Protein having a property of improving a stringiness of fermented milk, and fermented milk using the same and a method for manufacturing the same

Abstract

at least one protein selected from the group consisting of (a) to (d) below: (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (b) an amino acid sequence in which one or a plurality of amino acids, except for the tyrosine at position 40, in the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted, inserted and / or added, and which has a fermented milk stretchability-improving effect; (c) a protein consisting of an amino acid sequence having 80% or greater identity with the amino acid sequence represented by SEQ ID NO: 1, the amino acid corresponding to position 40 of the amino acid sequence represented by SEQ ID NO: 1 being tyrosine, and which has a fermented milk stretchability-improving effect; and (d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with the complementary strand of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2, the amino acid corresponding to position 40 of the amino acid sequence represented by SEQ ID NO: 1 being tyrosine, and which has a fermented milk stretchability-improving effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a protein having a fermented milk stringiness-improving effect, and a fermented milk using the same and a manufacturing method thereof, and more particularly, to a protein, DNA, vector, lactic acid bacteria and lactic acid bacteria composition having a fermented milk stringiness-improving effect, and a fermented milk, a fermented milk thickener using the same and a manufacturing method thereof, a method of improving fermented milk stringiness, and a method of evaluating lactic acid bacteria. BACKGROUND

[0002] Fermented milk is a food that is widely consumed, and is defined in "Regulations on Standards for Ingredients, etc. of Milk and Milk Products (Milk Etc. Regulations)" in Japan as "milk etc. containing milk or non-fat milk solid ingredients equal to or more than milk, which is fermented with lactic acid bacteria or yeast to become paste or liquid, or which is frozen". As representative examples of the fermented milk, for example, yogurt such as solid yogurt (solid fermented milk), soft yogurt (paste fermented milk), and beverage yogurt (liquid fermented milk) can be given. In recent years, with diversification of consumer preferences, there is a demand for diversification of fermented milk, and in particular, there is an increasing demand for thick fermented milk with high viscosity and high stringiness.

[0003] In the production of fermented milk such as yogurt, it is mainstream to inoculate lactic acid bacteria in raw milk and ferment it, and as the lactic acid bacteria, for example, Lactobacillus or Streptococcus thermophilus can be used. As a fermented milk stringiness-related component, exopolysaccharide (EPS) produced by lactic acid bacteria is known. For example, in Japanese Patent Application Publication No. 2016-178911 (Patent Literature 1), a method of producing a soy milk ferment characterized by fermenting soy milk using lactic acid bacteria that produces stringiness exopolysaccharide is described, and as the lactic acid bacteria, Lactococcus lactis subsp. cremoris FC (FERM P-20185) can be given. In addition, in Japanese Patent Application Publication No. 2018-143220 (Patent Literature 2), a fermented milk containing a lactic acid bacteria-derived sticky polysaccharide and having a median particle diameter of 1 μm or more and 30 μm or less is described, and as the lactic acid bacteria, Streptococcus thermophilus SBT0087 or the like can be given.

[0004] In addition, Japanese Patent Application Publication No. 2007-236227 (Patent Literature 3) describes a composition for preventing liver dysfunction, which contains lactic acid bacteria or a culture thereof as an effective ingredient, and inhibits water separation or whey separation by using Lactobacillus helveticus SR-1 (FERM P-20600) or the like as the lactic acid bacteria. In addition, Japanese Patent Application Publication No. 7-255465 (Patent Literature 4) describes Bifidobacterium longum SBT10013 as a Bifidobacterium strain characterized by producing a large amount of polysaccharide outside the bacterial cell. In addition, some studies have been made on proteins related to the stringiness of fermented milk or nucleotide sequences encoding the same. However, information on the proteins related to the stringiness is not sufficient, and there is a demand for providing a novel protein having an effect of improving the stringiness of fermented milk.

[0005] Prior Art Documents

[0006] Patent Literature

[0007] Patent Literature 1: Japanese Patent Application Publication No. 2016-178911

[0008] Patent Literature 2: Japanese Patent Application Publication No. 2018-143220

[0009] Patent Literature 3: Japanese Patent Application Publication No. 2007-236227

[0010] Patent Literature 4: Japanese Patent Application Publication No. 7-255465 SUMMARY

[0011] Problems to be Solved by the Invention

[0012] The present application has been made in view of the problems of the related art, and has an object to provide a novel protein having an effect of improving the stringiness of fermented milk, and fermented milk excellent in stringiness and a method for producing the same.

[0013] Solution to Problem

[0014] The present inventors have made intensive studies to achieve the above object and have clarified a protein that enhances the stringiness of fermented milk and a gene encoding the same. That is, it has been known that fermented milk fermented with Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (deposited under Accession No. FERM BP-10741) (hereinafter sometimes referred to as "R-1 strain") has a tendency to have higher stringiness than fermented milk fermented with other strains of lactic acid bacteria, but it has not been clear which gene or protein of the R-1 strain enhances the stringiness of fermented milk. Therefore, the present inventors have first confirmed that fermented milk fermented with the R-1 strain indeed has higher stringiness than fermented milk fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 (hereinafter sometimes referred to as "2038 strain") in order to clarify a novel protein having a fermented milk stringiness-enhancing effect.

