EA039996B1 - APPLICATION OF GLUCOSE-DEFICIENT STRAINS OF STREPTOCOCCUS THERMOPHILUS IN A METHOD OF OBTAINING FERMENTED DAIRY PRODUCTS - Google Patents

Description

Technical field

The present invention relates to the use of bacterial strains of Streptococcus thermophilus deficient in glucose metabolism in combination with a strain of Lactobacillus in a method for producing a fermented dairy product.

Prior Art

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

EP-A1-2957180 in one embodiment discloses a process for making a fermented dairy product using a combination of a glucose-deficient starter culture and conventional lactase to reduce the lactose content and the level of end acidification in the fermented dairy product.

WO 2013/160413 discloses a galactose-fermenting strain of Streptococcus thermophilus, wherein the strain carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, wherein said mutation inactivates the glucokinase protein or has a negative effect on gene expression. In a particular embodiment, the Streptococcus thermophilus strain also carries a mutation that reduces glucose transport into the cell, such as a mutation in the gene encoding a glucose transporter component, where the mutation inactivates the glucose transporter or has a negative effect on gene expression. WO 2013/160413 also discloses glucose-deficient strains of Lactobacillus delbrueckii subsp. bulgaricus. When used in a process for making a fermented dairy product, Streptococcus thermophilus strains release glucose to produce fermented dairy products with natural (intrinsic) sweetness, no added calories, increased sweetness, and reduced lactose levels.

In Sieuwerts et al., Applied and Environmental Microbiology, Aug. 2008, p. 4997-5007, disclosed a review study of microbial interactions during yogurt fermentation and reported that when Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are grown in co-culture, the growth of both species is stimulated. Sieuwerts also reports that Streptococcus thermophilus supplies Lactobacillus delbrueckii subsp. bulgaricus with growth stimulating factors such as formic acid, pyruvic acid and folic acid.

The standard definition of yogurt in many countries calls for Streptococcus thermophilus together with Lactobacillus delbrueckii subsp. bulgaricus. Both types create the required amounts of acetaldehyde, which, in addition to acidity, is an important flavoring ingredient in yogurt.

In some types of fermented dairy products containing Streptococcus thermophilus together with a strain of Lactobacillus, it is desirable to obtain a high level of the strain of Lactobacillus. Also in some countries, the standard definition of these types of fermented dairy products requires that the Lactobacillus strain be present in the product at a level of about 1.0E06 cfu/ml. For such fermented dairy products, the main technical problem is that the level of Lactobacillus cells obtained at the end of the fermentation is not as high as required. The present invention relates to the production of a fermented dairy product using Streptococcus thermophilus together with a strain of Lactobacillus, where an increased level of Lactobacillus cells can be obtained upon completion of the fermentation.

Brief summary of the invention

The present invention is directed to the use of a glucose metabolizing deficient strain of Streptococcus thermophilus to improve the growth of a Lactobacillus strain in a process for producing a fermented dairy product.

The present invention is based on the unexpected experimental discovery that strains of Streptococcus thermophilus with deficiency in glucose metabolism are capable of enhancing the growth of strains of lactic acid bacteria of the genus Lactobacillus. Thus, the application of the invention made it possible to obtain an increased level of Lactobacillus cells at the end of the fermentation or, alternatively, the use of a mixture of starter cultures with a reduced number of bacterial cells of Lactobacillus.

In addition, when used in a process for producing a fermented dairy product, strains of Streptococcus thermophilus with deficiency in glucose metabolism have the ability to excrete glucose to produce fermented dairy products with natural (intrinsic) sweetness without added calories, increased sweetness, and with reduced levels of lactose. Thus, in accordance with the present invention, it is possible to obtain fermented dairy products with a combination of an increased level of Lactobacillus bacterial cells and an increased level of natural sweetness.

The present invention also relates to a composition containing a strain of Streptococcus thermophilus with deficiency of glucose metabolism and a strain of Lactobacillus, where the ratio of CFU/g of strain

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Streptococcus thermophilus to CFU/g of Lactobacillus strain is at least 50.

In addition, the present invention relates to a method for producing a fermented dairy product, including the inoculation and fermentation of a dairy substrate with a composition according to the invention.

Detailed description of the invention

Definitions

As used herein, the term lactic acid bacterium refers to a Gram-positive, microaerophilic, or anaerobic bacterium that ferments sugars to produce acids, including lactic acid as the predominant acid produced, acetic acid, and propionic acid. The most industrially useful lactic acid bacteria are within the Lactobacillales group, which includes Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Brevibacterium spp., Enterococcus spp. and Propionibacterium spp. Lactic acid bacteria, including Lactobacillus sp. and Streptococcus thermophilus are commonly supplied to the dairy industry either as frozen or freeze-dried cultures for the production of starter cultures or as so-called direct application cultures (DVS). DVS cultures are intended for direct inoculation into a fermentation tank or bath to produce a dairy product, such as a fermented dairy product. Such cultures are generally referred to as starter cultures or sourdoughs.

The terms containing, having, including, and containing in itself are to be construed as non-limiting terms (i.e., meaning including, but not limited to), unless otherwise indicated. The listing of ranges of values in this specification is only intended to be used as a shorthand way of referring to each individual value falling within the specified range, unless otherwise indicated in the present specification, and each individual value is included in the description of the invention as if they have been separately set forth in the present description of the invention. All methods described in the present description of the invention, can be performed in any suitable order, unless otherwise indicated in the present description of the invention or otherwise does not conflict with the obvious way the context. The use of any and all examples, or exemplary expression (eg such as) provided herein, is intended only to better explain the invention, and does not constitute a limitation on the scope of the invention unless otherwise stated. In the description of the invention, no expression should be taken as indicating that any unclaimed element is essential to the practice of the invention.

A bacterial strain as used herein refers to a bacterium that remains genetically unchanged when grown or propagated. Includes a large number of identical bacteria.

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+2% galactose medium. Galactose-fermenting strains of Streptococcus thermophilus are defined herein as strains of Streptococcus thermophilus that lower the pH of M17 broth containing 2% galactose as the sole carbohydrate to 5.5 or lower when inoculated from an overnight culture at a concentration of 1% and incubated for 24 h at 37°C.

The term mutation inactivates the glucokinase protein as used herein refers to a mutation that results in an inactivated glucokinase protein, i.e. a glucokinase protein that, if present in a cell, is unable to perform its normal function, as well as mutations that prevent the formation glucokinase protein or lead to cleavage 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 the gene, where said mutation may include, without limitation, a deletion, a frameshift mutation, the inclusion of a stop codon, or a 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 the gene.

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 positions 1-966 in SEQ ID NO: 1, or a sequence that has at least 85% identity, such as at least 90% identity , such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, with a sequence corresponding to positions 1-966 of SEQ ID NO: 1.

