STREPTOCOCCUS THERMOPHILUS STRAIN, COMPOSITION, USES THEREOF, FERMENTED MILK PRODUCT, ITS PRODUCTION

专利摘要:
USE OF LACTIC ACID BACTERIA FOR PREPARATION OF FOOD PRODUCTS FERMENTED WITH INCREASED NATURAL SWEETENERS. The present invention relates to mutant strains of Streptococcus thermophilus and mutant strains of Lactobacillus delbrueckii subsp. bulgaricus that excrete glucose into the milk when the milk is inoculated and fermented with such strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus Thus, the present invention relates to strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus that secrete glucose into the milk substrate during fermentation, as well as mixed cultures comprising strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus, starter cultures comprising the strains and dairy products made from the cultures. The present method also relates to using the strains to decrease the lactose content of a fermented food product and to stimulate the growth of the probiotic BB-12(r).
公开号:BR112014026580B1
申请号:R112014026580-1
申请日:2013-04-25
公开日:2022-01-11
发明作者:Eric Johansen；Kim Ib Soerensen；Mirjana Curic-Bawden；Mette Pia Junge
申请人:Chr. Hansen A/S；
IPC主号:

专利说明:

Field of Invention
[0001] The present invention relates to strains of Streptococcus thermophilus bacteria and cultures with a sweetening property by excreting high levels of glucose formed by the degradation of lactose, strains of bacteria of Lactobacillus delbrueckii subsp. bulgaricus with a sweetening property by excreting high levels of glucose formed by the degradation of lactose, starter cultures comprising such strains and dairy products fermented with the cultures. The present invention also relates to a method of obtaining such strains and the use of such strains for the preparation of fermented milk products and for increasing the sweetness of fermented milk products while decreasing the lactose content of milk products. fermented. Background of the Invention
[0002] Pure fermented milk products are recognized for a sour or acidic taste as a result of the conversion of lactose to lactic acid by bacteria during fermentation. They are therefore often sweetened through the addition of fruit, honey, sugar or artificial sweeteners to accommodate customers' desire for a sweet tasting product.
[0003] The food industry has an increasing demand for low calorie food products with sweet taste so as to help overcome the problems of overweight and obesity that have become so common in the last 20 years. Sweetness, generally thought of as a pleasant sensation, is produced by the presence of sugars and certain other substances. The perception of sugars is very different. Using sucrose as a reference of 100, the sweetness of lactose is 16, of galactose is 32, and of 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.
[0004] Sugar in fermented foods is more often being replaced by sweeteners such as aspartame, acesulfame K, sucralose and saccharin, which can provide the sweetness with a lower calorie intake. However, the use of artificial sweeteners can result in an undesirable taste and several studies that indicate that consumption of artificial sweeteners are connected with disadvantages such as increased hunger, allergies, cancer, etc. fermented dairy products that only contain natural sweeteners or, preferably, contain no sweetener.
[0005] Thus, a special challenge lies in the development of fermented dairy products, where the natural (internal) sweetness is high.
[0006] The acidity of fermented dairy products will largely depend on the lactic acid bacteria present and the process parameters used to make the fermented dairy product.
[0007] The fermentation of lactose disaccharide is widely studied in lactic acid bacteria because it is the main source of carbon in milk. In many species, lactose is cleaved by β-galactosidase into glucose and galactose after absorption. Glucose is phosphorylated by glucokinase to glucose-6-phosphate and fermented by Embden-Meyerhof-Parnas (glycolysis) by most lactic acid-producing bacteria (Figure 1).
[0008] Streptococcus thermophilus is one of the most widely used lactic acid bacteria for commercial thermophilic dairy fermentation, where the organism is normally used as part of a mixed starter culture, the other component being Lactobacillus sp., e.g. Lactobacillus delbrueckii subsp. bulgaricus for yogurt or Lactobacillus helveticus for Swiss-type cheese.
[0009] The legal definition of yogurt in many countries requires Streptococcus thermophilus alongside Lactobacillus delbrueckii subsp. bulgaricus. Both species generate desirable amounts of acetaldehyde, an important flavor component in yogurt.
[00010] Lactose and sucrose are more easily fermented by Streptococcus thermophilus than their monosaccharide components. In the presence of excess galactose, only the glucose part of the lactose molecule is fermented, and galactose accumulates in fermented dairy products when Streptococcus thermophilus is used. 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.
[00011] However, galactose fermentation strains of both Streptococcus thermophllus and Lactobacillus delbrueckii subsp. bulgaricus have been reported by several researchers (Hutkins et al. (1986) J. Dairy Sci. 69(1) :1-8; Vaillancourt et al. (2002) J. Bacteriol. 184 (3); 785-793) and in WO 2011/026863 (Chr. Hansen) a method for obtaining strains of Streptococcus thermophilus which are galactose fermentation is described.
[00012] In order to meet the demands of the food industry, it has become relevant to propose new strains, in particular strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus, which provide more natural sweetness than extra calories directly in the fermented product (internal sweetness) by excreting glucose.
[00013] Pool et al. (2006. Metabolic Engineering 8 (5); 456-464) reveals a strain of Lactococcus lactis in which glucose metabolism is completely disrupted by deletion of genes encoding glucokinase, EII (man/glc) and the newly discovered glucose PTS EII (cell). The construction method is genetic recombination to generate all the mutations and the resulting strain is therefore a genetically modified organism (GMO) which at present cannot be used in food products.
[00014] Thompson et al. (1985. J Bacteriol. 162(1);217-223) studied lactose metabolism in Streptococcus lactis (now renamed Lactococcus lactis). In this work, 2-deoxyglucose was used to obtain a mutant in the mannose-PTS system. Subsequently, the mutant was mutated using UV mutagenesis followed by screening for glucose-negative colonies by plate replication. In this way, a double mutant (PTS mannose and glucokinase) was isolated. This double mutant was used to study the mechanisms involved in the regulation of lactose fermentation by "initiator" organisms. These mutants have several disadvantages compared to the mother strain that make them unsuitable for inclusion in a commercial starter culture. The cell yield of the mutants was half that of the mother strain per mole of fermented lactose and the doubling time was significantly increased in the mutants when cultured on lactose. Likewise, the lactic acid yield was half that of the mother strain per mole of lactose fermented. The behavior of these strains in milk has not been analyzed, but it is anticipated that the acidification rate would be significantly reduced.
[00015] Furthermore, Lactococcus lactis is generally not chosen for the production of acetaldehyde and does not contribute to meeting the requirements for the legal definition of yogurt.
[00016] Chervaux et al. (2000. Appl. And Environ. Microbiol., 66, 5306-5311 ), studied the physiology of strains of Lactobacillus delbrueckii subsp. bulgaricus in a 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 reported.
[00017] None of the above approaches solves the problem of providing Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp. bulgaricus with improved properties for natural sweetening of food products that are fermented using these strains alone or in conjunction with other strains of lactic acid bacteria.
[00018] Furthermore, none of the above approaches solves the problem of decreasing the lactose content in food products that are fermented using such strains to a tolerable level for individuals with lactose intolerance. Summary of the Invention
[00019] In contrast to the prior art described above, the present inventors have found that strains of Streptococcus thermophilus with a mutation in the glucokinase (glcK) gene can be selected by exposing galactose-fermenting Streptococcus thermophilus strains to 2-deoxyglixose and that these cells digest lactose and galactose, and excrete glucose into the medium when grown in a milk substrate.
[00020] Surprisingly, these strains of Streptococcus thermophilus are the only ones still fully capable of milk acidification, although the acidification time to pH 5 is delayed by 2 to 5 hours. They are therefore, as such, useful in fermented dairy applications.
[00021] However, glucose is used as a carbon source by many lactic acid bacteria and any glucose excreted can be consumed by other microorganisms present in the fermented milk product.
[00022] To overcome this problem, the present invention provides 2-deoxyglucose resistant mutants of Lactobaciflus delbrueckii subsp. bulgaricus, which must have lost the ability to grow on glucose as a carbon source or exhibit an impaired ability to grow under such conditions. Mutant strains of Lactobacillus delbrueckii subsp. bulgaricus, not only do not consume glucose excreted in the milk by other microorganisms that may be present, they also excrete large amounts of glucose into the surrounding medium and are, surprisingly, still fully capable of acidifying the milk, although the acidification time to pH 5 is delayed by 2 to 5 hours. They are therefore useful in fermented dairy applications as such.
[00023] Such food grade bacteria can be used to fortify fermented dairy products with glucose. Glucose has a greater perceived sweetness than both lactose and galactose and as such, the excretion of glucose into the milk substrate will result in a greater perceived sweetness (internal) in the fermented milk product.
[00024] The inventors of the present invention have found that when a dairy substrate is fermented with a strain of Streptococcus thermophilus and a strain of Lactobacillus delbrueckii subsp. bulgaricus according to the invention, the level of lactose in milk drops significantly.
[00025] Lactose intolerance is a condition caused by the inability to digest lactose. Most individuals with lactose intolerance can tolerate some amount of lactose in their diet, and the severity of their symptoms (including nausea, cramping, bloating, diarrhea, and flatulence) increases with the amount of lactose consumed.
[00026] Thus, it is of great importance in the industry to be able to produce food products that are either lactose-free or have a reduced lactose content.
[00027] There are no common threshold values defined so far in the EU for the lactose content of lactose-free and low-lactose food products, but the Finnish Food Safety Authority Evira sets the Nordic threshold values to be a lactose content of less than 10 mg/100 g or 100 ml for lactose-free foods and a lactose content of less than 1 g/100 g or 100 ml for low-lactose foods.
[00028] The dairy industry today faces a problem of providing an alternative to adding sweeteners to fermented dairy products in order to achieve the desired sweet taste without the additional calories. Furthermore, it would be highly advantageous to establish a method for reducing lactose from fermented dairy products to a level that will be acceptable to lactose intolerant consumers.
[00029] The above problems were solved by using mutant strains of Streptococcus thermophilus and mutant strains of Lactobacillus delbrueckii subsp. bulgaricus, which excrete glucose in milk when 9.5% B-milk is inoculated with 106 to 107 CFU/mL of a strain of Streptococcus thermophilus, according to the invention or with 106 to 107 CFU/mL of a strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention and fermented with strains of Streptococcus thermophilus and strain of Lactobacillus del-brueckii subsp. Bulgaricus, according to the invention, at 40°C for at least 20 hours. Preferably, such mutant strains alone will excrete at least 5 mg/mL glucose in B-milk when 9.5% of B-milk is inoculated with 10 6 to 10 7 CFU/mL of a Streptococcus thermophilus strain, according to the invention or with 106 to 107 CFU/ml of a strain of Lactobacillus delbrueckii subsp. bulgaricus, according to the invention and fermented with strains of Streptococcus thermophilus and strain of Lactobacillus delbrueckii subsp. bulgaricus, according to the invention, at 40°C for at least 20 hours. The strains are still able to acidify the milk, although the acidification time to pH 5 is delayed by 2 to 5 hours. The final fermented milk contains up to 15 mg/mL of lactose in the fermented milk. The final fermented milk consequently has a higher internal index of about 2 times or more.