[0015] Next, as a stringiness-related component, it has been known that exopolysaccharide (EPS) produced by lactic acid bacteria, and therefore the sequences of an EPS gene cluster considered to be involved in the biosynthesis of EPS were compared between the two. As a result, it was found that differences in the nucleotide sequences of epsC gene and epsF gene were observed between the R-1 strain and the 2038 strain. Furthermore, transformants into which the epsC gene or the epsF gene of the R-1 strain was introduced in the 2038 strain were each prepared, and as a result, the transformant into which only the epsC gene of the R-1 strain was introduced had improved stringiness, and thus it was found that the protein encoded by the epsC gene of the R-1 strain has a fermented milk stringiness-enhancing effect.

[0016] In addition, on February 5, 2020, the nucleotide sequence of epsC of the R-1 strain was searched, web Blast (parameters: default values) was performed on the nt database of NCBI, and as a result, the top hit sequence was the epsC gene of the 2038 strain, the query coverage was 100%, and the Per. Ident was 99.87%. Furthermore, the difference in the bases was also reflected in the difference in the amino acids. Therefore, the present inventors have found that the nucleotide sequence of the epsC gene of the R-1 strain and the protein as a product thereof are novel, and thus the present application has been completed.

[0017] That is, the present application relates to a protein having a fermented milk stringiness-enhancing effect and fermented milk using the same, a method for producing the same, and the like, and more specifically as shown below. [1]

[0019] at least one protein selected from the group consisting of proteins of (a) to (d) below.

[0020] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;

[0021] (b) an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence shown in SEQ ID NO: 1 except for the tyrosine at position 40 are substituted, deleted, inserted and / or added, and which has a protein having a fermentation milk stringiness-improving effect;

[0022] (c) an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and which has a protein having a fermentation milk stringiness-improving effect;

[0023] (d) an amino acid sequence encoded by a DNA hybridizing under stringent conditions with the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and which has a protein having a fermentation milk stringiness-improving effect.

[0024] [1']

[0025] A composition containing at least one protein selected from the group consisting of the proteins of (a) to (d) above (preferably a composition for improving the fermentation milk stringiness). [2]

[0027] A DNA encoding the protein of [1].

[0028] [2']

[0029] A composition containing at least one DNA selected from the group consisting of the DNAs encoding any of the proteins of (a) to (d) above (preferably a composition for improving the fermentation milk stringiness). [3]

[0031] A vector containing the DNA of [2].

[0032] [3']

[0033] A vector containing at least one DNA selected from the group consisting of the DNAs encoding any of the proteins of (a) to (d) above. [4]

[0035] A composition containing at least one selected from the group consisting of the protein of [1], the DNA of [2], and the vector of [3]. [5]

[0037] A lactic acid bacterium into which at least one selected from the group consisting of the DNA of [2] and the vector of [3] has been introduced.

[0038] [5']

[0039] A lactic acid bacterium into which the vector described in [3'] is introduced (preferably a lactic acid bacterium having a fermented milk stringiness-improving effect). [6]

[0041] A lactic acid bacterium having the DNA described in [2]. [7]

[0043] The lactic acid bacterium described in [6] having a fermented milk stringiness-improving effect. [8]

[0045] A lactic acid bacterium composition containing the lactic acid bacterium described in any one of [5] to [7].

[0046] [8']

[0047] A lactic acid bacterium composition containing the lactic acid bacterium described in [5'] (preferably a lactic acid bacterium composition for improving fermented milk stringiness). [9]

[0049] The lactic acid bacterium composition described in [8] or [8'] is fermented milk.

[10]

[0051] The lactic acid bacterium composition described in [8], [8'] or [9] contains exopolysaccharide from the lactic acid bacterium described in any one of [5] to [7] or [5'].

[11]

[0053] A method for producing fermented milk, comprising the step of adding the lactic acid bacterium described in any one of [5] to [7] or [5'] or the lactic acid bacterium composition described in any one of [8] to

[10] or [8'] to a prepared milk liquid containing raw milk and allowing it to ferment.

[12]

[0055] A method for improving fermented milk stringiness, comprising the step of adding the lactic acid bacterium described in any one of [5] to [7] or [5'] or the lactic acid bacterium composition described in any one of [8] to

[10] or [8'] to a prepared milk liquid containing raw milk and allowing it to ferment.

[13]

[0057] A method for evaluating a lactic acid bacterium, which evaluates whether or not it has a fermented milk stringiness-improving effect, using at least one DNA selected from the group consisting of DNAs encoding any of the proteins described in (a) to (d) below as an index,

[0058] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;

[0059] (b) an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence shown in SEQ ID NO: 1 except for the tyrosine at position 40 are substituted, deleted, inserted and / or added, and which has a fermented milk stringiness-improving effect;

[0060] (c) an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and which has a fermented milk stringiness-improving effect;

[0061] (d) an amino acid sequence encoded by a DNA hybridizing under stringent conditions with the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and which has a fermented milk stringiness-improving effect.