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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; 5873-5877); the algorithm of Myers and Miller (1988. CABIOS 4;11-17); algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48; 443-453); and the Pearson and Lipman algorithm (1988. Proc. Natl. Acad. Sci. USA 85; 2444-2448). A computer program is also available for determining nucleic acid or amino acid sequence identity based on these mathematical algorithms. For example, comparison of nucleotide sequences can be performed using the BLASTN program, score=100, wordlength=12. Comparison of amino acid sequences can be performed using the BLASTX program, score=50, wordlength=3. For other parameters of BLAST programs, you can use the standard parameters.

A mutant bacterium or mutant strain, as used herein, refers to a natural (spontaneous, naturally occurring) mutant bacterium or induced mutant bacterium containing one or more mutations in its genome (DNA) that are not present in the wild-type DNA. An induced mutant is a bacterium where the mutation has been induced by a human through treatment such as treatment with chemical mutagens, UV or gamma radiation, and so on. In contrast, a spontaneous mutant or a naturally occurring mutant has not been subjected to human mutagenesis. Mutant bacteria in the present description of the invention are not GMO (genetically modified microorganism), that is, not modified through genetic engineering.

A wild-type strain refers to the non-mutated form of a bacterium that is found in nature.

Terms such as strains with sweetening properties, strains that can produce desirable glucose accumulation in a fermented dairy product, and strains with enhanced natural sweetening properties of foods are used interchangeably herein to describe the beneficial effect of using the strains of the present invention in the fermentation of dairy products.

The term 2-deoxyglucose-resistant in the present description of the invention in relation to Streptococcus thermophilus is defined by the fact that a particular mutated bacterial strain has the ability to grow to a colony when streaked on a plate with M17 medium containing 20 mm 2-deoxyglucose, after incubation at 40 ° C in within 20 hours. The presence of 2-deoxyglucose in the culture medium will prevent the growth of unmutated strains, while the growth of mutated strains is not altered or not significantly altered. Non-mutated strains that can be used as sensitive reference strains in resistance assessment preferably include strains CHCC14994 and CHCC11976.

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

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

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

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 this context, the term strains derived from them 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, radiation and/or chemical processing. Strains derived from them may also be spontaneously occurring mutants. Preferably, the strains derived therefrom are functionally equivalent mutants, eg mutants that have substantially the same or improved properties (eg regarding glucose release) as their parent strain. Such strains derived from them are part of the present invention. In particular, the term strains derived therefrom refers to strains obtained by subjecting the strain of the invention to any conventional treatment for mutagenesis, including treatment with a chemical mutagen such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine. (NTG), UV radiation, or to a spontaneously appearing mutant. The mutant could be subjected to several mutagenesis treatments (a single treatment should be understood as one mutagenization step followed by a screening/selection step), but in the context of the present invention, it is preferred that no more than 20

- 3 039996 or not more than 10, or not more than 5 treatments (or screening/selection steps). In a preferred mutant in the context 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 compared to the parental strain has been replaced with a different nucleotide or removed.

The term 2-deoxyglucose resistant in relation to a strain of Lactobacillus delbrueckii subsp. bulgaricus means that a particular bacterial strain has the ability to grow to a colony after incubation at 40° C. for 20 hours when streaked onto a plate with MRS-IM medium containing 2% lactose and 20 mM 2-deoxyglucose. The presence of 2-deoxyglucose in the culture medium will prevent the growth of resistant strains, while the growth of resistant strains does not change or does not change significantly. Unstable strains of Lactobacillus delbrueckii subsp. Bulgaricus that can be used as sensitive reference strains in resistance assessment include strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC759, which is deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures; DSMZ) under accession number DSM 26419, and CHCC10019, which is deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under accession number DSM 19252.

The term glucose-deficient is used in the context of the present invention to characterize LAB (lactobacilli) that have partially or completely lost the ability to use glucose as a source for cell growth or to maintain cell viability. The corresponding deficiency in glucose metabolism can be caused, for example, by a mutation in a gene that inhibits or inactivates the expression or activity of a glucokinase protein or a glucose transporter protein. LAB with deficiency of glucose metabolism can be characterized as increasing the concentration of glucose in the culture medium when grown on lactose as a source of carbohydrate. The increase in glucose is caused by the secretion of glucose by glucose-deficient LABs. The increase in glucose concentration in the culture medium can be determined by HPLC (high performance liquid chromatography) analysis, for example using a Dionex CarboPac PA 20 3x150 mm column (Thermo Fisher Scientific, item number 060142).

The term glucose-positive is used in the context of the present invention to describe LABs that retain some or all of their ability to use glucose as a source for cell growth or cell viability.

The term genus means genus as defined on the following website:

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi

In relation to strains of the genus Lactobacillus, the term CFU denotes colony-forming units, which are determined by growth (colony formation) on an MRS agar plate incubated under anaerobic conditions at 37°C for 3 days. MRS agar has the following composition (g/l):

proteose peptone Bacto No. 3: 10.0;

beef extract Bacto: 10.0;

yeast extract Bacto: 5.0;

dextrose: 20.0;

sorbitan monooleate complex: 1.0;

ammonium citrate: 2.0;

sodium acetate: 5.0;

magnesium sulfate: 0.1;

manganese sulfate: 0.05;

dibasic potassium phosphate: 2.0;

Bacto agar: 15.0;

Milli-Q quality water: 1000 ml.

The pH value was adjusted to 5.4 or 6.5; The pH was adjusted to 6.5 for L. rhamnosus, L. casei and L. paracasei. For all other Lactobacillus species, the pH was adjusted to 5.4. In particular, the pH was adjusted to 5.4 for L. delbrueckii subsp. bulgaricus; L. acidophilus and L. helveticus. The pH was adjusted to 6.5 for L. rhamnosus, L. casei and L. paracasei.

In relation to Streptococcus thermophilus, the term CFU refers to colony forming units, which are determined by growth (colony formation) on an M17 agar plate incubated under aerobic conditions at 37°C for 3 days. Agar M17 has the following composition (g/l):

tryptone: 2.5 g;

peptic digest of meat: 2.5 g;

papain hydrolyzate of soy flour: 5.0 g;

yeast extract: 2.5 g;

meat extract: 5.0 g;

lactose: 5.0 g;

sodium glycerophosphate: 19.0 g;

magnesium sulfate, 7 H ₂ O: 0.25 g;

- 4 039996 ascorbic acid: 0.5 g;

agar: 15.0 g;

Milli-Q quality water: 1000 ml.

The pH value was adjusted to a final pH of 7.1±0.2 (25°C).