[00030] To provide the Streptococcus thermophilus strains, the present inventors have provided a method of isolating 2-deoxyglucose resistant mutant strains from a galactose-fermenting Streptococcus thermophilus mother strain, and preferably one with a mutation in the galactose operon which increases the expression of a previously modest expressed or non-expressed operon, wherein resistance to the 2-deoxyglucose phenotype is caused by a mutation in the glucokinase (glcK) gene, which partially or fully inactivates the encoded protein. The method comprises subjecting the mother strain to 2-deoxyglucose and selecting mutant strains that are capable of growing in the presence of 2-deoxyglucose on agar plates containing M17 medium + 2% galactose, as described in Example 1 herein. These mutants are screened and strains that have a growth rate that is higher on M17 + 2% galactose medium than on M17 + 2% glucose medium are chosen.
[00031] It was surprisingly found that mutant strains of Streptococcus thermophilus with a mutation in the glucokinase gene (glcK), but with an apparently normal functioning glucose transport system, were glucose secretors. These mutants were named, CHCC15757 and CHCC15887.
[00032] Furthermore, the inventors have found that strains of Streptococcus thermophilus with an even greater capacity for lactose fermentation and glucose excretion can be selected by subjecting strains of Streptococcus thermophilus with a mutation in the glucokinase gene to 2-deoxyglucose and selection of strains that are unable to grow in B-milk at 9.5%, except when sucrose is added to B-milk at a concentration of only 0.01%. A glucose-secreting and hyperlactose fermentation mutant was named CHCC16404.
[00033] CHCC16404 has been found to have a mutation in a glucose transporter gene (manM), resulting in the inactivation of a glucose transporter protein responsible for transporting glucose into the cell.
[00034] To provide the strains Lactobacillus delbrueckii subsp. bulgaricus, the present inventors have found a method of isolating 2-deoxyglucose resistant mutant strains from a Lactobacillus delbrueckii subsp. bulgaricus, which either has lost the ability to grow on glucose as a carbon source or has an impaired ability to grow on glucose as a carbon source. The method comprises subjecting the mother strain to 2-deoxyglucose and selecting mutant strains that are capable of growing in the presence of 2-deoxyglucose on agar plates containing MRS-IM medium containing 2% lactose, as described in Example 5 here. These mutants are screened and strains that have either completely lost or have an impaired ability to grow on MRS-IM containing 2% glucose as compared to the mother strain that can grow glucose are chosen.
[00035] In accordance with this surprising finding, the present invention relates to novel strains of lactic acid bacteria, such as, in particular, Streptococcus thermophllus with a mutation in the glcK gene or mutants of Lactobacillus delbrueckii subsp. bulgaricus, which excrete glucose into the fermented product to provide a natural sweetening power without extra calories, a method for producing the strains, fermented dairy products made using such strains, and use of such strains for the preparation of fermented dairy products with increased sweetness and decreased levels of lactose.
[00036] In addition, strains of Streptococcus thermophilus and Lactobacillus delbruecklí subsp. bulgaricus of the present invention were surprisingly found to increase the growth of the strain Bifidobacterium animalis subsp. lactis BB-12®, a probiotic bacterium that does not grow well when present alone in milk. Brief Description of Drawings
[00037] Figure 1 is a schematic representation of lactose catabolism in Streptococcus thermophilus, GLcK, glucokinase; LacS, lactose transporter; LacZ, β-galactosidase; GaIM, mutarotase; GalK, galactokinase; GaIT, galactose-1-phosphate uridyltransferase; GalE, UDP-glucose 4-epimerase; Gal1P, galactose-1-phosphate.
[00038] Figure 2 shows the DNA sequence (SEQ ID NO: 1.) of the Streptococcus thermophilus glucokinase (glcK) gene, as well as the sequence of an encoded amino acid (SEQ ID NO: 2.). The single nucleotide substitutions found in CHCC15757 and CHCC15887, respectively, are indicated.
[00039] Figure 3 represents the man operon, which encodes the glucose/mannose phosphotransferase (PTS) system in Streptococcus thermophilus. When compared to the parent strain, CHCC15757, the glucose-secreting and hyperlactose fermentation mutant CHCC16404 was found to have a mutation in the manM gene encoding the IICMan glucose/mannose PTS protein. The G to T mutation changes the codon GAA for glutamic acid at amino acid position 209 to a stop codon TAA (*) aborting translation in CHCC16404 and inactivating protein function.
[00040] Figure 4 depicts the DNA sequence (SEQ ID NO: 5) of the manM gene of Streptococcus thermophilus strain CHCC15757, as well as the encoded amino acid sequence (SEQ ID NO: 6). The single nucleotide substitution found in CHCC16404 is indicated. Detailed Description of the Invention
[00041] As used herein, the term "lactic acid bacterium" designates a gram-positive, microaerophilic or anaerobic bacterium, which ferments sugars with the production of acids including lactic acid as the predominantly produced acid, acetic acid and propionic acid. The most industrially useful acid bacteria are found 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 Lactobacillus sp. and Streptococcus thermophilus, are normally supplied to the dairy industry either as frozen or freeze-dried starter propagation cultures in bulk, or as so-called "Direct Vat Set" (DVS) cultures, intended for direct inoculation into a fermentation vessel or vat for production of a dairy product, such as a fermented dairy product. Such cultures are commonly referred to as "leavens" or "starters".
[00042] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be interpreted as encompassing both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The terms "comprising", "having", "including "and" containing "are to be interpreted as open-ended terms (ie meaning "including, but not limited to"), unless otherwise indicated. Recitation of ranges of values herein are merely intended to serve as a method of shorthand referring individually to each separate value that falls within the range, unless otherwise noted herein, and each separate value incorporated into the specification as a case. were cited here individually. All methods described herein may be performed in any order, unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein, is only intended to further illuminate the invention and does not represent a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any element not claimed to be essential to the practice of the invention.
[00043] In some countries, the legal definition of yogurt requires the presence of both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus Both species generate desirable amounts of acetaldehyde, an important component of yogurt flavor.
[00044] Cheese, such as mozzarella cheese and pizza, as well as feta cheese, can also be prepared by fermentation using both Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (H0ier et al. (2010) in The Technology of Cheesemaking, 2nd Ed. Blackwell Publishing, Oxford; 166-192).
[00045] In order to satisfy the demands of the food industry, it has become desirable to develop new strains, in particular strains of Lactobacillus delbruckii subsp. bulgaricus and Streptococcus thermophilus strains, which produce more natural sweeteners directly in the fermented product (inner sweetness) without contributing extra calories.
[00046] Streptococcus thermophilus is one of the most widely used lactic acid bacteria for commercial milk fermentation, where the organism is normally used as part of a mixed starter culture, the other component being a Lactobacillus sp., e.g. Lactobacillus delbrueckii subsp. bulgaricus for yogurt and Lactobacillus helveticus for Swiss type cheese.
[00047] Lactose and sucrose are more rapidly fermented by Streptococcus thermophilus than their monosaccharide components. Only the glucose part of the lactose molecule is fermented by Streptococcus thermophilus and galactose accumulates in fermented milk products when Streptococcus thermophilus is used. In yogurt, where high acid concentrations limit fermentation, free galactose remains, while the free galactose produced in the early stages of Swiss cheese making is later fermented by Lactobacillus helveticus. Lactococcus lactis found in many ferments used to make cheese is also able to consume the galactose produced by Streptococcus thermophilus.
[00048] In order to ensure Streptococcus thermophilus strains with as optimal growth performance as possible, the present inventors exposed the galactose-fermenting strains of Streptococcus thermophilus to the 2-deoxyglucose selective agent. Typically, 2-deoxyglucose resistant mutants have mutations in the gene encoding glucokinase and in the genes encoding for glucose transport. The isolated mutants, CHCC15757, CHCC15887 and CHCC16404, which are resistant to 2-deoxyglucose have mutations in their gicokinase gene (gfcK). In addition to the mutation in the glucokinase gene, those present found that CHCC16404 has a stop codon mutation in a glucose/mannose transporter gene that could explain why the exported glucose is not transported back into cells again.
[00049] Surprisingly, such mutants are the only ones still fully capable of acidifying milk, although the acidification time to pH 5 is delayed by 2 to 5 hours. They are therefore useful in fermented milk applications and have preserved the mother strains' ability to clot milk, which is characteristic of yogurt. Additionally, it was found that the mutants excreted more than 5 mg/mL of glucose when 9.5% B-milk was inoculated with 106 to 107 of CFU/mL of Streptococcus thermophilus strain CHCC15757 or CHCC15887 and fermented at 40° C for at least 20 hours without the need for isolation of glucose transport mutants, or when 9.5% milk-B with 0.05% sucrose was inoculated with 10 6 to 10 7 CFU/mL of the Streptococcus thermophilus strain CHCC16404 and fermented at 40°C for at least 20 hours. At the same time, as little as about 10 mg/mL lactose and less than 1.5 mg/mL lactose (limit of detection), respectively, remain in the fermented milk. Therefore, the use of such strains for the production of fermented milk products may be of importance for people with lactose intolerance.
[00050] Therefore, the final fermented milk has a higher inner sweetness index of at least 2.0, calculated as described by Godshall (1988. Food Technology 42(11): 71-78).
[00051] The first aspect of the present invention, therefore, relates to a galactose-fermenting mutant strain of Streptococcus thermophilus, wherein the mutant strain carries a mutation in the DNA sequence of the glcK gene encoding a glucokinase protein, in that the mutation inactivates the encoded glucokinase protein or has a negative effect on gene expression. Methods for measuring the level of glucokinase activity or the level of glucokinase gene expression are readily known (Porter et al. (1982) Biochim. Biophys. Acta, 709; 178-186) and include enzyme assays with commercially available kits. available and transcritical or quantitative PCR using materials that are readily available.
[00052] A bacterial "strain", as used herein, refers to a bacterium that remains genetically unaltered when cultivated or multiplied. The multitude of identical bacteria is included.
[00053] 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. Streptococcus thermophilus strains ferment - galactose strains are defined herein as strains of Streptococcus thermophilus, which lower the pH of M17 broth containing 2% galactose as the sole carbohydrate to 5.5 or less when inoculated from a 1% overnight culture and incubated for 24 hours at 37°C.
[00054] Galactose fermenting strains can be obtained by the method described in WO 2011/026863.
[00055] The term "inactivated mutation of glucokinase protein", as used herein, refers to a mutation that results in an "inactivated glucokinase protein", a glucokinase protein that, when present in a cell, is not capable of to perform its normal function, as well as mutations that prevent the formation of the glucokinase protein or result in the degradation of the glucokinase protein.
[00056] In particular, an inactivated glucokinase protein is a protein that, compared to a functional glucokinase protein, is not able to facilitate the phosphorylation of glucose to glucose 6-phosphate or facilitate 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 comprises a mutation in the open reading frame (ORF) of the gene, wherein said mutation may include, but is not limited to, a deletion , a frameshift mutation, introduction of a stop codon or a mutation that results in an amino acid substitution, which alters the functional properties of the protein, or a promoter mutation that reduces or suppresses transcription or translation of the gene.
[00057] In preferred embodiments, the mutation reduces the activity (the 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%, such as at least 90%.
[00058] Glucokinase activity can be determined by glucokinase enzymatic assays as described by Pool et al. (2006. Metabolic Engineering 8; 456-464).
[00059] The term "functional glucokinase protein" as used herein refers to a glucokinase protein which, when present in a cell, facilitates the phosphorylation of glucose to glucose-6-phosphate. In particular, a functional glucokinase protein can be encoded by a gene comprising an ORF that has a sequence that corresponds to position 1-966 in SEQ ID NO: 1 or a sequence that has at least 85% identity, such as at least at least 90% identity, such as at least 95% identity, such as at least 98% identity, such as at least 99% identity, with the sequence corresponding to position 1-966 of SEQ ID NO: 1.