[14]

[0063] A fermented milk containing a lactic acid bacterium having a fermented milk stringiness-improving effect as evaluated by the evaluation method for lactic acid bacteria described in

[13] .

[15]

[0065] A method for producing a lactic acid bacterium, comprising: an evaluation step of evaluating whether a lactic acid bacterium has a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacteria described in

[13] ; and a step of obtaining a lactic acid bacterium having a fermented milk stringiness-improving effect as evaluated in the evaluation step.

[16]

[0067] A method for producing a fermented milk, comprising: an evaluation step of evaluating whether a lactic acid bacterium has a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacteria described in

[13] ; and a fermentation step of adding a lactic acid bacterium having a fermented milk stringiness-improving effect as evaluated in the evaluation step to a prepared milk liquid containing raw milk and allowing it to ferment.

[17]

[0069] A method for improving the stringiness of a fermented milk, comprising: an evaluation step of evaluating whether a lactic acid bacterium has a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacteria described in

[13] ; and a fermentation step of adding a lactic acid bacterium having a fermented milk stringiness-improving effect as evaluated in the evaluation step to a prepared milk liquid containing raw milk and allowing it to ferment.

[18]

[0071] A fermented milk thickening agent containing an exocellular polysaccharide from a lactic acid bacterium described in any one of [5] to [7] or [5'] as an effective ingredient.

[19]

[0073] A method for producing an exocellular polysaccharide of a lactic acid bacterium, comprising the steps of: adding the lactic acid bacterium according to any one of [5] to [7] or [5'] or the lactic acid bacterium composition according to any one of [8] to

[10] or [8'] to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment; and collecting an exocellular polysaccharide contained in a fermentation product.

[20]

[0075] A method for producing an exocellular polysaccharide of a lactic acid bacterium, comprising: an evaluation step of evaluating whether or not a lactic acid bacterium has a fermentation milk stringiness improving effect by the evaluation method for lactic acid bacteria according to

[13] ; and a step of adding a lactic acid bacterium evaluated as having a fermentation milk stringiness improving effect in the evaluation step to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, and collecting an exocellular polysaccharide contained in a fermentation product.

[21]

[0077] A method for producing a fermentation milk thickener, comprising: a fermentation step of adding the lactic acid bacterium according to any one of [5] to [7] or [5'] or the lactic acid bacterium composition according to any one of [8] to

[10] or [8'] to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, to obtain a fermentation product containing an exocellular polysaccharide; and a step of obtaining a fermentation milk thickener containing the exocellular polysaccharide as an effective ingredient.

[22]

[0079] A method for producing a fermentation milk thickener, comprising: an evaluation step of evaluating whether or not a lactic acid bacterium has a fermentation milk stringiness improving effect by the evaluation method for lactic acid bacteria according to

[13] ; a fermentation step of adding a lactic acid bacterium evaluated as having a fermentation milk stringiness improving effect in the evaluation step to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, to obtain a fermentation product containing an exocellular polysaccharide; and a step of obtaining a fermentation milk thickener containing the exocellular polysaccharide as an effective ingredient.

[0080] Effects of the Invention

[0081] According to the present application, it is possible to provide a novel protein having a fermentation milk stringiness improving effect, and a fermentation milk excellent in stringiness and a method for producing the same. In more detail, it is possible to provide a novel protein having a fermentation milk stringiness improving effect, a DNA encoding the above protein, a vector containing the above DNA, a lactic acid bacterium containing the above DNA or the above vector and a lactic acid bacterium composition thereof, and a fermentation milk excellent in stringiness, a fermentation milk thickener, a method for producing the same, a method for improving the stringiness of a fermentation milk, and an evaluation method for lactic acid bacteria using the same.

[0082] For example, by introducing DNA encoding the novel protein of the present application into various lactic acid bacteria, it is possible to easily produce fermented milk having higher stringiness and thickness than in the past. In addition, by using the DNA sequence encoding the novel protein of the present application as a selection criterion, it is possible to easily select lactic acid bacteria that produce fermented milk having higher stringiness and thickness than in the past. Furthermore, the novel protein of the present application can improve the stringiness of fermented milk, and as a result, the amount of free whey can be reduced, and the appearance of the fermented milk can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0083] Figure 1 is a graph showing the stickiness time (sec) of fermented milk fermented by 2038 strain or R-1 strain, which was measured by <Stickiness Evaluation 1>.

[0084] Figure 2 is a schematic diagram of EPS gene cluster 1 and EPS gene cluster 2 in <Comparison of EPS gene cluster regions>.

[0085] Figure 3 is a photograph showing the appearance of fermented milk fermented by 2038 strain, R-1 strain, 2038-epsC strain or 2038-epsF strain, which was prepared in <Stickiness Evaluation 2>.

[0086] Figure 4 is a graph showing the stickiness time (sec) of fermented milk fermented by 2038 strain, R-1 strain, 2038-epsC strain or 2038-epsF strain, which was measured in <Stickiness Evaluation 2>. DETAILED DESCRIPTION

[0087] The present application will be described in detail below by means of preferred embodiments thereof.