The expression ratio CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain refers to the number of cells of the Streptococcus thermophilus strain in CFU/g contained in the composition divided by the number of cells of the Lactobacillus strain in CFU/g contained in said composition. If more than one strain of Streptococcus thermophilus is present in the composition, the number of cells indicated is the sum of all Streptococcus thermophilus bacteria present. If more than one strain of Lactobacillus is present in the composition, the number of cells indicated is the sum of all Lactobacillus present.

The term X,YEZZ stands for X,Yx10 ^ZZ

Streptococcus thermophilus strain in use according to the invention

In a particular embodiment of the use according to the invention, the strain of Streptococcus thermophilus is a galactose fermenter and carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, where the mutation inactivates the glucokinase protein or has a negative effect on the expression of said 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 enzyme analysis using commercially available kits and transcriptomics or quantitative PCR (polymerase chain reaction ) using substances that are readily available.

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 enzyme assays as described in Pool et al. (2006. Metabolic Engineering 8; 456-464).

Galactose fermenting strains can be obtained by the method described in WO 2011/026863.

In a particular embodiment of the use according to the invention, the strain of Streptococcus thermophilus carries a mutation that reduces the transport of glucose into the cell. In a specific embodiment, the strain of Streptococcus thermophilus carries a mutation in the gene encoding a glucose transporter component, where the mutation inactivates the glucose transporter or negatively affects the expression of said gene. In a more specific embodiment, the Streptococcus thermophilus strain carries a mutation in the DNA sequence of the manM gene encoding the IICMan protein of the glucose/mannose phosphotransferase system, where the mutation inactivates the IICMan protein or has a negative effect on gene expression. In another specific 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 ^Man IID of the glucose/mannose phosphotransferase system, where the mutation inactivates the IID ^Man protein or has a negative effect on gene expression.

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%.

The transport of glucose into a cell can be determined by the glucose uptake assay, which is described in Cochu et al. (2003. Appl Environ Microbiol 69(9); 5423-5432).

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

The component may be any component of a glucose transport protein that is critical for glucose transport. For example, inactivation of any component of the glucose/mannose phosphotransferase system in Streptococcus thermophilus is expected to result in inactivation of the glucose transporter function.

In particular, an inactivated glucose transport protein is a protein that, compared to a functional glucose transport 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 transport protein, as compared to the gene encoding a functional glucose transport protein, contains a mutation in the open reading frame (ORF) of the gene, where said mutation may include, without limitation, deletion, frameshift mutation, inclusion a stop codon, or a 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.

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

- 5 039996 at least 70%, for example at least 80%, for example at least 90%.

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

In a specific embodiment of the use according to the invention, a strain of Streptococcus thermophilus increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into 9.5% B-milk at a concentration of 1.0E06-1.0E07 cfu/ml and grown at 40°C for 20 h.

In a specific embodiment of the application of the invention, a strain of Streptococcus thermophilus increases the amount of glucose in 9.5% B-milk containing 0.05% sucrose to at least 5 mg/ml when inoculated into 9.5% B-milk containing 0.05 % sucrose, at a concentration of 1.0E06-1.0E07 CFU/ml and grown at 40°C for 20 hours.

In this context, 9.5% B-milk is heat-treated milk made using low-fat skimmed milk powder, reconstituted to a dry weight of 9.5%, and pasteurized at 99°C for 30 minutes followed by cooling to 40 °C.

In more preferred embodiments, 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 e.g. up to at least 10 mg/mL, e.g. at least up to 11 mg/mL, e.g. at least up to 12 mg/mL, e.g. at least up to 13 mg/mL, e.g. at least up to 14 mg/mL , for example at least up to 15 mg/ml, for example at least up to 20 mg/ml, for example at least up to 25 mg/ml.

In a particular embodiment of the use according to the invention, the Streptococcus thermophilus strain is selected from the group consisting of the Streptococcus thermophilus strain CHCC15757, which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) under accession number DSM 25850, the Streptococcus thermophilus strain CHCC15887, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 25851, Streptococcus thermophilus CHCC16404, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26722, Streptococcus thermophilus CHCC16731, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 28889, Streptococcus thermophilus CHCC19216, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 32227, and a mutant strain obtained from them, where the mutant strain mm is obtained by using one of these deposited strains as a starting material and where the mutant retains or further improves the lactose fermentation property and/or the glucose secretion property of the specified deposited strain.

In the most preferred embodiment of the present invention, the mutant strain is a naturally occurring mutant or an induced mutant.

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

Lactobacillus strain in use according to the invention

The bacterial strain of Lactobacillus according to the invention may be any bacterial strain of the genus Lactobacillus. In a specific embodiment, the Lactobacillus strain is selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus acidophilus. In a preferred embodiment of the invention, the Lactobacillus strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus paracasei and Lactobacillus acidophilus.

In a specific embodiment, the Lactobacillus bacterial strain of the invention is Lactobacillus delbrueckii subsp. bulgaricus. Lactobacillus delbrueckii subsp. bulgaricus is a lactic acid bacterium that is often used in commercial lactic fermentation, where the microorganism is usually used as part of a mixed starter culture.

In one embodiment of the invention, the Lactobacillus bacterial strain of the invention is glucose deficient. In an alternative embodiment of the invention, the Lactobacillus bacterial strain of the invention is glucose positive.

In particular, the strain Lactobacillus delbrueckii subsp. bulgaricus according to the invention is a strain that is resistant to 2-deoxyglucose. In a specific embodiment of the invention, the Lactobacillus strain is a strain of Lactobacillus delbrueckii subsp. bulgaricus, where the strain is resistant to 2-deoxyglucose and where the strain increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated in 9.5% B-milk at a concentration of 1.0E06-1, 0E07 cfu/ml and grown at 40°C for at least 20 hours.

In a specific embodiment of the invention, the Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of a strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26420, strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160, which was deposited with Deutsche

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Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 26421, and a mutant strain obtained from them, where the mutant strain is obtained by using one of these deposited strains as a starting material and where the mutant retains or further improves the lactose fermentation property and/or the secretion property glucose of the specified deposited strain.

In another specific embodiment of the invention, the Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of a strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC4351, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the accession number DSM22586, and a mutant strain obtained from them, where the mutant strain was obtained by using one of the deposited strains as a starting material.

In a specific embodiment of the invention, the Lactobacillus paracasei strain is selected from the group consisting of the CHCC2115 strain, which has been deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under DSM accession number 19465, and a mutant strain derived therefrom, wherein the mutant strain is obtained by using one of deposited strains as starting material.

In a specific embodiment of the invention, the Lactobacillus acidophilus strain is selected from the group consisting of the CHCC2169 strain, which has been deposited as DSM13241 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, and a mutant strain derived therefrom, wherein the mutant strain is obtained by using one of the deposited strains as original source material.