[00060] The percent identity of the two sequences can be determined using mathematical algorithms, such as the algorithm of Karlin and Altschul (1990. Proc. Natl. Acad. Sci. USA 87; 2264), the modified algorithm described in Karlin and Altschul (1993. Proc. Natl. Acad. Sci. USA 90; 5873-5877); The algorithm of Myers and Miller (1988. CABIOS 4; 11-17); the algorithm of Needleman and Wunsch (1970. J. Mol. Biol. 48; 443-453); and algorithm of Pearson and Lipman (1988. Proc. Natl. Acad Sci. USA 85;2444-2448). Computer software for determining nucleic acid or amino acid sequence identity based on these mathematical algorithms is also available. For example, nucleotide sequence comparison can be performed with the BLASTN program, score = 100, word length = 12. Amino acid sequence comparison can be performed with the BLASTX program, score = 50, word length = 3. For the remaining parameters of BLAST programs, the default parameters can be used.
[00061] In many countries, the use of genetically modified organisms (GMOs) for fermented milk products is not accepted. The present invention therefore provides a method for obtaining naturally occurring or induced mutant strains which can provide a desirable accumulation of glucose in the fermented milk product.
[00062] Thus, in a most preferred embodiment of the present invention, the mutant strain is a naturally occurring mutant or an induced mutant.
[00063] A "mutant bacterium" or a "mutant strain", as used herein, refers to a single (naturally occurring, spontaneous) mutant bacterium or an induced mutant bacterium that comprises one or more mutations in its genome (DNA) , which are absent 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 "naturally occurring mutant" has not been mutated by man. Mutant bacteria are here non-GMO (non-genetically modified organism), ie not modified by recombinant DNA technology.
[00064] "Wild-type strain" refers to the unmutated form of a bacterium as found in nature.
[00065] Terms such as "strains with a sweetening property", "strains that can provide a desirable accumulation of glucose in the fermented milk product" and "strains with improved properties for the natural sweetness of food products" are used interchangeably herein to characterize a advantageous aspect of using the strains of the present invention in the fermentation of dairy products.
[00066] In a preferred embodiment, the mutant strain of Streptococcus thermophilus, according to the present invention, increases the amount of glucose in 9.5% B-milk by at least 5 mg/mL when inoculated at 9, 5% B-milk at a concentration of 106 to 107 CFU/ml and grown at 40°C for at least 20 hours.
[00067] 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 at a sucrose concentration with 0.05% sucrose of 106 to 107 CFU/ml and grown at 40°C for at least 20 hours.
[00068] In the present context, 9.5% B-milk is boiled milk made with low-fat skim milk reconstituted powder to a dry matter level of 9.5% and pasteurized at 99°C for 30 min. followed by cooling to 40°C.
[00069] In more preferred embodiments of the invention, the mutant strain leads to an increase in the amount of 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 at least 9 mg/mL, such as at least 10 mg/mL, such as at least 11 mg/mL, such as at least 12 mg/mL, such as at least 13 mg/mL, such as at least 14 mg/mL , such as at least 15 mg/ml, such as at least 20 mg/ml, such as at least 25 mg/ml.
[00070] In another embodiment of the invention, the mutant strain of Streptococcus thermophilus is resistant to 2-deoxyglucose.
[00071] The term "resistant to 2-deoxyglucose" here is defined because a particular mutated bacterial strain has the ability to grow to a colony when seeded on a plate of M17 medium containing 20 mM 2-deoxyglucose after incubation at 40° C for 20 hours. The presence of 2-deoxyglucose in the culture medium will prevent non-mutated strains from growing, while the growth of mutated strains is unaffected or not significantly affected. Non-mutated strains that can be used as reference susceptible strains in the evaluation of resistance preferentially include strains CHCC14994 and CHCC11976.
[00072] Examples 1 and 2 described herein exemplify the isolation of mutant strains of Streptococcus thermophilus that are resistant to 2-deoxyglucose.
[00073] In yet another embodiment, the mutant strain according to the invention may be characterized by its growth pattern. This is illustrated by the finding that the growth rate of the mutant strain is higher in M17 medium + 2% galactose than in M17 medium + 2% glucose. The growth rate is measured as the development of the optical density of the exponentially growing culture at 600 nanometers (OD600) with time, as described in Example 2 here.
[00074] In a preferred embodiment, the growth rate is at least 5% greater, such as at least 10% greater, such as at least 15% greater, such as at least 20% greater, on M17 + 2% medium of galactose than in the M17 + 2% glucose medium.
[00075] In a preferred embodiment, the mutation results in the replacement of the serine coding codon with the proline coding strand in SEQ ID NO: 2. Preferably, the mutation in the glcK gene results in the substitution of a T , with a C at position 214 in SEQ ID NO: 1.
[00076] In another preferred embodiment, the mutation results in the replacement of the threonine coding codon with the isoleucine coding codon at position 141 in SEQ ID NO: 2.
[00077] Preferably, the mutation in the glcK gene results in the substitution of a C for a T at position 422 in SEQ ID NO: 1.
[00078] It should be emphasized that the glcK gene of a Streptococcus thermophilus can be inactivated by other types of mutations at other sites in the glcK gene.
[00079] In a preferred embodiment, the Streptococcus thermophilus strain carries a mutation that reduces the transport of glucose into the cell.
[00080] The term "a mutation that reduces the transport of glucose into the cell", as used herein, refers to a mutation in a gene encoding a protein involved in the transport of glucose that results in an accumulation of glucose in the environment. of the cell. The glucose level in the culture medium of a strain of Streptococcus thermophilus can be easily measured by methods known to the person skilled in the art and as described in Example 4, also herein, when the culture medium is a milk substrate.
[00081] 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% .
[00082] The transport of glucose into the cell can be determined by the glucose uptake assay, as described by Cochu et al. (2003. Appl Environ Microbiol 69 (9); 5423-5432).
[00083] Preferably, the Streptococcus thermophilus strain carrying a mutation in a gene encoding a component of a glucose transporter, wherein the mutation inactivates the glucose transporter protein or has a negative effect on gene expression .
[00084] The component can be any component of a glucose transporter protein, which is critical for the transport of glucose. For example, it is contemplated that inactivation of any component of the glucose/mannose PTS in Streptococcus thermophilus depicted in Figure 3 will result in inactivation of glucose transporter function.
[00085] The term "inactivated glucose transporter mutation", as used herein, refers to a mutation that results in an "inactivated glucose transporter", a glucose transporter protein which, if present in a cell, is not capable of exerting its normal function as well as mutations that prevent glucose transport protein formation or result in glucose transport protein degradation.
[00086] In particular, an inactivated glucose transporter protein is a protein that, compared to a functional glucose transporter protein, is not able to facilitate the transport of glucose across a plasma membrane or facilitate the transport of glucose along the plasma membrane. across a plasma membrane at a significantly reduced rate. The gene encoding such an activated glucose transporter protein compared to the gene encoding a functional glucose transporter protein comprises a mutation in the open reading frame (ORF) of the gene, wherein said mutation may include, but is not limited to a, a deletion, a frameshift mutation, introduction of a stop codon, or a mutation that results in an amino acid substitution, which alters the functional properties of the protein, or a promoter mutation that reduces or suppresses the transcription or translation of the gene.
[00087] In preferred embodiments, the mutation reduces the activity (the rate of glucose transport) of glucose transporter protein by at least 50%, such as at least 60%, such as at least 70%, such as at least least 80%, such as at least 90%.
[00088] Glucose transporter activity can be determined by the glucose uptake assay as described by Cochu et al. (2003. Appl Environ Microbiol 69 (9); 5423-5432).
[00089] The term "functional glucose transporter protein" as used herein refers to a transporter protein which, if present in a cell, facilitates the transport of glucose across a plasma membrane.
[00090] More preferred, the Streptococcus thermophilus strain, carrying a mutation in the DNA sequence of the manM gene encoding the IICMan protein of the glucose/mannose phosphotransferase system, wherein the mutation inactivates the IICMan protein or has a negative effect on gene expression.
[00091] In an even more preferred embodiment, the mutation results in the substitution of the codon coding for glutamic acid with a stop codon at position 209 of SEQ ID NO: 6 of the IICMan protein of the glucose/mannose phosphotransferase system. Preferably, the mutation results in the replacement of a G with a T at position 625 of SEQ ID NO: 5.
[00092] A second aspect of the invention relates to a Streptococcus thermophilus strain selected from the group consisting of the Streptococcus thermophilus strain CHCC15757, which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 25850, a Streptococcus thermophilus strain CHCC15887 which has been deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 25851, Streptococcus thermophilus strain CHCC16404 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 26722 and strains derived therefrom.
[00093] In the present context, the term "strains derived therefrom" shall be understood as derived strains, or strains which can be derived from a strain (or its parent strain) of the invention by, for example, genetic engineering, radiation and/or chemical treatment. The "strains derived therefrom" may be spontaneously occurring mutants. It is preferred that the "strains derived therefrom" are functionally equivalent mutants, e.g., mutants that have substantially the same or improved properties (e.g., with respect to glucose excretion) as their parent strain. Such "strains derived therefrom" are part of the present invention. Especially, the term "strains derived therefrom," refers to strains obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment, including treatment with a chemical mutagen such as methane sulfonate ethane (EMS) or N-methyl- N'-nitro-N-nitroguanidine (NTG), UV light or a spontaneously occurring mutant. A mutant may have undergone several mutagenization treatments (a single treatment should be understood as a mutagenization step followed by a screening/selection step), but it is presently preferred that not more than 20, or not more than 10, or no more than 5 treatments (or screening/selection steps) are performed. In a presently 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 were replaced. -tuated by another nucleotide, or excluded, compared to the mother strain.
[00094] Thus, in a preferred embodiment, 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 under No. accession DSM 25850, a strain of Streptococcus thermophilus CHCC15887 which has been deposited with the Deutsche Sammlung von Mikroorganis und Zellkulturen under No. accession DSM 25851, a strain of Streptococcus thermophilus CHCC16404 which has been deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen under No. accession DSM 26722 and a mutant strain derived therefrom, wherein the mutant strain is obtained using one of the deposited strains as starting material, and wherein the mutant has retained or further improved the property of fermenting lactose and/or the secretory property of glucose of said deposited strain.
[00095] Lactobacillus delbrueckii subsp. bulgaricus is a lactic acid bacterium that is often used for commercial fermentation of milk, where the organism is normally used as part of a mixed starter culture.
[00096] Lactose is fermented more easily than the monosaccharides of glucose, fructose and mannose by Lactobacillus delbrueckii subsp. bulgaricus and strains of these species do not normally grow on galactose (Buchanan RE, Gibbons NE, eds (1974): Bergey's manual of determinative bacteriology (The Williams & Wilkins Co. Baltimore, Md), 8th ed.) during fermentation of lactose by Lactobacillus delbrueckii subsp. bulgaricus, only the glucose part of the lactose molecule is fermented and thus accumulates galactose in fermented milk products.
[00097] In order to obtain the strains of Lactobacillus delbrueckii subsp. bulgaricus that are unable to grow on glucose as a carbon source, the present inventors have exposed strains of Lactobacillus delbrueckii subsp. bulgaricus to 2-deoxyglucose. The isolated mutants were resistant to 2-deoxyglucose and able to grow in a milk substrate without the use of glucose as a carbon source. The mutants were found to increase the glucose content of milk. Therefore, fermented milk products produced using these strains are characterized by a higher amount of glucose, which makes the products sweeter to the taste.