[0088] <Protein, DNA, vector and composition containing them>

[0089] The protein of the present application is a protein having a fermented milk stringiness improving effect, and is at least one protein selected from the group consisting of the following (a) to (d),

[0090] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;

[0091] (b) a protein consisting of an amino acid sequence in which one or a plurality of amino acids other than the tyrosine at position 40 in the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted, inserted and / or added, and having a fermented milk stringiness improving effect;

[0092] (c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and having a fermented milk stringiness-improving effect; and

[0093] (d) a protein encoded by a DNA hybridizing under stringent conditions with the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and having a fermented milk stringiness-improving effect.

[0094] The protein of the present application is a protein having a fermented milk stringiness-improving effect (hereinafter referred to as "stringiness-improving protein" depending on the case). In the present application, the "fermented milk stringiness" means a stringiness characteristic exhibited by the viscosity and / or elasticity of fermented milk, and the "fermented milk stringiness-improving effect" means an effect of imparting the above-mentioned stringiness to fermented milk or of improving the above-mentioned stringiness of fermented milk (hereinafter referred to as "stringiness-improving effect" depending on the case). The reason why the protein of the present application has the above-mentioned stringiness-improving effect is not clear, but it is presumed by the present inventors that it is because the protein of the present application acts in the process of EPS biosynthesis by lactic acid bacteria to produce EPS having a structure with high stringiness.

[0095] In the present application, the fermented milk stringiness can be evaluated, for example, by the time (stickiness time) during which the filamentous shape is maintained when the fermented milk is stretched. The stickiness time is evaluated, for example, using a creep meter (model: RE2-33005S (Yamato Scientific Co., Ltd.), container: cylinder-shaped container with a height of 35 mm, grips: height: 25 mm, speed: 10 mm / sec, return distance: 10 mm, amount of fermented milk: 10 g), and the time until the fermented milk attached to the grips is completely separated from the fermented milk in the container is measured. The longer the stickiness time, the higher the fermented milk stringiness, and it can be evaluated as excellent.

[0096] The DNA of the present application is a DNA encoding the above-mentioned stringiness-improving protein (hereinafter referred to as "stringiness-improving DNA" depending on the case). That is, the DNA of the present application is at least one DNA selected from the group consisting of the following (a') to (d'),

[0097] (a') a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1;

[0098] (b') a DNA encoding a protein consisting of an amino acid sequence in which one or more of the amino acids except for the tyrosine at position 40 in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, inserted and / or added, and having a fermentation milk stringiness-improving effect;

[0099] (c') a DNA encoding a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and having a fermentation milk stringiness-improving effect; and

[0100] (d') a DNA encoding a protein consisting of an amino acid sequence encoded by a DNA hybridizing under stringent conditions with the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, the amino acid corresponding to position 40 in the amino acid sequence shown in SEQ ID NO: 1 being tyrosine, and having a fermentation milk stringiness-improving effect.

[0101] "(a) the amino acid sequence shown in SEQ ID NO: 1" is the amino acid sequence encoded by the epsC gene of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (depositional number: FERM BP-10741) (R-1 strain). As "(a') a DNA encoding the amino acid sequence shown in SEQ ID NO: 1", there is no particular limitation as long as it encodes the amino acid sequence, and a nucleotide sequence shown in SEQ ID NO: 2 is preferred. The nucleotide sequence shown in SEQ ID NO: 2 is the nucleotide sequence of the epsC gene of the R-1 strain. As described above, the present inventors have found that the protein encoded by the epsC gene of the R-1 strain has a fermentation milk stringiness-improving effect. With respect to the amino acid sequence shown in SEQ ID NO: 1, it is particularly important that the amino acid at position 40 is tyrosine. If this amino acid is substituted with another amino acid (e.g., the 2038 strain and the 2038-epsF strain in the examples), even if the other sequences are identical, it cannot exert excellent stringiness with respect to fermented milk. Hereinafter, the amino acid sequence shown in SEQ ID NO: 1 will be referred to as "Rl-EpsC", and the nucleotide sequence shown in SEQ ID NO: 2 will be referred to as "Rl-epsC".

[0102] In addition, in nature, the amino acid sequence of a protein encoded by a nucleotide sequence can be varied by variation of the nucleotide sequence. Furthermore, at the present technical level, if a person skilled in the art obtains information on the nucleotide sequence of the epsC gene of the R-1 strain (Rl-epsC) or the amino acid sequence of the protein encoded thereby (Rl-EpsC), it is possible to vary the nucleotide sequence to produce a stringiness-improving protein having a stringiness-improving effect maintained or further improved, although the encoded amino acid sequence is different.