Fermented dairy products obtained by the use according to the invention

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

The term milk substrate can be any raw and/or processed milk raw material that can be fermented according to the process of the invention. Thus, suitable dairy substrates include, without limitation, solutions/suspensions of any dairy 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. Evidently, the milk substrate may be derived from any mammal, for example essentially pure mammalian milk or reconstituted 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.

Homogenization as used herein means vigorous mixing to form a soluble suspension or emulsion. If the homogenization is carried out prior to fermentation, it may be carried out in such a way as to reduce the milk fat to smaller sizes so that it no longer separates from the milk. This can be achieved by passing milk under high pressure through small holes.

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 predetermined temperature for a predetermined period of time. The set temperature is usually reached by heating. The temperature and duration can be selected to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.

Fermentation in the methods of the present invention means the conversion of carbohydrates into alcohols or acids caused by the metabolism of the microorganism. Preferably, the fermentations in the methods of the invention include the conversion of lactose to lactic acid.

Fermentation processes for use in making fermented dairy products are well known and one skilled in the art will know how to select suitable process conditions such as temperature, amount of oxygen, amount and characteristics of the microorganism(s), and process time. Obviously, the fermentation conditions are chosen so as to facilitate the implementation of the present invention, that is, to obtain a dairy product in solid or liquid form (fermented dairy product).

The term fermented dairy product as used in the present description of the invention refers to a food or feed product, where fermentation of a milk substrate by lactic acid bacteria is involved in the production of a food or feed product. The fermented dairy product as used herein includes, without limitation, products such as yogurt and cheese.

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Examples of cheeses that are obtained by fermentation with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus include mozzarella and pizza cheese (H0ier et al. (2010) in The

Technology of Cheesemaking, ^2nd Ed. Blackwll 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.

Use according to the invention

In one embodiment of using a glucose metabolizing deficient strain of Streptococcus thermophilus to improve the growth of a Lactobacillus strain in a process for producing a fermented dairy product, the ratio of Streptococcus thermophilus strain to Lactobacillus strain is selected at a normal level, resulting in an increased level of Lactobacillus strain in the fermented dairy product. In particular, the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is from 10 to 500, preferably from 50 to 400, more preferably from 80 to 300 and most preferably from 100 to 200.

In a second embodiment of using a glucose metabolizing deficient strain of Streptococcus thermophilus to improve the growth of a Lactobacillus strain in a method for producing a fermented dairy product, the ratio of Streptococcus thermophilus strain to Lactobacillus strain is selected at a reduced level, and as a result, a normal level of Lactobacillus strain in the fermented dairy product is obtained. In particular, at the start of fermentation, the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is at least 50, preferably at least 80, more preferably at least 100, more preferably at least 150, more preferably at least 200, more preferably at least 250, more preferably at least 300, more preferably at least 350, more preferably at least 400, more preferably at least 450, more preferably at least 500, more preferably at least 550 , more preferably at least 600, more preferably at least 650, more preferably at least 700, more preferably at least 750, more preferably at least 800, more preferably at least 850, more preferably at least 900 and most preferably at least 950. In this case, the ratio of CFU/g of Strep strain tococcus thermophilus to CFU/g of Lactobacillus strain is below 2000, more preferably below 1800, more preferably below 1600, more preferably below 1400, more preferably below 1200 and most preferably below 1000.

In a specific embodiment of the use according to the invention, the strain of Streptococcus thermophilus increases the amount of the Lactobacillus strain to at least 5.0E06 CFU/g in the fermented dairy product at the end of fermentation when inoculated into the milk substrate at a concentration of 1.0E07-1.0E08 CFU/g in the milk substrate and grown at 43°C until a pH of 4.55 is reached, where the milk substrate is a mixture of fresh skimmed milk with a fat content of 0.1%, fresh semi-skimmed milk with a fat content of 1.5% and skimmed milk powder and where the milk substrate contains 4 .7% protein, and 1.0% fat, and 0.05% sucrose.

Composition according to the invention

The present invention further relates to a composition comprising a glucose metabolizing deficient strain of Streptococcus thermophilus and a Lactobacillus strain, wherein the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is at least 50.

The composition according to the invention is based on the unexpected experimental discovery that strains of Streptococcus thermophilus with insufficient glucose metabolism are able to enhance the growth of strains of lactic acid bacteria of the genus Lactobacillus and that it is therefore possible to ferment milk with a mixture of starter cultures having a reduced level of the strain of Lactobacillus relative to the strain of Streptococcus thermophilus , and still get an acceptable level of Lactobacillus strain at the end of the fermentation.

In a particular embodiment of the composition of the invention, the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is at least 80, preferably at least 100, more preferably at least 150, more preferably at least 200, more preferably at least 250, more preferably at least 300, more preferably at least 350, more preferably at least 400, more preferably at least 450, more preferably at least 500, more preferably at least 550, more preferably at least 600 , more preferably at least 650, more preferably at least 700, more preferably at least 750, more preferably at least 800, more preferably at least 850, more preferably

- 8 039996 at least 900 and most preferably at least 950.

In a particular embodiment of the composition of the invention, the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is below 2000, more preferably below 1800, more preferably below 1600, more preferably below 1400, more preferably below 1200 and most preferably below 1000.

In a specific embodiment, the composition according to the invention contains from 1.0E04 to 1.0E12 CFU/g of Streptococcus thermophilus strain, for example from 1.0E05 to 1.0E11 CFU/g, for example from 1.0E06 to 1.0E10 CFU/g, or for example from 1.0E07 to 1.0E09 CFU/g of Streptococcus thermophilus strain.

In particular, the strain of Streptococcus thermophilus of the composition of the invention is a strain of Streptococcus thermophilus as described above with respect to the use of the invention. In particular, the strain of Lactobacillus of the composition of the invention is a strain of Lactobacillus as described above with respect to the use of the invention.

In a preferred composition of the present invention, the Streptococcus thermophilus strain is selected from the group consisting of the Streptococcus thermophilus strain CHCC15757, which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) under accession number DSM 25850, the Streptococcus thermophilus strain CHCC15887, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 25851, Streptococcus thermophilus strain CHCC16404, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under the accession number DSM 26722, Streptococcus thermophilus CHCC16731, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 28889, Streptococcus thermophilus CHCC19216, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 32227, and a mutant strain obtained from them, where the mutant The th strain is obtained by using one of said deposited strains as a starting material, and wherein the mutant retains or further improves the lactose fermentation property and/or the glucose secretion property of said deposited strain.

In one embodiment of the invention, the Lactobacillus bacterial strain of the invention is glucose deficient. In an alternative embodiment of the invention, the Lactobacillus bacterial strain of the invention is glucose positive.

In a preferred composition of the present invention, the Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of a strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26420, strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26421, and a mutant strain obtained from them, where the mutant strain is obtained by using one of the said deposited strains as a starting material and where the mutant retains or further improves lactose fermentation property and/or glucose secretion property of said deposited strain.