[00098] Surprisingly, strains of Lactobacillus delbrueckii subsp. bulgaricus of the invention alone are still fully capable of milk acidification, although the acidification time to pH 5 is delayed by hours. Furthermore, as demonstrated in the Examples, the strains were found to excrete approximately 5 mg/mL or more of glucose while less than approximately 10 mg/mL of lactose remained in the fermented milk, when 9.5% B-milk was inoculated with 106 to 107 of CFU/mL of a strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention, and fermented with the strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention, at 40°C for at least 20 hours. Therefore, the use of such strains for the production of fermented milk products may be of importance for people with lactose intolerance.
[00099] Therefore, the final fermented milk has a high internal sweetness index of approximately 2 or greater, calculated as described by Godshall (1988. Food Technology 42(11): 7178), such as 2.5 or greater, such as 3 or higher.
[000100] The third aspect of the present invention relates to a strain of Lactobacillus delbrueckii subsp. bulgaricus, wherein said strain is resistant to 2-deoxyglucose.
[000101] The term "2-deoxyglucose resistant" in relation to a strain of Lactobacillus delbrueckii subsp. bulgaricus is defined by a particular bacterial strain, which has the ability to grow to a colony after incubation at 40°C for 20 hours when seeded on a plate of 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 non-resistant strains, while the growth of resistant strains is unaffected or not significantly affected. Lactobacillus delbrueckii subsp. bulgaricus non-resistant strains, which can be used as sensitive reference strains in the evaluation of resistance, include strains of Lactobacillus del-brueckii subsp. bulgaricus CHCC759, which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under No. accession DSM 26419 and CHCC10019, which has been deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen (DSMZ) under No. Access DSM 19252.
[000102] In the case of MRS-IM agar plates containing 2% lactose and, in addition, plate containing 20 mM 2-deoxyglucose is full of colonies, which is suitable for increasing the concentration of 2-deoxyglucose in the plates, for example to 30 mM or even 40 mM or more. In case no colonies are obtained, it is appropriate to decrease the concentration of 2-deoxyglucose on the plates, for example, to 15 mM or 10 mM, or even less. If deemed necessary, the mutation rate can be enhanced through appropriate physical or chemical mutagenesis protocols.
[000103] Preferably, the strain of Lactobacillus delbrueckii subsp. bulgaricus of the invention increases the amount of glucose in 9.5% of B-milk to at least 5 mg/mL when inoculated into 9.5% of B-milk at a concentration of 106 to 107 CFU/ ml and grown at 40°C for at least 20 hours, such as for between 20 to 30 hours, such as for 20 hours.
[000104] In more preferred embodiments of the invention, the mutant strain leads to an increase in the amount of 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 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.
[000105] A fourth aspect of the invention relates to a strain of Lactobacillus delbrueckii subsp. Bulgaricus, which is selected from the group consisting of the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, which was deposited with the Deutsche SammLung von und Mikroorganismen Zellkulturen (DSMZ) under No. accession DSM26420, the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16160, which was deposited with the Deutsche SammLung von und Mikroorganismen Zellkulturen (DSMZ) under No. accession DSM26421, and strains derived therefrom.
[000106] The term "strains derived therefrom" shall be understood as defined above.
[000107] Thus, in a preferred embodiment, the strain of Lactobacillus delbrueckii subsp. bulgaricus is selected from the group consisting of the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159, which was deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen (DSMZ) under No. accession DSM26420, a strain of Lactobacillus delbrueckii subsp. bulgaricus to CHCC16160, which was deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen (DSMZ) under No. accession DSM26421 and a mutant strain derived therefrom, where the mutant strain is obtained using one of the deposited strains as a starting material, and where the mutant has maintained or further improved the lactose fermentation property and/or the secreting glucose of said deposited strain.
[000108] A fifth aspect of the present invention relates to a composition comprising 104 to 1012 CFU (colony forming units)/g of a strain of Streptococcus thermophilus, according to the first or second aspect of the invention, such as from from 105 to 1011 CFU/g, such as from 106 to 1010 CFU/g, or, such as from 107 to 10 9 CFU/g Streptococcus thermophilus strain.
[000109] In a preferred embodiment, the Streptococcus thermophilus strain 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 B is inoculated with 106 to 107 CFU/mL of the Streptococcus thermophilus strain and incubated for 14 hours at 40°C, and the composition further comprises an amount of a compound, which can cause acidification of 9.5% of milk-B by the Streptococcus thermophilus strain CHCC16404, which was deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen under No. accession DSM 26722, defined as resulting in a pH reduction of 1.0 or more when 9.5% B-milk is inoculated with 106 to 107 CFU/mL of the Streptococcus thermophilus strain and incubated for 14 hours at 40°C.
[000110] Preferably, the compound is sucrose.
[000111] Preferably, the amount of sucrose is from 0.000001% to 2%, such as from 0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001 % to 0.05%.
[000112] In an especially preferred embodiment, the composition further comprises from 104 to 1012 CFU/g of a strain of Lactobacillus delbrueckii subsp. bulgaricus, according to the invention, such as from 105 to 1011 CFU/g, such as 106 to 1010 CFU/g, or such as from 107 to 109 CFU/g of the Lactobacillus delbrueckii subsp strain . bulgaricus
[000113] A preferred composition of the present invention comprises, for example, the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 and/or the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 in combination with Streptococcus thermophilus strain CHCC15757. An even more preferred composition comprises Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 and/or the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 in combination with Streptococcus thermophilus strain CHCC15887. An even more preferred composition comprises the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16159 and/or the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 in combination with Streptococcus thermophilus strain CHCC16404.
[000114] Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are commonly used as starter cultures serving a technological purpose in the production of various foods, such as in the dairy industry, such as for fermented milk products. Thus, in another preferred embodiment, the composition is suitable as a starter culture.
[000115] Starter cultures can be provided as frozen or dried starter cultures in addition to liquid starter cultures. Thus, in yet another preferred embodiment, the composition is in frozen, lyophilized or liquid form.
[000116] As disclosed in WO 2005/003327, it is advantageous to add certain cryoprotective agents to a starter culture. Thus, a starter culture composition in accordance with the present invention may comprise one or more cryoprotective agent(s) 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 derivatives of any such compounds.
[000117] A sixth aspect of the invention is directed to a method for producing a fermented milk product comprising inoculating and fermenting a milk substrate with at least one strain of Streptococcus thermophilus, according to the first or second aspect of the invention.
[000118] The term "milk" is to be understood as the milky secretion obtained by milking any mammal, such as a cow, a sheep, a goat, a buffalo or a camel. In a preferred embodiment, the milk is cow's milk.
[000119] The term "milk substrate" can be any raw milk and/or processed material, which can be subjected to fermentation, in accordance with the method of the invention. Thus, useful milk substrates include, but are not limited to, solutions/suspensions of any milk or products such as milk that comprise protein, such as whole or low fat milk, skimmed milk, reconstituted milk powder, condensed milk, milk in powder, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream. Obviously, the milk substrate can be from any mammal, for example, the mammalian milk being substantially pure or reconstituted milk powder.
[000120] Preferably, at least part of the milk protein in the substrate is naturally occurring milk proteins, such as casein or whey protein. However, some of the protein may be proteins that are not naturally occurring in milk.
[000121] Prior to fermentation, the milk substrate can be homogenized and pasteurized according to methods known in the art.
[000122] "Homogenization" as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is carried out before fermentation, it can be carried out to break the milk fat into smaller pieces that no longer separate from the milk. This can be achieved by forcing the milk at high pressure through small holes.
[000123] "Pasteurization", as used herein, means the treatment of the milk substrate to reduce or eliminate the presence of living organisms, such as microorganisms. Preferably, pasteurization is achieved by maintaining a specified temperature for a specified period of time. The specified temperature is generally reached by heating. The temperature and duration can be selected to inactivate or kill certain bacteria, such as harmful bacteria. A rapid cooling step may follow.
[000124] "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 comprises the conversion of lactose to lactic acid.
[000125] Fermentation processes to be used in the production of fermented milk products are well known and the person skilled in the art will know how to select suitable process conditions such as temperature, oxygen, quantity and characteristics of microorganism(s) and process time. Obviously, the fermentation conditions are selected in order to support the realization of the present invention, that is, to obtain a dairy product in solid or liquid form (fermented milk product).
[000126] The term "fermented milk product", as used herein, refers to a human or animal food product, wherein the preparation of the human or animal food product involves the fermentation of a milk substrate with bacteria from the lactic acid. "Fermented milk product" as used herein includes, but is not limited to, products such as yogurt, cheese, sour cream and buttermilk, as well as fermented whey.
[000127] In a preferred embodiment, the concentration of inoculated Streptococcus thermophilus cells is from 104 to 109 CFU of Streptococcus thermophilus cells per ml of milk substrate, such as from 104 CFU to 108 CFU of milk cells. Streptococcus thermophilus per mL of milk substrate.
[000128] In another preferred embodiment, the Streptococcus thermophilus strain is unable to acidify 9.5% B-milk, defined as the result of a pH decrease of less than 1.0 when 9.5% milk -B is inoculated with 106 107 CFU/mL of the Streptococcus thermophilus strain incubated for 14 hours at 40°C and the milk substrate is added in an amount of an effective compound capable of acidification of 9.5% of the milk -B by the Streptococcus thermophilus strain CHCC16404 which was deposited with the Deutsche SammLung von und Mikroorganismen Zellkulturen under No. accession DSM 26722, defined as resulting in a pH decrease of 1.0 or more when 9.5% B-milk is inoculated with 10 6 to 10 7 CFU/mL of the Streptococcus thermophilus strain and incubated for 14 hours at 40°C.
[000129] Preferably, the compound is sucrose.
[000130] Preferably, the amount of sucrose is from 0.000001% to 2%, such as from 0.00001% to 0.2%, such as from 0.0001% to 0.1%, such as from 0.001 % to 0.05%.
[000131] A seventh aspect of the invention is directed to a method for producing a milk product comprising fermenting and inoculating a milk substrate with at least one strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the third or fourth aspect of the present invention.
[000132] In a preferred embodiment, the concentration of Lactobacillus delbrueckii subsp. bulgaricus inoculated is 104 to 109 CFU of Lactobacillus delbrueckii subsp. bulgaricus per ml of milk substrate, such as from 10 4 CFU to 10 8 CFU of Lactobacillus delbrueckii subsp. bulgaricus per mL of milk substrate.
[000133] In a preferred embodiment, the method for producing the fermented milk product comprises inoculating and fermenting a milk substrate with at least one strain of Streptococcus thermophilus, according to the present invention, at least one strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the present invention.
[000134] In another preferred embodiment, the fermented milk product is a yogurt or a cheese.
[000135] Examples of cheeses that are prepared 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. Blackwell Publishing, Oxford; 166-192).
[000136] Preferably, the fermented milk product is a yogurt.
[000137] In the present context, a yogurt starter culture is a bacterial culture comprising at least one strain of Lactobacillus delbrueckii subsp. bulgaricus and at least one strain of Streptococcus thermophilus. Accordingly, the term "yogurt" refers to a fermented milk product, obtained by inoculating and fermenting milk, with a composition comprising a strain of Lactobacillus delbrueckii subsp. bulgaricus and a strain of Streptococcus thermophilus.
[000138] In an eighth aspect, the present invention relates to a fermented milk product obtainable by the method according to the sixth or seventh aspect of the invention.
[000139] In a ninth aspect, the present invention relates to a fermented milk product comprising at least one strain of Streptococcus thermophllus, according to the first or second aspect of the invention.