[0103] Thus, as another aspect of the "protein that improves the stretchability" of the present application, there is included a "protein that improves the stretchability of fermented milk, which is composed of an amino acid sequence in which one or more of the amino acids other than the tyrosine at position 40 in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, inserted and / or added". Further, as another aspect of the "DNA that improves the stretchability" of the present application, there is included a "DNA that encodes a protein that improves the stretchability of fermented milk, which is composed of an amino acid sequence in which one or more of the amino acids other than the tyrosine at position 40 in the amino acid sequence shown in SEQ ID NO: 1 are substituted, deleted, inserted and / or added". Thus, the "one or more" means the number of amino acid changes of the protein (altered body) after the substitution, deletion, insertion and / or addition (hereinafter collectively referred to as "alteration") within the range having the stretchability-improving effect, and is generally within 100, 1 to 80, preferably 1 to 40, more preferably 1 to 20, and further preferably 1 to several (e.g., 1 to 10, 1 to 8, 1 to 4, 1 to 2).

[0104] The polynucleotide encoding such an altered body can be prepared by a person skilled in the art, for example, using a known site-directed mutagenesis method or the like, based on the nucleotide sequence (Rl-epsC) information of the epsC gene of the R-1 strain.

[0105] In addition, at the present technical level, a polynucleotide (homologous gene) encoding a protein that enhances stringiness can be obtained from other microorganisms other than the R-1 strain by hybridization techniques (Southern, E. M., J. Mol. Biol., 98: 503, 1975) or polymerase chain reaction (PCR) techniques (Saiki, R. K., et al. Science, 230: 1350-1354, 1985; Saiki, R. K. et al. Science, 239: 487-491, 1988) and the like, with the nucleotide sequence of the epsC gene of the R-1 strain (Rl-epsC) information. Therefore, the "protein that enhances stringiness" of the present application also includes "(d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes to the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions, in which the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and which has a protein having a fermentation milk stringiness-enhancing effect". In addition, in another aspect of the "DNA that enhances stringiness" of the present application, it also includes "(d') a DNA encoding a protein consisting of an amino acid sequence encoded by a DNA that hybridizes to the complementary strand of the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions, in which the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and which has a protein having a fermentation milk stringiness-enhancing effect". Furthermore, in the present application, the "amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1" means an amino acid that is aligned with the tyrosine at the 40th position in Rl-EpsC when the nucleotide sequence and the amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) and the like (for example, parameters: default values (i.e., initial settings)) are used to align the amino acid sequence shown in SEQ ID NO: 1 (Rl-EpsC).

[0106] When isolating homologous genes, the hybridization reaction is usually performed under stringent conditions. As "stringent conditions", there are washing of the membrane after hybridization under a low salt concentration solution at a high temperature, for example, washing conditions in 2 x SSC concentration (1 x SSC: 15 mM sodium citrate, 150 mM sodium chloride), 0.5% SDS solution at 60°C for 20 minutes. In addition, the hybridization can be performed according to the method described in the attached instruction manual of the known ECL direct DNA / RNA labeling / detection system (manufactured by Amersham Pharmacia Biotech). The more stringent the hybridization conditions, the more highly identical DNA can be expected to be isolated. However, the above conditions are merely examples, and the necessary stringency (stringent conditions) can be achieved by appropriately combining the concentration of DNA, the length of DNA, the reaction time of hybridization, and the like.

[0107] Further, the protein encoded by the homologous gene obtained by such a method or the like usually has a high identity with the amino acid sequence shown in SEQ ID NO: 1 (R1-EpsC). Therefore, the "protein having a stringiness-improving effect" of the present application also includes "(c) a protein consisting of an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and having a fermented milk stringiness-improving effect". In addition, the "stringiness-improving DNA" of the present application also includes "(c') DNA encoding a protein consisting of an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1, the amino acid corresponding to the 40th amino acid of the amino acid sequence shown in SEQ ID NO: 1 is tyrosine, and having a fermented milk stringiness-improving effect".

[0108] The identity of the amino acid sequence can be determined using the above BLAST or the like (for example, parameters: default values (i.e., initial set values)). In addition, the identity with the amino acid sequence (R1-EpsC) described in SEQ ID NO: 2 is usually 80% or more, preferably 90% or more, and more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more).

[0109] The filamentation-enhancing protein encoded by the homologous gene can be a protein encoded by a gene isolated from a microorganism other than a lactic acid bacterium, but is preferably one isolated from a lactic acid bacterium. As the lactic acid bacterium, for example, there can be mentioned Streptococcuaceae, Lactobacillaceae, Leuconostocaceae, etc., and more specifically, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Liquorilactobacillus, Limosilactobacillus, Levilactobacillus, Lentilactobacillus, Weissella, etc. lactic acid bacteria; Pediococcus, Leuconostoc, Lactococcus, Streptococcus, Enterococcus, etc. lactococci; Bifidobacterium, etc. Among them, Lactobacillus is preferred, and Lactobacillus delbrueckii (including subspecies) is more preferred, and Lactobacillus delbrueckii subsp. bulgaricus is further preferred.

