Microorganisms and Cell Cultures under Accession No. DSM 32227, the Streptococcus thermophilus strain CHCC16731, which has been deposited to the German Collection of Microorganisms and Cell Cultures (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, June 4, 2014, Accession No. DSM 28889, Streptococcus thermophilus strain CHCC15757, which has been deposited with the German Collection of Microorganisms and Cell Cultures, Accession No. DSM 25850, Streptococcus thermophilus strain CHCC15887, which has been deposited with German Collection of Microorganisms and Cell Cultures Accession No. DSM 25851, Streptococcus thermophilus strain CHCC16404 which has been deposited with the German Collection of Microorganisms and Cell Cultures Accession No. DSM 26722, and a mutant strain derived therefrom, wherein the mutant strain is a functionally equivalent strain specified deposited strains and in which less than 1% of the nucleotides in the bacterial genome has been replaced by another nucleotide or subjected to a deletion compared to the parent strain. 8. A method for producing a fermented dairy product, comprising inoculating and fermenting a dairy substrate with a composition according to any one of claims 1 to 7. 9. A fermented dairy product containing a composition according to any one of claims 1 to 7. 10. The use of a composition according to any one of claims 1 to 7 for the production of a fermented dairy product. -