In another specific embodiment of the composition according to the invention, the strain Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of a strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC4351, which is deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under the accession number DSM22586, and a mutant strain obtained from it, where the mutant strain is obtained by using one of the deposited strains as a starting material and where the property of the mutant is retained or further improved fermenting glucose of said deposited strain.

Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are commonly used as starter cultures serving a technological purpose, for example in the dairy industry, in the production of various food products, for example for fermented dairy products. Thus, in another preferred embodiment, the composition is suitable as a starter culture.

Starter cultures can be offered as frozen or dry starter cultures in addition to liquid starter cultures. Thus, in another preferred embodiment, the composition is in frozen, lyophilized or liquid form.

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

Method for obtaining a fermented dairy product

The present invention also relates to a process for producing a fermented dairy product, comprising inoculating and fermenting a dairy substrate with a composition of the invention.

Below, as non-limiting examples, embodiments of the present invention are described.

Examples

Substances and Methods

Agar medium for cell count of Lactobacillus delbrueckii subsp. bulgaricus

CFU was determined by growth (colony formation) on an MRS agar plate incubated under anaerobic conditions at 37° C. for 3 days. MRS agar has the following composition (g/l):

proteose peptone Bacto No. 3: 10.0;

meat extract Bacto: 10.0;

yeast extract Bacto: 5.0;

dextrose: 20.0;

sorbitan monooleate complex: 1.0;

ammonium citrate: 2.0;

sodium acetate: 5.0;

magnesium sulfate: 0.1;

manganese sulfate: 0.05;

dipotassium phosphate: 2.0;

Bacto agar: 15.0;

Milli-Q quality water: 1000 ml.

The pH was adjusted to 5.4.

Agar medium for cell count of Streptococcus thermophilus strains

For Streptococcus thermophilus, the medium used is M17 medium known to the person skilled in the art. Bacteria were grown under aerobic conditions at 37°C for 3 days.

M17 agar medium has the following composition (g/L):

tryptone: 2.5 g;

peptic digest of meat: 2.5 g;

papain hydrolyzate of soybean meal: 5.0 g;

yeast extract: 2.5 g;

meat extract: 5.0 g;

lactose: 5.0 g;

sodium glycerophosphate: 19.0 g;

magnesium sulfate, 7 n ₂ about: 0.25 g;

ascorbic acid: 0.5 g;

agar: 15.0 g;

Milli-Q quality water: 1000 ml.

The pH was adjusted to a final pH of 7.1±0.2 (25°C).

Example 1 Effect of glucose-deficient strains of Streptococcus thermophilus on the growth of Lactobacillus strains during milk fermentation.

A total of 4 different mixtures of bacterial strains, each consisting of several strains of Streptococcus thermophilus and one strain of Lactobacillus delbrueckii subsp. Bulgaricus were tested in the process of obtaining yogurt from a milk substrate in order to determine the effect of glucose-deficient strains of Streptococcus thermophilus on the growth of strains of Lactobacillus delbrueckii subsp. bulgaricus.

The two mixtures according to the use of the invention consisted of a mixture of glucose-deficient strains of Streptococcus thermophilus in combination with either a glucose-deficient strain of Lactobacillus delbrueckii subsp. Bulgaricus, or with a glucose-positive strain of Lactobacillus delbrueckii subsp. bulgaricus. For comparison, 2 mixtures consisted of a mixture of glucose-positive strains of Streptococcus thermophilus in combination with either a glucose-deficient strain of Lactobacillus delbrueckii subsp. bulgaricus, or with a glucose-positive strain of Lactobacillus delbrueckii subsp. bulgaricus. The components and compositions of 4 mixtures are visible from the table. 1.

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Table 1 Composition and growth of mixtures of Lactobacillus delbrueckii subsp. bulgaricus and glucose-positive or glucose-deficient Streptococcus thermophilus

Mixture components in % mass, (g/g) Blend 1: Glu(-)St Glu(-)Lb Blend 2: Glu(-)St Glu(+)Lb Blend 3: Glu(+)St Glu(-)Lb Blend 4: Glu(+)St Glu(+)Lb Glu(-)St 90.91 94.44 Glu(-)Lb 9.09 9.09 Glu(+)St 90.91 94.44 Glu(+)Lb 5.56 5.56 St to Lb ratio 95.18 88.47 95.18 88.47 Cell count for Lb at the end fermentation (CFU/g) 5.69E07 6.75E08 l,20E06 l,57E06

Glu(-): glucose-deficient

Glu(+): glucose positive

St: Streptococcus thermophilus

Lb: Lactobacillus delbrueckii subsp. bulgaricus

The level of total inoculation of the culture was 0.018 wt.% (g/ml). The milk substrate used was a mixture of fresh skimmed milk (0.1% fat), fresh semi-skimmed milk (1.5% fat), Lactalis skimmed milk powder and Ingredia ProMilk 802 (soluble milk protein isolate). The final milk substrate contained 4.7% protein and 1.0% fat and 0.05% sucrose. Milk fermentation was carried out in 200 ml containers at 43° C. until a final pH of 4.55 was reached.

In table. 1 also indicates for each of mixtures 1-4 the ratio of the sum of the number of cells for Streptococcus thermophilus to the number of cells for Lactobacillus delbrueckii subsp. bulgaricus at the start of fermentation, as well as the number of cells (CFU/g) of Lactobacillus delbrueckii subsp. bulgaricus at the end of fermentation. The results shown in table. 1 represent the mean of the determination in replicates.

Two fermentations using glucose-positive strains of Streptococcus thermophilus resulted in cell numbers of 1.20E06 and 1.57E06 for glucose-deficient and glucose-positive L. delbrueckii subsp. bulgaricus, respectively. The use of glucose-deficient strains of Streptococcus thermophilus increased the level to 5.69E07 and 6.75E08 for glucose-deficient L. delbrueckii subsp. bulgaricus and glucose-positive L. delbrueckii subsp. bulgaricus, respectively. Thus, the use of glucose-deficient strains of Streptococcus thermophilus had a significantly enhanced effect on the growth of both glucose-deficient and glucose-positive, L. delbrueckii subsp. bulgaricus.

Example 2 Effect of glucose-deficient strains of Streptococcus thermophilus on the growth of Lactobacillus strains during milk fermentation using low initial levels of Lactobacillus cells.

Mixtures of bacterial strains with different ratios of a mixture of glucose-deficient Streptococcus thermophilus strains to a glucose-positive strain of Lactobacillus delbrueckii subsp. bulgaricus was tested during the production of yogurt from a milk substrate in order to determine the effect of glucose-deficient strains of Streptococcus thermophilus on the growth of strains of Lactobacillus delbrueckii subsp. bulgaricus. The composition of these mixtures and these different ratios are visible from the table. 2.