[0110] In the present application, the fact that each protein has the above-described filamentation-enhancing effect can be confirmed, for example, by the following method. When a DNA encoding each protein or a vector containing the above-described DNA is introduced expressibly into at least one lactic acid bacterium selected from the group consisting of Lactobacillus delbrueckii, preferably Lactobacillus delbrueckii subsp. bulgaricus (but, even if one of the above-described (a) to (d) or even if one of the above-described (a') to (d') is not possessed, the same applies), assuming that the adhesion time of fermented milk obtained using the lactic acid bacterium before the introduction under the same fermentation conditions (for example, conditions under which the lactic acid bacterium before the introduction can be fermented) is 1, the adhesion time of fermented milk obtained using the lactic acid bacterium (transformant) after the introduction is 2 or more, preferably 3 or more, and more preferably 4 or more. As the lactic acid bacterium into which the above-described DNA or vector is to be introduced, specifically, for example, Lactobacillus delbrueckii subsp. bulgaricus 2038 strain (2038 strain) is preferred. The 2038 strain can be isolated by spreading a diluted solution of Meiji Bulgarian yogurt LB81 (Meiji Co., Ltd.) on a BCP agar medium, and after culturing at 37°C for 48 hours, picking up a rough colony to isolate.

[0111] The tack time can be measured as follows: using the creep tester under the above conditions, the time until the fermented milk adhering to the clamp is completely separated from the fermented milk in the container is measured after the second compression of each fermented milk. In addition, each lactic acid bacterium does not have this property even when the DNA of (a') to (d') above is introduced, which can be confirmed by the nucleotide sequence of the DNA of each lactic acid bacterium by a known method or a method based thereon, for example, by the detection method of the DNA described in the evaluation step of the <Evaluation method of lactic acid bacteria> below. Furthermore, the method of introducing the DNA or the vector encoding the protein into the lactic acid bacterium can be appropriately selected from a known method or a method based thereon, for example, a method in which the lactic acid bacterium is used as a host cell.

[0112] [Protein for improving stringiness]

[0113] The protein for improving stringiness of the present application can be obtained by a known method or a method based thereon. For example, it can be obtained by a production method including the steps of culturing a host cell into which at least one selected from the group consisting of the DNA encoding the protein for improving stringiness and the vector containing the DNA is introduced, and collecting the protein expressed in the host cell. More specifically, first, the DNA encoding the protein for improving stringiness (DNA for improving stringiness) is obtained from a target microorganism having at least one of the DNA of (a') to (d') above in the form of isolated DNA by a conventional method. As the isolated DNA, it can be chemically synthesized DNA in which the DNA for improving stringiness is artificially synthesized chemically. Next, an expression vector containing the DNA (isolated DNA) or the DNA is prepared, introduced into a host cell, and the transformant obtained is cultured, whereby the protein for improving stringiness of the present application is expressed in the transformant, and the protein is obtained in the form of a recombinant protein from the culture.

[0114] As the method of obtaining the isolated DNA from the target microorganism, for example, a method in which genomic DNA extracted from the microorganism or cDNA synthesized based on mRNA extracted from the microorganism is ligated with a plasmid vector, a phage vector, a cosmid vector, a BAC vector, a PAC vector, or the like to prepare a DNA library or a cDNA library, and the desired genomic DNA or cDNA is isolated from the library by hybridization using a probe prepared based on the nucleotide sequence of the DNA for improving stringiness (e.g., R1-epsC); a method in which a primer prepared based on the nucleotide sequence of the DNA for improving stringiness (e.g., R1-epsC) is used to perform PCR using genomic DNA of the target microorganism or the cDNA as a template, and the amplified DNA fragment is ligated with an appropriate vector as needed to isolate the desired genomic DNA can be given.

[0115] The expression vector described above is a vector which can replicate in a host cell and which contains the polynucleotide sequence in a state in which the encoded protein can be expressed in the host cell. The expression vector can be constructed, for example, as a self-replicating vector, i.e., a plasmid which exists as an extrachromosomal element and whose replication is independent of the replication of the chromosome. Alternatively, the expression vector described above can be constructed as a bacteriophage DNA which is integrated into the genome of the host cell and which replicates together with the integrated chromosome. As the plasmid described above, there can be mentioned, for example, plasmids from E. coli (pET22, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), plasmids from yeast (YEp13, YEp24, YCp50, etc.), plasmids from Bacillus (pUB110, pTP5, etc.), and a shuttle vector for E. coli and lactic acid bacteria (pGMβl, etc.). As the bacteriophage DNA described above, there can be mentioned, for example, bacteriophages (Charon 4A, Charon 21A, EMBL3, EMBL4, λgtlO, λgtl l, λZAP, etc.).

[0116] The steps and methods for constructing the expression vector described above can be appropriately selected from known methods or methods based thereon. For example, when the drawability-improving DNA described above is inserted into a vector, the following methods can be used: first, the isolated DNA described above is cleaved with an appropriate restriction enzyme, and inserted into a restriction enzyme site or a multiple cloning site of an appropriate plasmid and ligated to the plasmid; etc.

[0117] In the expression vector described above, in order to actually introduce it into a host cell and express the drawability-improving protein, it is preferable to contain, in addition to the DNA (drawability-improving DNA) encoding the drawability-improving protein of the present application, a polynucleotide sequence for controlling the expression thereof, a polynucleotide sequence for inducing the expression other than the polynucleotide sequence for controlling the expression described above, and a genetic marker for selecting cells, etc.