[000140] In a tenth aspect, the present invention relates to a milk product fermented with at least one strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the third or fourth aspect of the invention.
[000141] In a preferred embodiment, the fermented milk product comprises at least one strain of Streptococcus thermophilus, according to the invention, and at least one strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention.
[000142] In another preferred embodiment, the fermented milk product is a yogurt or a cheese. Preferably, the fermented product is a yogurt.
[000143] In an eleventh aspect, the present invention relates to the use of a strain of Streptococcus thermophilus, according to the first or second aspect of the invention for the preparation of a fermented milk product.
[000144] In a twelfth aspect, the present invention relates to the use of a strain Lactobacillus delbrueckii subsp. bulgaricus, according to the third or fourth aspect of the invention, for the preparation of a fermented milk product.
[000145] In a thirteenth aspect, the present invention relates to the use of a strain of Streptococcus thermophilus according to Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention, for the preparation of a fermented milk product.
[000146] In a fourteenth aspect, the present invention relates to the use of a strain of Streptococcus thermophilus, according to the first or second aspect of the invention, to increase the sweetness of a fermented milk product.
[000147] In a fifteenth aspect, the present invention relates to the use of a strain of Lactobacillus delbrueckii subsp. bulgaricus, according to the third and fourth aspects of the present invention, to increase the sweetness of a fermented milk product.
[000148] In a sixteenth aspect, the present invention relates to the use of a strain of Streptococcus thermophilus, according to the first or second aspect of the invention, and a strain of Lactobacillus delbrueckii subsp. bulgaricus to increase the sweetness of a fermented milk product.
[000149] Especially, children as a consumer group have a preference for sweet-tasting foods and it is contemplated that the Streptococcus thermophilus strain of the invention and the Lactobacillus delbrueckii subsp. bulgaricus of the invention can be particularly useful for increasing the sweetness of a fermented milk product intended for children.
[000150] A seventeenth aspect of the present invention relates to a fermented milk product in accordance with the invention for use in reducing calorie intake.
[000151] The fermented milk product according to the invention is thought to be especially useful for the diet of people suffering from overweight or obesity.
[000152] Thus, in a preferred embodiment, the fermented milk product according to the invention is for use in reducing the calorie intake of a person suffering from overweight or obesity.
[000153] Overweight and obesity are medical conditions defined by the World Health Organization (WHO) as abnormal or excessive fat accumulation that presents a health risk. The Body Mass Index (BMI) can be used as a guide to classify overweight and obesity in adults, and is calculated as a person's weight in kilograms divided by the square of his/her height in meters (kg/ m2). The WHO definition predicts that a BMI greater than or equal to 25 is overweight and a BMI greater than or equal to 30 is obese.
[000154] An eighteenth aspect of the present invention relates to the use of a strain of Streptococcus thermophilus, according to the first or second aspect of the invention, to decrease the lactose content in a fermented milk product.
[000155] In a nineteenth aspect, the present invention relates to the use of a strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the third or fourth aspect of the invention, for decreasing the lactose content in a fermented milk product.
[000156] A twentieth aspect of the present invention relates to the use of a Streptococcus thermophilus, according to the first or second aspect of the invention, and a strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the third or fourth aspect of the invention, for decreasing the lactose content in a fermented milk product.
[000157] A twenty-first aspect of the present invention is directed to a fermented milk product in accordance with the invention for use in preventing the symptoms of lactose intolerance.
[000158] A twenty-second aspect relates to a composition of the invention for use as a medicament.
[000159] In a twenty-third aspect of the invention, it is directed to the use of a strain of Streptococcus thermophllus according to the invention to enhance the growth of a strain of Bifidobacterium.
[000160] In a preferred embodiment, the Bifidobacterium strain belongs to a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Blfldobacterium lactis, Bifidobacterium brevis, Bifidobacterium animalis, Bifidobacterium adolescentis and Bifidobacterium infantil, such as a strain selected from the group consisting of the strain of Bifidobacterium animalis subsp. lactis BB-12® deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under No. accession DSM 15954, Bifidobacterium animalis strain deposited with the DSMZ under No. accession DSM 15954, strain of Bifidobacterium longum subsp. infantile filed with the DSMZ under No. accession DSM 15953 and strain of Bifidobacterium longum subsp. longum deposited with the DSMZ under No. accession DSM 15955. The most preferred strain of Bifidobacterium is Bifidobacterium animalis subsp. lactis BB-12® deposited with the DSMZ under No. Access DSM 15954.
[000161] In a twenty-fourth aspect, the invention relates to the use of a strain of Lactobacillus delbrueckii subsp. bulgaricus, according to the invention, to enhance the growth of a strain of Bifidobacterium.
[000162] In a preferred embodiment, the Bifidobacterium strain belongs to a species selected from the group consisting of Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium lactis, Bifidobacterium brevis, Bifidobacterium animalis, Bifidobacterium adolescentis and Blfldobacterium infantil, such as a strain selected from the group consisting of the strain of Bifidobacterium animalis subsp. lactis BB-12® deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) under No. accession DSM 15954, Bifidobacterium animalis strain deposited with the DSMZ under No. accession DSM 15954, strain of Blfldobacterium longum subsp. infantile filed with the DSMZ under No. accession DSM 15953 and strain of Bifi-dobacterium longum subsp. longum deposited with the DSMZ under No. accession DSM 15955. The most preferred strain of Bifidobacterium is Bifi-dobacterium animalis subsp. lactls BB-12® deposited with the DSMZ under No. Access DSM 15954.
[000163] A twenty-fifth aspect relates to the use of the Streptococcus thermophilus strain, according to the invention, a strain of Lactobacillus delbrueckii subsp. Bulgaricus, according to the invention, to improve the growth of the strain of Bifidobacterium animalis subsp. lactis BB-12® which was deposited with the Deutsche Sammlung von und Mikroorganismen Zellkulturen under No. Access DSM 15954.
[000164] 2-deoxyglucose and a determination of the growth pattern of bacteria in M17 medium + 2% galactose relative to M17 medium + 2% glucose is used for the selection of bacteria that have a mutation in the glucokinase gene (glcK) .
[000165] In a twenty-sixth aspect of the present invention, a method for screening and for isolating a strain of Streptococcus thermophilus with a mutated glcK gene is provided. The method comprises the following steps: a) providing a galactose-fermenting Streptococcus thermophilus mother strain; b) selecting and isolating, from a set of mutant strains of Streptococcus thermophilus derived from the mother strain, a set of mutant strains of Streptococcus thermophilus that are resistant to 2-deoxyglucose; and c) select and isolate, from the set of mutant strains of Streptococcus thermophilus that are resistant to 2-deoxyglucose, a mutant strain of Streptococcus thermophilus, if the growth rate of the mutant strain of Streptococcus thermophilus is higher on M17 + 2% medium of galactose than in the M17 medium + 2% glucose.
[000166] The term "2-deoxyglucose resistant" here is defined because a particular mutated bacterial strain has the ability to grow from a colony when seeded onto M17 medium plates containing 2% lactose or 2% galactose and containing 20 mM 2-deoxyglucose, after incubation at 40°C for 20 hours. The presence of 2-deoxyglucose in the culture medium will prevent the growth of non-mutated strains, while the growth of mutant strains is unaffected or not significantly affected. Unmutated strains, which can be used as reference susceptible strains in the evaluation of resistance, include strains CHCC14994 and CHCC11976.
[000167] Examples 1 and 2 herein exemplify the isolation of mutant strains of Streptococcus thermophilus, which are resistant to 2-deoxyglucose.
[000168] In a preferred embodiment, the method further comprises step a1) subjecting the mother strain to mutagenization, such as subjecting the mother strain to a physical and/or chemical mutagen.
[000169] In another preferred embodiment, the method further comprises a step d) selecting and isolating a set of 2-deoxyglucose resistant Streptococcus thermophilus strains derived from the Streptococcus thermophilus strain selected in step c) a Streptococcus thermophilus strain if the rate The growth rate of the Streptococcus thermophilus strain is high on M17 + 2% sucrose medium, but zero or at least 0 to 50% reduced compared to the growth rate of the mother strain on M17 + 2% glucose medium.
[000170] The galactose-fermenting Streptococcus thermophilus mother strains are capable of growing on/in M17 medium + 2% galactose and are defined herein as having the ability to lower the pH in M17 broth containing 2% galactose as single carbohydrate of 5.5 or less when inoculated from a 1% overnight culture and incubated for one hour at 37°C. Such galactose-positive strains were described in the prior art in WO2011/026863 (Chr. Hansen A/S) describes a method for obtaining such strains.
[000171] In a most preferred embodiment, the mother strain is selected from the group consisting of the Streptococcus thermophilus strain CHCC14994 which has been deposited with the Deutsche SammLung von Mikroorganismen und Zellkulturen under No. accession DSM 25838, Streptococcus thermophilus strain CHCC11976 which has been deposited with Deutsche SammLung von Mikroorganismen und Zellkulturen under No. accession DSM 22934, and the strains derived therefrom.
[000172] In the present context, the term "strains derived therefrom" shall be understood as derived strains or strains which can be derived from galactose-fermenting Streptococcus thermophilus mother strains, by means of, for example, genetic engineering, radiation and/or chemical treatment. The "strains derived therefrom" can also be spontaneously occurring mutants. It is preferred that the "strains derived therefrom" are functionally equivalent mutants, e.g., mutants that have substantially the same, or improved, properties (e.g., with respect to galactose fermentation) as their parent strain. Such "strains derived therefrom" are part of the present invention. Especially, the term "strains derived therefrom" refers to strains obtained by treating a strain of the invention by any conventionally used mutagenization treatment, including treatment with a chemical mutagen such as methane sulfonate, ethane (EMS), or N -methyl-N'-nitro-N-nitroguanidine (NTG), UV light, or a spontaneously occurring mutant. A mutant may have undergone several mutagenization treatments (a single treatment should be understood to be a mutation step, followed by a selection/screening step), but it is currently preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening/selection steps) are performed. In a presently preferred mutant 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 were replaced with other nucleotides, or deleted, compared to the mother strain.
[000173] In a twenty-seventh aspect, a mutant strain of Streptococcus thermophilus obtainable by the method, in accordance with the twenty-seventh aspect, is herein understood.
[000174] In a twenty-eighth aspect of the present invention, a method for screening and isolating a strain of Lactobacillus delbrueckii subsp. bulgaricus with impaired glucose metabolism is provided. The method comprises the following steps: a) providing a mother strain of Lactobacillus delbrueckii subsp. bulgaricus; b) select and isolate, from a set of mutant strains of Lactobacillus delbrueckii subsp. bulgaricus derived from the mother strain, a set of strains of Lactobacillus delbrueckii subsp. bulgaricus that are resistant to 2-deoxyglucose; and c) select and isolate, from the set of strains of Lactobacillus delbrueckii subsp. bulgaricus that are resistant to 2-deoxyglucose, a strain of Lactobacillus del-brueckii subsp. Bulgaricus, if the growth rate of the Lac-tobacillus delbrueckii subsp. bulgaricus is higher in the MRS-IM + 2% lactose medium than in the MRS-IM + 2% glucose medium.
[000175] Isolation of mutant strains of Lactobacillus delbrueckii subsp. bulgaricus that are resistant to 2-deoxyglucose is described in detail in the Examples. Based on the 2-deoxyglucose resistance screening assay of Example 5, the skilled artisan can routinely test for a specific strain of interest (e.g., one from a relevant commercial product), whether that specific strain of interest has the resistance relevant here to 2-deoxy-glucose. Based on resistance to the 2-deoxyglucose mutant growth pattern of Example 6, the skilled person can routinely test for a specific strain of interest (e.g., one from a relevant commercial product) whether this specific strain of interest has the relevant growth pattern, which is a property of the selected mutants.