The milk substrate used was a mixture of fresh skimmed milk (0.1% fat), fresh semi-skimmed milk (1.5% fat), Lactalis skimmed milk powder and Ingredia ProMilk 802 (soluble milk protein isolate). The final milk substrate contained 4.7% protein and 1.0% fat and 0.05% sucrose. Milk fermentation was carried out in 200 ml containers at 43° C. until a final pH of 4.55 was reached.

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Table 2 Growth of Lactobacillus delbrueckii subsp. bulgaricus in the presence of glucose-deficient Streptococcus thermophilus in different proportions

The composition of the culture Lb cell count after fermentation Glu(-) St % (g/g) Glu(+) Lb % (g/g) Ratio of St to Lb Test A: Cell count Lb (CFU/g) Test B: Cell count Lb (CFU/g) 1 94.0 6.0 119.32 1.9E08 1.9E08 2.0E08 2.0E08 2 94.5 5.5 130.86 1.9E08 2.0E08 1.9E08 1.9E08 3 95.0 5.0 144.71 1.9E08 1.9E08 1.8E08 1.9E08 4 95.5 4.5 161.64 1.7E08 1.7E08 1.7E08 1.8E08 5 96.0 4.0 182.79 1.5E08 1.6E08 1.7E08 1.6E08 6 96.5 3.5 210.00 1.7E08 1.7E08 1.6E08 1.6E08 7 97.0 3.0 246.26 1.8E08 1.7E08 1.7E08 1.8E08 8 97.5 2.5 297.04 1.4E08 1.4E08 1.7E08 1.7E08 9 98.0 2.0 373.20 2.1E08 1.9E08 1.7E08 1.6E08 10 98.5 1.5 500.14 1,ZE08 1.1E08 1.6E08 1.5E08 AND 99.0 ι, ο 754.02 1.0E08 8.9E07 1.5E08 1.5E08 12 99.5 0.5 1515.66 7.0E07 6.7E07 1,ZE08 1.2E08

Glu(-): glucose-deficient

Glu(+): glucose positive

St: Streptococcus thermophilus

Lb: Lactobacillus delbrueckii subsp. bulgaricus

Two tests A and B were performed to obtain a double independent determination. In each test, A and B were determined in replication for each mixture, and the number of cells (CFU/g) was determined at the end of fermentation. The results are visible from the table. 2. As can be seen from the results, the number of Lactobacillus delbrueckii subsp. bulgaricus at the end of fermentation was at a very high level, i.e. from 6.7E07 to 2.1E08 for all blends. In addition, a decrease in the level of Lactobacillus delbrueckii subsp. bulgaricus in relation to the sum of glucose-deficient strains of Streptococcus thermophilus in the mixtures used for inoculation, had very little effect on the number of cells of Lactobacillus delbrueckii subsp. bulgaricus at the end of fermentation for most blends. Thus, with the ratio of Streptococcus thermophilus to Lactobacillus delbrueckii subsp. bulgaricus from 119.32 to 373.20, the number of Lb cells after fermentation was at a higher level from 1.4E08 to 2.1E08. At ratios of St to Lb from 500.14 to 1515.66, the number of Lb cells after fermentation was at a slightly lower level from 6.7E07 to 1.6E08, which is an unexpectedly high level, considering the extremely low level of Lb in the mixture for inoculation. Also, the indicated level of the number of Lb cells obtained after fermentation, which is from 6.7E07 to 1.6E08, is significantly higher than the level obtained with glucose-positive strains compared with comparative example 3.

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Thus, this experiment showed that glucose-deficient strains of Streptococcus thermophilus increase the growth of Lactobacillus delbrueckii subsp. bulgaricus to a high degree and regardless of the initial level of Lactobacillus delbrueckii subsp. bulgaricus.

Comparative Example 3 Effect of glucose-positive strains of Streptococcus thermophilus on the growth of Lactobacillus strains during milk fermentation using low amounts of Lactobacillus cells.

For the purpose of comparison with the results of Example 2, mixtures of bacterial strains with different mixture ratios of glucose-positive strains of Streptococcus thermophilus to glucose-positive strains of Lactobacillus delbrueckii subsp. bulgaricus was tested during the production of yogurt from a milk substrate in order to determine the effect of glucose-deficient strains of Streptococcus thermophilus on the growth of strains of Lactobacillus delbrueckii subsp. bulgaricus. The composition of these mixtures and these different ratios are visible from the table. 3.

Fermentations were performed using the same process and conditions as defined in Example 2.

Table 3 Growth of Lactobacillus delbrueckii subsp. bulgaricus in the presence of glucose-positive Streptococcus thermophilus at different ratios

The composition of the culture Number of Lb cells after fermentation Glu(+) St % (g/g) Glu(+) Lb % (g/g) Attitude St to Lb Trial A: Lb Cell Count (CFU/g) Test B: number of cells Lb (CFU/g) 1 94.0 6.0 97.85 1.6E07 1.6E07 1.5E07 1.5E07 2 94.5 5.5 107.31 1.0E07 9.5E06 1.2E07 1DE07 3 95.0 5.0 118.67 9.4E06 9.0E06 1.0E07 1DE07 4 95.5 4.5 132.55 8.4E06 8.4E06 9.0E06 8.7E06 5 96.0 4.0 149.90 6.9E06 7.2E06 7.5E06 7.4E06 6 96.5 3.5 172.20 7.0E06 7.4E06 7,ZE06 7.6E06 7 97.0 3.0 201.94 6.5E06 6.8E06 6.1E06 6.5E06 8 97.5 2.5 243.58 5.8E06 5.8E06 6.4E06 6.4E06 9 98.0 2.0 306.04 4.4E06 4.7E06 5.2E06 4.9E06 10 98.5 1.5 410.13 2.8E06 3.2E06 4.2E06 4.6E06 AND 99.0 ι, ο 618.32 2.4E06 2.8E06 3.1E06 3,ZE06 12 99.5 0.5 1242.89 2.1E06 2,ZE06 2.8E06 2.7E06

Glu(-): glucose-deficient

Glu(+): glucose positive

St: Streptococcus thermophilus

Lb: Lactobacillus delbrueckii subsp. bulgaricus

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Two tests A and B were performed to obtain a double independent determination. In each test, A and B were determined in replication for each mixture and the number of cells (CFU/g) was determined at the end of fermentation. The results are visible from the table. 3. As can be seen from the results, the number of Lactobacillus delbrueckii subsp. bulgaricus at the end of fermentation was at an average level of 2.1E06 to 1.6E07. In addition, low levels of Lactobacillus delbrueckii subsp. bulgaricus relative to the sum of glucose-positive strains of Streptococcus thermophilus in mixtures used for inoculation, clearly cause a significantly greater reduction in the number of Lactobacillus delbrueckii subsp. Bulgaricus obtained at the end of fermentation than in the case of glucose-deficient strains of Streptococcus thermophilus according to example 2. Thus, the results of these experiments confirm that glucose-deficient strains of Streptococcus thermophilus significantly increase the growth of Lactobacillus delbrueckii subsp. bulgaricus compared with glucose-positive strains of Streptococcus thermophilus.