[0118] As the polynucleotide sequence for controlling the expression described above, there can be mentioned, for example, a promoter, a terminator, and a polynucleotide sequence encoding a signal peptide, and it can be one of them or a combination of two or more of them. The promoter described above is not particularly limited as long as it shows transcriptional activity in a host cell, and it can be a polynucleotide sequence for controlling the expression of a gene encoding a protein which is the same species as the host cell or a different species. As the polynucleotide sequence for inducing the expression described above, when the host cell is a bacterium, there can be mentioned, for example, a lactose operon which can induce the expression of a gene disposed downstream by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG). As the genetic marker described above, it can be appropriately selected depending on the method for selecting transformants, and, for example, a gene encoding drug resistance or a complementation gene for an auxotroph can be used.

[0119] As the host cell, there is no particular limitation, and microorganisms such as filamentous fungi, yeasts, Escherichia coli, actinomycetes, lactic acid bacteria, and the like are preferable. As the host cell in the production method of the stringiness-improving protein of the present application, there is no particular limitation, and lactic acid bacteria are preferable when the host cell into which the above-described DNA has been introduced is directly used in the <manufacturing method of fermented milk>, <method for improving stringiness of fermented milk>, <manufacturing method of exocellular polysaccharide of lactic acid bacteria>, or <manufacturing method of thickener for fermented milk> described below. As the host cell, a transformant or a mutant in which a specific function has been deleted as needed can also be used.

[0120] As the method for introducing the above-described DNA or expression vector into these host cells, a publicly known method or a method according to the same can be appropriately used, and examples thereof include heat shock method, electroporation method, spheroplast method, lithium acetate method, and conjugation method as the method for introducing into lactic acid bacteria. In addition, as the method for introducing into plant cells, a method using Agrobacterium or particle gun method can be used, as the method for introducing into insect cells, a method using Baculovirus or electroporation method can be used, and as the method for introducing into animal cells, calcium phosphate method, lipofection method, or electroporation method can be used.

[0121] The stringiness-improving protein of the present application can be collected from the culture (e.g., cultured microbial cells) of the transformant into which the above-described DNA or expression vector has been introduced into the host cell by culturing the transformant in a suitable medium. Thus, the present application can also provide a production method of the stringiness-improving protein of the present application, which includes the steps of culturing the above-described transformant and collecting the stringiness-improving protein expressed in the transformant.

[0122] As the culture conditions of the above-described transformant, for example, the culture conditions of the host cell can be applied, and the temperature, whether or not to add air, the concentration of oxygen, the concentration of carbon dioxide, the pH of the culture medium, the culture temperature, the culture time, the humidity, and the like can be appropriately adjusted by the person skilled in the art according to the kind of the host cell, the culture medium used, and the like. In addition, as the method for collecting the above-described filamentous protein from the culture, for example, the following method can be used: the filamentous protein is expressed in the host cell (for example, Escherichia coli), after the culture of the transformant is completed, the cultured cells are recovered by centrifugation or filtration, and the like, the cells are broken, and the obtained liquid is obtained as a crude purified product. Further, the supernatant can be concentrated by an ultrafiltration method, or the like, and a preservative, or the like, is added to become a concentrated crude purified product. In addition, the above-described crude purified product or the above-described concentrated crude purified product is purified, for example, by using a salting-out method, an organic solvent precipitation method, a membrane separation method, a chromatographic separation method alone, or a combination of two or more thereof. Alternatively, the filamentous protein to which a purification tag is added can be expressed in the host cell (for example, Escherichia coli), and the crude extract is passed through a purification column for a tag-carrying protein, and the tag-carrying protein is eluted to be purified.

[0123] The filamentous protein of the present application is directly or indirectly linked to another compound. As the linkage, there is no particular limitation, and linkage at the genetic level is possible, and chemical linkage is also possible. In addition, there is no particular limitation on the site of addition, and either of the amino-terminal end (hereinafter also referred to as "N-terminal end") and the carboxyl-terminal end (hereinafter also referred to as "C-terminal end") of the filamentous protein of the present application is possible, and both are also possible. Linkage at the genetic level can be achieved by using a DNA in which a DNA encoding another protein is linked in a reading frame to a DNA encoding the filamentous protein of the present application (filamentous protein-encoding DNA). As the "another protein" thus linked, there is no particular limitation, and, for example, when the filamentous protein of the present application is more easily purified, a purification tag protein such as a polyhistidine (His-) tag protein, a FLAG-tag protein (registered trademark, Sigma-Aldrich Corporation), glutathione-S-transferase (GST), and the like is preferably used, and, for example, when the filamentous protein of the present application is more easily detected, a detection tag protein such as a fluorescent protein such as GFP, a chemiluminescent protein such as luciferase, and the like is preferably used. Chemical linkage can be a covalent bond, and can also be a non-covalent bond. As the "covalent bond", there is no particular limitation, and, for example, an amide bond between an amino group and a carboxyl group, an alkyl amine bond between an amino group and an alkyl halide group, a disulfide bond between thiols, a thioether bond between a thiol group and a maleimide group or an alkyl halide group can be given. As the "non-covalent bond", for example, the binding between biotin and avidin can be given.