[000176] In a preferred embodiment, the method further comprises step a1) subjecting the mother strain to mutagenization, such as subjecting the mother strain to a chemical and/or physical mutagen.
[000177] The mother strains of Lactobacillus delbrueckii subsp. bulgaricus are able to grow on/on MRS-IM + 2% lactose medium and are defined here so they have the ability to lower the pH in MRS-IM broth containing 2% lactose as the sole carbohydrate to 5. 5 or less when inoculated from a 1% overnight culture and incubated for 24 hours at 37°C.
[000178] In a most preferred embodiment, the mother strain is selected from the group consisting of a strain of Lactobacillus del-brueckii subsp. bulgaricus CHCC759 which was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 26419, the strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC10019 has been deposited with the Deutsche Sammlung von Mikroorganis und Zellkulturen under No. accession DSM 19252, and the strains derived therefrom.
[000179] In a twenty-ninth aspect, a strain of Lactobacillus delbrueckii subsp. bulgaricus obtainable by the method, according to the twenty-eighth aspect, is understood here.
[000180] Embodiments of the present invention are described below, by way of non-limiting examples. EXAMPLES Materials and MethodsMedium
[000181] For Streptococcus thermophilus, the medium used is M17 medium, known to persons skilled in the art.
[000182] M17 agar medium has the following composition per liter of H2O: agar, 12.75 ascorbic acid, 0.5 gpeptone casein (tryptic), 2.5 gβ-disodium glycerophosphate pentahydrate, 19 g magnesium sulfate hydrate, 0.25 gpeptone of meat, 5 gpeptone of meat (peptic), 2.5 gglycerophosphate of sodium, 19 gpeptone of soy (papain), 5 gtryptone, 2.5 g of yeast, 2.5 gpH final 7.0 ± 0.2 (25°C) and M17 broth has the following composition per liter of H2O: ascorbic acid, 0.5 g magnesium sulfate, 0.25 g beef extract, 5 gpeptone from beef (peptic), 5 gglycerophosphate sodium, 19 g soy peptone (papain), 5 g tryptone, 2.5 g yeast extract, 2.5 g final pH 7.0 ± 0.2 (25°C).
[000183] The carbon sources are added to sterilized lactose 20 g/l, glucose 20 g/l or galactose 20 g/l.
[000184] As is known to the skilled person, M17 medium is a medium that is considered to be suitable for the growth of Streptococcus thermophilus. Further, as understood by the person skilled in the art in the present context, an M17 concentrate can be supplied from different suppliers and irrespective of the specific supplier, one will (within the standard measurement uncertainty) obtain the same relevant 2-deoxyglucose resistance result for a cell of interest here relevant.
[000185] The medium used for the culture of Lactobacillus delbrueckii subsp. bulgaricus was the MRS-IM medium. MRS-IM was used in the form of agar plates or broth.
[000186] MRS-M agar medium had the following composition per liter of H2O: Tryptone Oxoid L 42 10.0gOxoid Yeast Extract L 21 5.0gTween 80 Merck No. 8.22187 1.0gK2HPO4 Merck No. 105104 2, 6 g Merck Na Acetate No. 106267 5.0 g Merck Diammonium Hydrogen Citrate No. 101154 2.0 gMgSO4, 7 H2O Merck No. 105882 0.2 gMnSO4, H2O Merck No. 105941 0.05 agar SO-BI-GEL 13, 0 g
[000187] The pH was adjusted after autoclaving to 6.9 ± 0.1 at 25°C.
[000188] The MRS-IM broth used in the examples below for liquid cultures had the following composition per liter of H2O: Tryptone Oxoid L 42 10.0gOxoid Yeast Extract L 21 5.0 gTween 80 Merck n° 8.22187 1.0 gK2HPO4 Merck n. 105104 2.6 g Merck Na Acetate No. 106267 5.0 g Merck Diammonium Hydrogen Citrate No. 101154 2.0 gMgSO4, 7 H2O Merck No. 105882 0.2 gMnSO4, Merck H2O No. 105941 0.05 g
[000189] The pH is adjusted after autoclaving to 6.9 ± 0.1 at 25°C. The carbon sources, lactose 20g/l or glucose 20g/l, were first filtered, sterilized and then added to the autoclaved broth.
[000190] The above MRS-IM medium can vary to some extent without affecting the medium's ability to support the growth of Lactobacillus delbrueckii subsp. bulgaricus Furthermore, as will be understood by the person skilled in the art, a concentrate of MRS-IM or the various components described above can be obtained from different suppliers and used for the preparation of an MRS-IM medium. These means will likewise be used in the examples below, in particular, in the 2-deoxyglucose resistance selection assay. mother strains
[000191] Streptococcus thermophilus CHCC11976 (galactose fermenting strain with a Galk gene mutation and exopolysaccharide production as described in WO 2011/026863).Streptococcus thermophilus CHCC14994 (galactose fermenting strain).Lactobacillus delbrueckii subsp. bulgaricus CHCC759.Lactobacillus delbrueckii subsp. bulgaricus CHCC10019.2-deoxyglucose resistant strainsStreptococcus thermophilus CHCC15757 (2-deoxyglucose resistant CHCC14994).Streptococcus thermophilus CHCC15887 (2-deoxyglucose resistant mutant CHCC11976). subsp. bulgaricus CHCC16159 (2-deoxyglucose resistant mutant of CHCC759).Lactobacillus delbrueckii subsp. bulgaricus CHCC16160 (2-deoxyglucose resistant mutant of CHCC10019).EXAMPLE 1: Use of 2-deoxyglucose to isolate Streptococcus thermophilus glucose kinase mutants with increased glucose excretion.
[000192] In order to isolate mutants of Streptococcus thermophilus strain CHCC11976 and Streptococcus thermophilus strain CHCC14994, cells derived from the growth of a single colony were inoculated into 10 mL of M17 broth containing 2% lactose and grown overnight at 40°C.
[000193] The following day, the strains were plated in serial dilutions on M17 agar plates containing 2% galactose and a concentration of 2-deoxyglucose either 20 mM (CHCC14994) or 30 mM (CHCC11976) and incubated for 20 hours at 40°C. Resistant colonies were dried on the same type of agar plates when they were selected. Survivors were used to inoculate fresh M17 broth containing 2% lactose, 2% galactose or 2% glucose and growth was measured.
[000194] From this, a number of mutants that were able to grow faster on galactose than on glucose were identified, as outlined in Example 2. Two such mutants were CHCC15757 and CHCC15887, which are derived from CHCC14994 and CHCC11976 , respectively.EXAMPLE 2: Growth pattern of 2-deoxyglucose resistant mutant
[000195] To ensure the selection of 2-deoxyglucose resistant mutants that can grow on galactose, two strains were used that were selected from a collection of galactose fermenting strains. Although these galactose fermenting strains still grow at least 10% faster in the exponential phase in M17 broth + 2% glucose than in M17 broth + 2% galactose, derivatives of 2-deoxyglucose resistant mutants of CHCC11976 and CHCC14994, such as CHCC15757 and CHCC15887, on the other hand, are characterized by growing faster in the exponential phase in ML7 broth + 2% galactose than in M17 broth + 2% glucose.
[000196] Exponential phase growth is measured here as the development in optical density of the exponential growth culture at 600 nanometers (OD600) with time at 40°C.
[000197] As is known to the person skilled in the art, it may vary from species to species when the culture is exponentially growing. The skilled person will know how to determine exponential phase growth, for example, between OD600 0.1 to 1.0.
[000198] The optical density (OD) of the culture is measured in a spectrophotometer. Conclusion:
[000199] Based on the growth pattern of the 2-deoxyglucose resistant mutant of this Example 2, for a specific strain of interest (e.g. one from a relevant commercial product) - the skilled person can routinely test whether this specific strain of interest has the growth pattern relevant here, which is a property of the selected mutants.Example 3: Assay for mutation analysis of the gene encoding glucose kinase.
[000200] Total DNA was isolated from the mutants identified in Example 1 to perform the assay for mutation analysis of the gene encoding glucose kinase. Glucose kinase gene sequencing revealed that the gene in CHCC15757 contains a non-conserved mutation at codon 141 generating an isoleucine rather than a threonine codon. Sequencing of the CHCC15887 mutant gene revealed a mutation at codon 72, which results in a non-conserved amino acid change from serine to proline (Figure 2)
[000201] The 2-deoxyglucose resistant strains meet the conditions specified in Examples 1 and 2 and isolated, as described in Example 1, are characterized by having a mutation in the gene encoding glucose kinase (glcK). The mutation may result from an amino acid change of the encoded enzyme, or result in the generation of a stop codon, which will truncate the encoded enzyme.
[000202] To reveal the mutation in the glcK gene, the specific strain of interest is cultured in liquid broth (M17), to which 2% lactose is added at 40°C overnight. After isolation of chromosomal DNA, the DNA was subjected to PCR analysis using two primers complementary to a conserved region just and precisely downstream of the gene encoding glucose kinase. The primer sequences are as follows: GK1F: 5' CTT GGG TAA AAG GCT CTA TG 3' (SEQ ID NO: 3.)GK1R: 5' CGT TTT TCA ACA AAA AAG TGC TACC 3' (SEQ ID NO: 4. )
[000203] The conditions for the PCR reactions were as specified by the manufacturer of the PCR amplification kit (Roche), eg 2 μl chromosomal DNA1 μl Primer GK1F1 μl Primer GK1R25 μl Mix Master21 μl H2O
[000204] Program-PCR: (94°C-1.5 min, 50°C-1 min, 72°C 1.5 min) X 30
[000205] PCR amplification generates a 1168 bp fragment. After purification with a Biorad PCR purification kit, the PCR fragment was subjected to DNA sequencing in Macrogen using the same two primers that were used for amplification. After sequencing, the DNA sequence was compared to that of the mother strain. Example 4. Carbohydrate analysis of fermented milk
[000206] In another experiment, the sugar concentrations of the relevant sugar were determined in milk fermented with CHCC14994, CHCC11976, CHCC15757 and CHCC15887, respectively. 9.5% B-milk was inoculated with 1% (10 6 to 10 7 CFU/ml) of a culture grown overnight in M17 broth, to which 2% galactose is added. Acidification was followed with an INTAB PC logger and EasyView software. After 30 hours of acidification at 40°C, the milk samples were taken for HPLC analysis to obtain the relevant sugar and acid content. The acidification curves showed that the mutants had a slightly delayed initiation of acidification, but ended at a similar pH. HPLC data are shown in Table 1.
[000207] From Table 1, it is evident that the two glcK mutant strains, CHCC15757 and CHCC15887 consume at least 71% lactose, while the mother strains consume about 28% lactose. For most lactose fermenting mutants, CHCC15887, as little as 11.9 mg/mL remains in the fermented milk, indicating a role for this product even for people with lactose intolerance. Very significantly, the two glcK mutants excreted between 8.3 and 11.3 mg/mL of glucose, while the glucose secretion of the mother strains is below the detection level. At the same time, both mutant strains also secrete more galactose than the mother strains: between 34 and 52%. Assuming that sucrose is a reference of 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74.3, a calculation of the relative sweetness of the final fermented product suggests a sweetness. 2.0 times sweeter when fermented with the best mutant CHCC15757 than the corresponding mother strain CHCC14994.Table 1: HPLC data from milk samples.