Example 4 Effect of glucose-deficient Streptococcus thermophilus strains on the growth of Lactobacillus acidophilus and Lactobacillus paracasei strains during milk fermentation.

Composition of cultures for inoculation Table 4. Composition of reference culture mixtures

La % (g/g) Lc % (g/g) Glu(+) St % (g/g) Glu(+) Lb % (g/g) Reference 1: La high+Lb+St Mix 1 32.0 64.0 4.0 Reference 2: Lc high+Lb+St Mix 1 32.0 64.0 4.0 Reference 3: La high+Lb+St Mix 2 31.6 61.3 7.1 Reference 4: Lc high+Lb+St Mix 2 31.6 61.3 7.1 Reference 5: La low+lb+St Blend 1 8.2 86.8 5.0 Reference 6: Lc low +lb+St Mix 1 8.2 86.8 5.0 Reference 7: La low+Lb+St Mix 2 8.5 82.0 9.5 Reference 8: Lc low +Lb+St Mix 2 8.5 82.0 9.5

La: Lactobacillus acidophilus, LA-5, CHCC2169

Lc: Lactobacillus paracasei, LC-01, CHCC2115

Glu(+): Glucose positive

St: Streptococcus thermophilus

Lb: Lactobacillus delbrueckii subsp. bulgaricus

St mix 1 contains two glucose-positive strains of St

St mix 2 contains three glucose-positive strains of St

Table 5. The composition of culture mixtures according to the present invention

La % (y/y) Lc % (y/y) Glu(-) St % (g/g) Glu(-) Lb % (g/g) 1: La high +Lb+St Mix 3 31.7 62.7 5.6 2: Lc high +Lb+St Mix 3 31.7 62.7 5.6 3: La low +Lb+St Mix 3 8.5 84.0 7.5 4: Lc low +Lb+St Mix 3 8.5 84.0 7.5

La: Lactobacillus acidophilus, LA-5, CHCC2169

Lc: Lactobacillus paracasei, LC-01, CHCC2115

Glu(-): glucose-deficient

St: Streptococcus thermophilus

Lb: Lactobacillus delbrueckii subsp. bulgaricus

St Mix 3 contains three glucose-deficient strains of St

0.05% sucrose was added to all fermentations using glucose-deficient St. Analysis.

1. Acidification curves during fermentation (not shown).

2. Number of cells at the end of fermentation.

3. Day 1: pH and cell count. Storage at 6°C.

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4. Day 14: pH and cell count. Storage at 6°C.

5. Day 28: pH and cell count. Storage at 6°C.

6. Day 42: pH and cell count. Storage at 6°C.

Acidification curves were measured by Cinac system (from Alliance Instruments, AMS Company Brand).

All fermentations were performed in duplicate.

Fermentations were performed in 9.5% B-milk (milk substrate).

Cell counts were performed by plating on Petri dishes using a standardized agar medium adapted for growing Lactobacillus acidophilus strain and Lactobacillus paracasei strain. Used the method described above under the heading Substances and methods for strains of Lactobacillus delbrueckii subsp. bulgaricus.

Results after Acidification Table 6. pH for Reference Culture Mixes

pH Day 1 Day 14 Day 28 Reference 1: La high +Lb+St Mix 1 4.50 4.35 4.35 Reference 2: Lc high +Lb+St Mix 1 4.49 4.35 4.35 Reference 3: La high +Lb+St Mix 2 4.49 4.34 4.33 Reference 4: Lc high +Lb+St Mix 2 4.50 4.34 4.34 Reference 5: La low +lb+St Mix 1 4.48 4.35 4.35 Reference 6: Lc low +lb+St Mix 1 4.49 4.35 4.36 Reference 7: La low +Lb+St Mix 2 4.49 4.36 4.33 Reference 8: Lc low +Lb+St Mix 2 4.48 4.35 4.33

Table 7. pH of culture mixtures according to the present invention

pH Day 1 Day 14 Day 28 1: La high + Lb + St Blend 3 4.51 4.35 4.37 2: Lc high +Lb+St Blend 3 4.49 4.35 4.35 3: La low +Lb+St Blend 3 4.48 4.35 4.35 4: Lc low +Lb+St Blend 3 4.49 4.35 4.36

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Cell count results

Table 8. Cell Counts for Reference Culture Mixes

cfu/g Day 1 Day 14 Day 42 The amount of La for reference 1: La high + Lb + St Blend 1 5.8E06 4.9E06 7.6E06 6.6E06 1.7E06 2.8E06 Number of Lc for reference 2: Lc high +Lb+St Blend 1 1.2E06 2.8E06 8.4E05 7.7E05 7.0E05 1.7E06 The amount of La for reference 3: La high + Lb + St Blend 2 6.4E06 5.2E06 8.2E06 6.4E06 3.4E06 1.05E06 Number of Lc for reference 4: Lc high +Lb+St Blend 2 3.0E06 1.6E06 2DE06 2DE06 2DE06 1.8E06 The amount of La for reference 5: La low +lb+St Blend 1 2.5E06 2DE06 2.57E06 2.6E06 1.5E06 1.5E06 Number of Lc for reference 6: Lc low +lb+St Blend 1 1.0E06 8.0E05 6.7E05 2.8E05 5.0E05 2.5E05 The amount of La for reference 7: La low +Lb+St Blend 2 9.2E05 2.7E06 1.05E06 1.6E06 3.5E05 2DE05 Number of Lc for reference 8: Lc low +Lb+St Blend 2 1.4E06 1,ZE05 5.4E05 4.0E04 ND 1.8E04

ND: no data

Table 9 Cell Counts for Culture Mixes of the Invention

cfu/g Day 3 Day 14 Number of La for 1: La high+Lb+St Mix 3 1.6E07 3.2E07 6.6E06 1.7E07 Lc quantity for 2: Lc high+Lb+St Mix 3 2,ZE07 6,ZE07 3.8E07 3.4E07 Number of La for 3: La low +Lb+St Mix 3 8.0E06 8,ZE06 6.5E05 ND Number of Lc for 4: Lc low +Lb+St Mix 3 1.2E07 1.5E07 9.5E06 1.0E07

ND: no data

The discussion of the results.

Culture mixture 1 according to the invention is comparable to standards 1 and 3.

Culture mixture 2 according to the invention is comparable to standards 2 and 4.

Culture mixture 3 according to the invention is comparable to standards 5 and 7.

Culture mixture 4 according to the invention is comparable to standards 6 and 8.

As can be seen from Table. 8 and 9, a mixture of glucose-deficient Streptococcus thermophilus (St mixture 3 from tables 8 and 9) increased the number of cells (see day 3) of both the Lactobacillus acidophilus strain and the Lactobacillus paracasei strain (see day 1). The number of cells increased for both low and high levels of Lactobacillus acidophilus strain or Lactobacillus paracasei strain, in a mixture of cultures inoculated into a milk substrate. The increase in the number of cells was of the order of magnitude from about half to about one and a half logarithmic orders.

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Deposit and permissions for experts

The Applicant requires that a sample of the deposited micro-organisms claimed below may only be made available to the examiner until the expiration of the term for which the patent has been granted.

Streptococcus thermophilus strain CHCC19216 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, December 8, 2015 under DSM 32227.

Streptococcus thermophilus CHCC16731 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, June 4, 2014 under DSM accession number 28889.

Streptococcus thermophilus CHCC15757 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, April 3, 2012 under DSM 25850

Streptococcus thermophilus CHCC15887 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, April 3, 2012 under DSM 25851

Streptococcus thermophilus CHCC 16404 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, December 12, 2012 under DSM accession number 26722.

Streptococcus thermophilus CHCC 14994 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124

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Braunschweig, Germany, April 3, 2012 under accession number DSM 25838.

Streptococcus thermophilus strain CHCC11976 was deposited with Deutsche

Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124

Braunschweig, Germany, September 8, 2009 under accession number DSM 22934.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC759 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, September 6, 2012 under DSM accession number 26419.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, April 3, 2007 under DSM accession 19252.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, September 6, 2012 under DSM 26420.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, September 6, 2012 under accession DSM 26421.

The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC4351 was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, May 19, 2009 under DSM accession 22586.

The Lactobacillus acidophilus La-5 strain, CHCC2169, was deposited as

DSM13241 from 2003-09-30 in Deutsche Sammlung von Mikroorganismen und Zellkulturen

GmbH Maschroder Weg lb, D-38124 Braunschweig.

Lactobacillus paracasei strain CHCC2115, LC-01 deposited at DSMZ-Deutsche

Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstr. 7B, D-38124

Braunschweig. Germany, June 27, 2007 under DSM 19465.

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

Links.

W02011/026863

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

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.

Claims (13)

CLAIM 1. A method for producing a fermented dairy product, including inoculation and fermentation of a milk substrate with a composition containing a Streptococcus thermophilus strain with glucose metabolism deficiency and a Lactobacillus strain, where the Lactobacillus strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus paracasei, and wherein the ratio of CFU/g of Streptococcus thermophilus strain to CFU/g of Lactobacillus strain is at least 80. 2. The method according to claim 1, where the strain of Streptococcus thermophilus is a galactose fermenter and carries a mutation in the DNA sequence of the glcK gene encoding the glucokinase protein, where the mutation inactivates the glucokinase protein or has a negative effect on the expression of said gene. 3. The method according to claim 1 or 2, wherein the strain of Streptococcus thermophilus carries a mutation in a gene encoding a protein involved in glucose transport, which leads to the accumulation of glucose in the environment of the cell. 4. The method of claim 3, wherein the Streptococcus thermophilus strain carries a mutation in a gene encoding a glucose transporter component, wherein the mutation inactivates the glucose transporter or has a negative effect on the expression of said gene. 5. The method according to claim 4, where the Streptococcus thermophilus strain carries a mutation in the DNA sequence of the manM gene encoding the IIC ^Man protein of the glucose/mannose phosphotransferase system, where the mutation is not - 18 039996 mutates the PS ^Map protein or has a negative effect on gene expression. 6. The method according to any one of claims 1 to 5, wherein the strain of Streptococcus thermophilus increases the amount of glucose in 9.5% B-milk to at least 5 mg/ml when inoculated into 9.5% B-milk at a concentration of 1.0E06 -1.0E07 cfu (colony forming units)/ml and grown at 40°C for 20 hours, where 9.5% B-milk is heat-treated milk made using low-fat skimmed milk powder reconstituted to dry weight 9.5%, and pasteurized at 99°C for 30 min, followed by cooling to 40°C. 7. The method according to any one of claims 1 to 6, wherein the strain of Streptococcus thermophilus is selected from the group consisting of the strain of Streptococcus thermophilus CHCC15757, which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) under accession number DSM 25850 , Streptococcus thermophilus strain CHCC15887, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under DSM accession number 25851, Streptococcus thermop hilus strain CHCC16404, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under DSM accession number 26722, Streptococcus deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 28889, Streptococcus thermophilus CHCC19216, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 32227, and a mutant strain obtained from them, where the mutant strain is obtained by using one of said deposited strains as a starting material, and wherein the mutant retains or further improves the lactose fermentation property and/or the glucose secretion property of said deposited strain. 8. The method according to claim 1, where the strain of Lactobacillus is a strain of Lactobacillus delbrueckii subsp. bulgaricus. 9. The method according to any one of claims 1 to 8, where the strain of Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of a strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26420, strain Lactobacillus delbrueckii subsp. bulgaricus CHCC16160, which was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under accession number DSM 26421, and a mutant strain obtained from them, where the mutant strain is obtained by using one of the said deposited strains as a starting material and where the mutant retains or further improves lactose fermentation property and/or glucose secretion property of said deposited strain. 10. The method according to any one of claims 1-9, where the Streptococcus thermophilus strain increases the amount of the Lactobacillus strain to at least 5.0E06 CFU/g in the fermented dairy product at the end of fermentation when inoculated into a milk substrate at a concentration of 1.0E07-1, 0E08 cfu/g in milk substrate and grown at 43°C up to pH 4.55, where the milk substrate is a mixture of fresh skimmed milk with a fat content of 0.1%, fresh semi-skimmed milk with a fat content of 1.5% and powdered skim milk and where the milk substrate contains 4.7% protein, and 1.0% fat, and 0.05% sucrose. 11. The method according to any one of claims 1 to 10, wherein an increased level of Lactobacillus strain cells is obtained in the fermented dairy product. 12. A composition for producing a fermented dairy product containing a strain of Streptococcus thermophilus with insufficient glucose metabolism and a strain of Lactobacillus, where the ratio of CFU/g of the Streptococcus thermophilus strain to CFU/g of the Lactobacillus strain is at least 80 and where the Lactobacillus strain is selected from the group consisting of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus paracasei. 13. Composition according to π.12 containing from 1.0E04 to 1.0E12 CFU/g of Streptococcus thermophilus strain.