Calculated sweetness: mg/mL glucose * 74 + mg/mL lactose * 16 + mg/mL galactose * 32.
[000208] As the two glcK mutant strains, CHCC15757 and CHCC15887, excrete high levels of glucose, it is contemplated that mutations in the glcK gene inactivate the encoded glucokinase protein.Example 5: Selection of strains of Lactobacillus delbrueckii subsp. bulgaricus resistant to 2-deoxyglucose.Selection of mutants resistant to 2-deoxyglucose
[000209] Two strains of Lactobacillus delbrueckii subsp. bulgaricus of interest, CHCC759 and CHCC10019, were independently inoculated into 10 ml of the above-described MRS-IM broth containing 2% lactose and incubated anaerobically at 40°C overnight. In the next step, samples from these cultures containing about 3 X 10 8 cells were plated on MRS-IM agar plates containing 2% lactose and still containing 20 mM 2-deoxyglucose. Colonies on the resulting plates were purified by streaking single colonies on MRS-IM agar plates containing 2% lactose and in addition containing 20 mM 2-deoxyglucose and further characterized as described below. -deoxyglucose:
[000210] The following method is suitable for determining whether or not a strain of interest is resistant to 2-deoxyglucose. Strains of interest are inoculated into 10 ml of the above-described MRS-IM broth containing 2% lactose and anaerobically incubated at 40°C overnight. In the next step, diluted samples of these cultures containing about 104 to 105 cells are seeded on MRS-IM agar plates containing 2% lactose and, in addition, with the same concentration of 2-deoxyglucose that was used for the selection of the mutant. resistant (typically 20 mM but other concentrations can be used). The agar plates are incubated under anaerobic conditions for 20 hours at 40°C and inspected. The strains of Lactobacillus delbrueckii subsp. bulgaricus that are not resistant to 2-deoxyglucose will produce few, if any colonies, whereas strains that are resistant to 2-deoxyglucose will produce large numbers of colonies. Appropriate controls include strains of Lactobacillus delbrueckii subsp. bulgaricus, CHCC759 and CHCC10019, which are sensitive to 2-deoxyglucose at a concentration of 20 mM and CHCC16159 and CHCC16160, which are resistant to 20 mM of 2-deoxyglucose.
[000211] Result: Several clones that were able to grow under selective conditions in the presence of 2-deoxyglucose were isolated by this approach. Mutant strains that showed rapid growth on 2-deoxyglucose plates were designated CHCC16159 (derived from mother strain CHCC759) and CHCC16160 (derived from mother strain CHCC10019). These mutant strains were deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH.EXAMPLE 6: Growth pattern of 2-deoxyglucose resistant mutants of Lactobacillus delbrueckii subsp. bulgaricus
[000212] To ensure that 2-deoxyglucose resistant mutants of Lactobacillus delbrueckii subsp. bulgaricus lost either the ability to grow on glucose or have an impaired ability to grow on glucose as a carbon source, the growth pattern of the mutants was compared to the parent strains by growth of both the parent strains, CHCC759 and CHCC10019, and the mutants. , CHCC16159 and CHCC16160, in MRS-IM broth containing 2% glucose.
[000213] While the two mother strains, CHCC759 and CHCC10019, grew exponentially in MRS-IM broth added 2% glucose with a doubling time of less than 10 hours, the two 2-deoxyglucose resistant mutants, CHCC16159 and CHCC16160, did not grow or grew very slowly on this medium. Exponential phase growth was monitored by measuring the optical density of the exponential growth culture at 600 nanometers (OD600) at 40°C in a spectrophotometer. The exponential growth phase was reached between OD 600 0.1 to 1.0.EXAMPLE 7: Analysis of carbohydrate in milk fermented with strains of Lactobacillus delbrueckii subsp. bulgaricus
[000214] In another experiment, sugar concentrations of different sugars were determined in milk fermented with strains of Lactobacillus delbrueckii subsp. bulgaricus CHCC759, CHCC10019, CHCC16159, and CHCC16160, respectively. 9.5% B-milk was inoculated with 1% (10 6 to 10 7 CFU/ml) of a liquid culture grown overnight anaerobically in MRS-IM broth containing 2% lactose. Acidification was followed with an INTAB PC logger and EasyView software. After 30 hours of acidification at 40°C, milk samples were taken for HPLC analysis to measure amounts of various sugars and organic acids. The acidification curves showed that the mutants had a slightly delayed initiation of acidification and ended at a slightly higher pH. HPLC data are shown in Table 2.
[000215] As can be seen from Table 2, the two 2-deoxyglucose resistant mutant strains, CHCC16159 CHCC16160, consumed at least about 94% lactose, while the mother strains consumed at least about 37 % of lactose. For the mutant showing the highest lactic acid production, CHCC16160, as little as 2.9 m/mL lactose remained in the fermented milk. Fermented milk products with such low levels of lactose may be suitable for consumption by people with lactose intolerance.
[000216] Significantly, the two mutant strains, CHCC16159 and CHCC16160, excreted between 15.2 and 15.8 of glucose, while the glucose secretion of the mother strains is below the detection level for CHCC10019 and 4.2 mg/mL for CHCC759, respectively. At the same time, both mutant strains also secreted more galactose than the mother strains. If the reference value for the sweetness of sucrose is 100, and the sweetness of lactose is 16, the sweetness of galactose is 32, and the sweetness of glucose is 74, a calculation of the relative sweetness of the final fermented product suggests that the mutant CHCC16160 produces a fermented milk product that is 2.5 times sweeter than the corresponding parent strain CHCC10019.Table 2. HPLC data from milk samples.
Calculated sweetness: mg/mL glucose * 74 + mg/mL lactose * 16 + mg/mL galactose * 32.EXAMPLE 8: Selection of a glucose-secreting mutant and a hyperlactose fermenter from Streptococcus thermophilus
[000217] In order to isolate a glucose-secreting mutant and a hyperlactose fermenter from the Streptococcus thermophilus strain CHCC15757, cells derived from the growth of a single colony were inoculated into 10 mL of M17 broth containing 2% galactose and grown overnight at 40°C.
[000218] The next day, the strain was seeded in serial dilutions on M17 agar plates containing 2% galactose and a concentration of 2-deoxyglucose of 30 mM and incubated for 20 hours at 40°C. Resistant colonies were reseeded onto the same type of agar plates that were selected. Survivors were used to inoculate fresh M17 broth containing 2% lactose, 2% galactose, 2% sucrose or 2% glucose.
[000219] From this, we were able to isolate a CHCC16404 mutant, derived from CHCC15757, which was unable to grow on B-milk, but able to grow on M17 added to 2% sucrose at 40°C. Furthermore, CHCC16404 was unable to grow on M17 added to 2% glucose.
[000220] The exponential phase growth is measured here while the growth in optical density of the exponentially growing culture at 600 nanometers (OD600), with time at 40°C.EXAMPLE 9: Growth pattern of the secretory mutant Streptococcus thermophilus CHCC16404 of glucose and hyperlactose fermenter:
[000221] To ensure the maintenance and adequate growth of the mutant CHCC16404, the strain was grown at 40°C in M17 added with 2% sucrose. To our surprise, we observed that the mutant strain CHCC16404 was able to acidify 9.5% of B-milk only when the strain was inoculated with 1% (106 to 107 CFU/mL) of a culture grown overnight in M17 broth added 2% sucrose or when sucrose was added to milk. We observed that addition of only 0.01% of sucrose to milk enabled CHCC16404 to acidify 9.5% of B-milk. Furthermore, CHCCl6404 was unable to grow on M17 plus 2% glucose at 40°C, as opposed to the mother strain CHCC15757, which could grow on M17 plus glucose under the same conditions. Taken together, these results indicate that the 2-deoxyglucose treatment of CHCC15757, which generated CHCC16404, selected a mutation that inactivates the glucose uptake system, disabling the uptake of secreted glucose from the medium.Example 10: Analysis of milk carbohydrate fermented with Streptococcus thermophilus mutant CHCC16404 glucose secretor and hyperlactose fermenter
[000222] In another experiment, sugar concentrations of relevant sugars were determined in milk fermented with CHCC16404. Vials containing 9.5% B-milk with 01%, 0.02%, 0.03% and 0.05% sucrose, respectively, were inoculated with 1% (106 to 107 of CFU/mL) of a grown overnight in M17 broth, to which 2% sucrose is added. Acidification was followed with an INTAB PC logger and EasyView software. After 30 hours of acidification at 40°C, milk samples were taken for HPLC analysis to obtain the relevant sugar and acid content.
[000223] From Table 3, it is evident that the glucose-secreting, hyperlactose-fermenting mutant CHCC16404 surprisingly consumes all of the lactose at all concentrations of added sucrose tested in this experiment. It was also observed that when a higher concentration of sucrose was added (eg >0.1 mg/mL), lactose fermentation is not completed before the final pH is reached. Furthermore, Table 3 also shows that all of the lactose is converted to glucose and galactose, and that only a part of the galactose is used for fermentation at all added sucrose concentrations. Interestingly, the secreted glucose is not taken up, thus leaving more than 23.9 mg/mL of glucose in the milk. Since about 25% of galactose is fermented, more than 16 mg/mL of galactose remains in the milk after fermentation. These data indicate that CHCC16404, in addition to the glcK mutation inherited from the mother strain CHCC15757, also harbors a mutation that inactivates glucose uptake, disabling the uptake of glucose secreted from the medium. Comparing the data in Table 3 with those in Table 1 and taking into account that sucrose is a reference of 100, and the sweetness of lactose is 16, the sweetness of galactose is 32 and the sweetness of glucose is 74.3, a calculation of sweetness relative to the final fermented product yields about 3.5 times sweeter sweetness when fermented with CHCC16404 with strain CHCC14994.Table 3: HPLC data from milk samples.
Calculated sweetness: mg/mL glucose * 74 + mg/mL lactose * 16 + mg/mL galactose * 32.Example 11: Carbohydrate analysis of milk fermented with a combination of Streptococcus thermophilus strains and Lactobacillus delbrueckii subsp strains . bulgaricus
[000224] In this experiment, the concentrations of sugars and organic acids were determined in milk fermented with combinations of a strain of Lactobacillus delbrueckii subsp. bulgaricus (selected from CHCC759, CHCC10019, CHCC16159 and CHCC16160) and a strain of Streptococcus thermophilus, which was CHCC14994, CHCC15757 or CHCC16404.
[000225] Yogurt production typically involves the use of a mixed starter culture that contained both Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. Typically, the milk substrate used in the production of yogurt is inoculated with 1 part of Lactobacillus delbrueckii subsp. bulgaricus and 9 parts of Streptococcus thermophilus. In order to analyze the secretory uptake of glucose in a standard configuration for yogurt production, 9.5% of B-milk was inoculated with 0.1% of a culture of Lactobacillus delbrueckii subsp. bulgaricus grown overnight anaerobically in MRS-IM broth containing 2% lactose and 0.9% of a Streptococcus thermophilus culture grown overnight in M17 broth containing 2% galactose (CHCC15757) or 2% sucrose (CHCC16404) . Acidification was followed with an INTAB PC logger and EasyView software. After 30 hours of acidification, milk samples were taken for HPLC analysis to measure the amount of different sugars and acids. The HPLC results are shown in Table 4.
[000226] It can be taken from Table 4 that the use of the 2-deoxyglucose resistant CHCC15757 and CHCC16404 mutant strain of Streptococcus thermophilus results in the secretion of glucose into milk, regardless of whether it is combined with a mother strain of Lactobacillus delbrueckii subsp. bulgaricus or with a 2-deoxyglucose resistant mutant thereof. However, glucose concentrations are higher when a combination of 2-deoxyglucose resistant mutant strains of both species are used, for example, a combination of CHCC15757 and CHCC16159, or a combination of CHCC15757 and CHCC16160. When using these mixed cultures, at least 82% of the lactose was found to be consumed, and between 11.8 mg/mL and 14.1 mg/mL of glucose was found to be excreted. Similar results are obtained when the Streptococcus thermophilus strain is CHCC15887, which is also a 2-deoxyglucose resistant mutant, with a mutant in the glcK gene.
[000227] At the same time, the presence of a 2-deoxyglucose resistant mutant strain in the starter culture also resulted in the excretion of more galactose. When a combination of mutant strains, e.g. CHCC15757 and CHCC16159 is used, galactose excretion is about 3 times higher (17.1 mg/mL) compared to a starter culture comprising the corresponding mother strains CHCC14994 and CHCC10019 .
[000228] Glucose secretion is even more efficient when a combination of the glucose-secreting Streptococcus thermophilus hyperlactose fermenter mutant and CHCC16404 secretory mutant and the 2-deoxyglucose resistant mutant strains of Lactobacillus delbrueckii subsp. bulgaricus are used, for example, a combination of CHCC16404 and CHCC16159, or a combination of CHCC16404 and CHCC16160.
[000229] Taking into account that the sweetness of sucrose is 100 (reference value), and the sweetness of lactose is 16, the sweetness of galactose is 32 and the sweetness of glucose is 74, a calculation of the relative sweetness of the product fermented final, shown in the last row of the table, suggests that the combination of different strains allows the definition of the final concentration of residual lactose, glucose and galactose in the final fermented product. If a yogurt with maximum internal sweetness due to high concentrations of secreted glucose and galactose and no residual lactose is desired, then the most efficient combination of strains is CHCC16404 and CHCC16160. This combination provided a yogurt that is 3.6 times sweeter than the corresponding combination of 2-DG sensitive strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, CHCC14994 and CHCC10019 and has no detectable lactose left in the fermented milk.Table 4: HPLC data from fermented milk samples with mixed cultures
* addition of 0.05% sucrose Calculated sweetness: mg/mL of glucose * 74 + mg/mL of lactose * 16 + mg/mL of galactose * 32.Example 12. Improvement of the growth of Bifidobacteria by the use of secreting mutants of glucose from Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus.
[000230] Some of the best known probiotic bacteria like Bifidobacterium, animalis subsp. lactis BB-12® (commercially available from Chr. Hansen A/S, Hoersholm, Denmark) did not grow well when present alone in lactose based media such as milk. In this experiment, we have therefore investigated the effect of combining Bifidobacterium animalis subsp. lactls BB-12® with a glucose-secreting strain of Streptococcus thermophilus or Lactobacillus delbrueckii subsp. Bulgaricus.Experiment 1:
[000231] CHCC 5445 (BB-12®) was grown under anaerobic conditions overnight at 40°C in MRS + 0.05% cysteine chloride. CHCC14994 was grown overnight at 40°C in M17 with 2% galactose.CHCC 15757 was grown overnight at 40°C in M17 with 2% galactose. Experiment 2:
[000232] CHCC 5445 (BB-12®) was grown anaerobically overnight at 40°C in MRS + 0.05% cysteine chloride. CHCC10019 was grown anaerobically overnight at 40°C in MRS with 2% lactose. CHCC15159 was grown anaerobically at 40°C in MRS with 2% lactose.
[000233] Six flasks with 200 ml of milk-B were inoculated at 40°C overnight, with a total of 1% of the cultures grown having similar optical densities: 1. 1% CHCC 5445 (BB-12®).2 . 0.5% CHCC 5445 (BB-12®) + 0.5% CHCC149943. 0.5% CHCC 5445 (BB-12®) + 0.5% CHCC157574. 1% CHCC5445 (BB-12®),5. 0.5% CHCC 5445 (BB-12®) + 0.5% CHCC100196. 0.5% CHCC 5445 (BB-12®) + 0.5% CHCC15159
[000234] Only bottles 2 to 3 and 5 to 6 acidified due to poor performance of BB-12 in milk. After fermentation, 100 μl of each culture was plated at different dilutions (10-5, 10-6, 10-7, 10-8) on agar plates selected for the growth of BB-12® specifically, that is, MRS + 0.05% cysteine chloride + tetracycline added 10 μg/mL of tetracycline and then incubated anaerobically overnight at 40°C. The number of colonies was subsequently determined by counting and an average of the results is given in Table 5 as CFU/mL (colony forming units per milliliter).Table 5. CFU/mL of BB-12 in fermented cultures

[000235] Only when the glucose-secreting mutants of Streptococcus thermophilus, CHCC15757 and Lactobacillus delbrueckii subsp. bulgaricus, CHCC16159 are present along with BB-12®, the growth of BB-12® is boosted and the total cell count presented as CFU/mL is approximately 10x (1 log) higher in these cultures. The result suggests that glucose-secreting strains can be used to enhance the content of BB-12® probiotic strains when mixed together in cultures.EXAMPLE 13. Comparison of the genome of CHCC15757 and CHCC16404 and identification of a mutation in the gene manM encoding the IICMan protein of the glucose/mannose phosphotransferase (PTS) system.
[000236] Genomic DNA preparations of CHCC15757 and CHCC16404 were sequenced at Beijing Genomics Institute (BGI, Beijing, China) and assembled and finished using CLC genomic bench software (CLCBio, Aarhus, Denmark). The genome sequences of CHCC15757 and CHCC16404 were aligned using the annotated genome sequence of CHRZ1066 as a reference using Mauve 2.3.1 software. After alignment, analysis of a single nucleotide polymorphism (SNP) was performed using the free software Mauve 2.3.1 on both CHCC15757 and CHCC16404. This allowed the identification of a G to T mutation in the codon GAA (glutamic acid) at amino acid position 209 of the manM gene that encodes the IICMan protein of the PTS glucose/mannose PTS. This change introduced a TAA stop codon at position 209 of the protein (Figure 3) resulting in the production of a truncated and therefore non-functional IICMan protein. Therefore, it is considered that this mutation results in a prevention for the transport of glucose into the cell via the PTS glucose/mannose. Deposits and Specialized Solutions
[000237] applicant applicant requests that a sample of the deposited microorganisms set forth below may only be made available to a specialist, until the date the patent is granted.
[000238] Streptococcus thermophilus strain CHCC15757 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH1 Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on April 3, 2013 under No. access DSM 25850
[000239] Streptococcus thermophilus strain CHCC15887 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on April 3, 2012 under No. Access DSM 25851.
[000240] Streptococcus thermophilus strain CHCC16404 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on December 12, 2012, under Accession No. DSM 26722.
[000241] Streptococcus thermophilus strain CHCC14994 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on April 3, 2012 under No. Access DSM 25838.
[000242] Streptococcus thermophilus strain CHCC11976 was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on September 8, 2009 under No. Access DSM 22934.
[000243] The strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC759 has already been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany, on September 6, 2012 under No. Access DSM 26419.
[000244] The strain of Lactobacillus delbrueckii subsp. bulgaricus CHCC759 has already been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany, on April 3, 2007, under No. Access DSM 19252.
[000245] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC759 has already been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany, on September 6, 2012 under No. Access DSM 26420.
[000246] The strain Lactobacillus delbrueckii subsp. bulgaricus CHCC759 has already been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124Braunschweig, Germany, on September 6, 2012 under No. Access DSM 26421.
[000247] The strain of Bifidobacterium animalis subsp. lactls CHCC5445 (BB-12®) was deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Inhoffenstr. 7B, D-38124 Braunschweig, Germany, on September 30, 2003, under No. Access DSM 15954.
[000248] The deposits were made in accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of the patent procedure.
WO 2011/026863 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.

权利要求:
Claims (14)
[0001]
1. Streptococcus thermophilus strain, characterized in that the strain is selected from the group consisting of the Streptococcus thermophilus strain CHCC15757 which was deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 25850, the Streptococcus thermophilus strain CHCC15887 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. accession DSM 25851, the Streptococcus thermophilus strain CHCC16404 which has been deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen under No. Access DSM 26722.
[0002]
2. Composition, characterized in that it comprises from 104 to 1012 CFU/g of a Streptococcus thermophilus strain as defined in claim 1.
[0003]
3. Composition according to claim 2, characterized in that it further comprises from 104 to 1012 CFU/g of at least one mutant strain of Lactobacillus delbrueckii subsp. Bulgaricus, selected from DSM 26420 and DSM 26421, in which said strain is resistant to 2-deoxyglucose so that it is able to grow in a colony plated in MRS-IM medium containing 2% (w/v) of lactose and 20 mM 2-deoxygicose after incubation at 40°C for 20 hours.
[0004]
4. Method for producing a fermented milk product, characterized in that it comprises the inoculation and fermentation of a milk substrate with at least one strain of Streptococcus thermophilus, as defined in claim 1.
[0005]
5. Method for producing a fermented milk product, characterized in that it comprises inoculation and fermentation of a milk substrate with the composition as defined in claim 2 or 3.
[0006]
6. Fermented milk product, characterized in that it comprises at least one strain of Streptococcus thermophilus, as defined in claim 1.
[0007]
7. Fermented milk product, characterized in that it comprises the composition as defined in claim 2 or 3.
[0008]
8. Fermented milk product according to claim 6 or 7, characterized in that it is for use in preventing the symptoms of lactose intolerance.
[0009]
9. Use of a composition as defined in claim 2 or 3, characterized in that it is in the preparation of a fermented milk product.
[0010]
10. Use of a composition as defined in claim 2 or 3, characterized in that it is to increase the sweetness of a fermented milk product.
[0011]
11. Use of a composition as defined in claim 2 or 3, characterized in that it is to decrease the lactose content in a fermented milk product.
[0012]
12. Use of a strain of Streptococcus thermophilus, as defined in claim 1, characterized in that it is to enhance the growth of a strain of Bifidobacterium in milk containing lactose.
[0013]
13. Use of a composition as defined in claim 2 or 3, characterized in that it is for enhancing the growth of a Bifidobacterium strain in milk.
[0014]
14. Method for the screening and isolation of a Streptococcus thermophilus strain with mutated glcK gene, characterized in that it comprises the following steps: a) providing a galactose-fermenting Streptococcus thermophilus mother strain; b) selecting and isolating, from from a set of Streptococcus thermophilus strains derived from the mother strains, a set of Streptococcus thermophilus strains that are resistant to 2-deoxyglucose so that it is able to grow into a colony when plated on M17 medium containing 2% (w/w) v) lactose or 2% (w/v) galactose, and 20 mM 2-deoxyglucose after incubation at 40°C for 20 hours; c) select and isolate from a set of Streptococcus thermophilus strains that are resistant to 2-deoxyglucose a strain of Streptococcus thermophilus, if the growth rate of the Streptococcus thermophilus strain is higher on M17 + 2% galactose medium than on M17 + 2% glucose medium.

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法律状态:
2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-09-03| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-06| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 25/04/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP12165517.9|2012-04-25|

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EP12165517|2012-04-25|

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EP12198766|2012-12-20|

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EP12198766.3|2012-12-20|

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PCT/EP2013/058655|WO2013160413A1|2012-04-25|2013-04-25|Use of lactic acid bacteria for preparing fermented food products with increased natural sweetness|

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