[0124] [filamentous protein-encoding DNA]

[0125] The drawing-increasing DNA of the present application can be DNA encoding the amino acid sequence of the drawing-increasing protein of the present application described above, and can be DNA into which a variation has been introduced in natural DNA, or DNA composed of a nucleotide sequence designed artificially, and in addition, can be composed of a part or all of unnatural nucleotides. In addition, there is no particular limitation on the form, and for example, cDNA, genomic DNA, and chemically synthesized DNA described above as the isolated DNA in the drawing-increasing protein are included.

[0126] In addition, from the viewpoint of further improving the expression efficiency of the drawing-increasing protein encoded in the host cell, the drawing-increasing DNA of the present application can be in a form of DNA encoding the drawing-increasing protein of the present application, which is codon-optimized according to the kind of the host cell.

[0127] [Vector]

[0128] As the drawing-increasing DNA of the present application, in order to be able to replicate the DNA in a host cell, a vector into which the DNA is inserted can also be adopted. Therefore, the present application also provides a vector containing the drawing-increasing DNA of the present application. As the vector of the present application, including preferred forms thereof, the expression vector described above as the drawing-increasing protein can be cited.

[0129] [Composition]

[0130] The present application provides a composition containing at least one of the drawing-increasing protein, the drawing-increasing DNA, and the vector of the present application described above. The composition of the present application is a composition for improving the drawing property of fermented milk, which contains at least one of the drawing-increasing protein, the drawing-increasing DNA, and the vector of the present application as an effective ingredient, and for example, the composition of the present application is introduced into various lactic acid bacteria to produce the lactic acid bacteria of the present application described below, and fermented milk is produced using the same, whereby fermented milk having a higher and more intense drawing property than in the past can be obtained.

[0131] As the composition of the present application, other ingredients can be further contained. As the other ingredients described above, there is no particular limitation, and for example, sterile water, physiological saline, vegetable oil, a surfactant, a lipid, a dissolution aid, a buffer, a DNase inhibitor, a preservative can be cited, and only one of them can be contained, or two or more of them can be contained in combination.

[0132] <Lactic acid bacteria and lactic acid bacteria composition>

[0133] The present application also provides a transformant into which the drawing-increasing DNA of the present application described above, or the vector of the present application containing the drawing-increasing DNA described above is introduced to the host cell described above. As the transformant, the transformant described above as the drawing-increasing protein can be cited.

[0134] In the present application, as the host cell of the above-mentioned transformant, a lactic acid bacterium is preferred, and "the lactic acid bacterium of the present application" includes: a lactic acid bacterium into which at least one selected from the group consisting of the above-mentioned DNA for improving stringiness of the present application and the above-mentioned vector of the present application containing the DNA for improving stringiness of the present application has been introduced; and a lactic acid bacterium having the above-mentioned DNA for improving stringiness of the present application. Further, "the lactic acid bacterium of the present application" also includes a lactic acid bacterium into which the protein itself for improving stringiness of the present application has been introduced. The lactic acid bacterium of the present application thus achieves the effect of improving stringiness of fermented milk.

[0135] In addition, the lactic acid bacterium of the present application can be in the form of a lactic acid bacterium composition, and the present application also provides: a lactic acid bacterium composition containing at least one of these lactic acid bacterium of the present application. The above-mentioned lactic acid bacterium composition can be a lactic acid bacterium composition that can be used for the production of fermented milk with improved stringiness, the improvement of stringiness of fermented milk, the production of exocellular polysaccharide of lactic acid bacteria, or the production of a thickener for fermented milk, in addition to the production of the above-mentioned protein for improving stringiness.

[0136] [Lactic acid bacterium]

[0137] As the lactic acid bacteria that are host cells into which the protein for improving drawability, the DNA for improving drawability, or the vector of the present application is introduced, there is no particular limitation, and examples include Streptococcuaceae, Lactobacillaceae, Leuconostocaceae, and the like, and more specifically, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Liquorilactobacillus, Limosilactobacillus, Levilactobacillus, Lentilactobacillus, Weissella, and the like; Pediococcus, Leuconostoc, Lactococcus, Streptococcus, Enterococcus, and the like; and Bifidobacterium. Among these, Lactobacillus is preferred, and Lactobacillus delbrueckii (including subspecies) is more preferred, and Lactobacillus delbrueckii subsp. bulgaricus is further preferred. The lactic acid bacteria that are host cells can already have at least one of the proteins of (a) to (d) above or at least one of the DNAs of (a') to (d') above. When this lactic acid bacteria is used as a host cell, a further effect of improving drawability can be expected.

[0138] As the method for introducing the above protein for improving drawability, the above DNA for improving drawability, or the above vector into these lactic acid bacteria, the methods described above as the method for introducing DNA or an expression vector in the [protein for improving drawability] can be suitably used, and for example, it is preferred to introduce the above DNA for improving drawability or the above vector using at least one selected from the group consisting of a heat shock method, an electroporation method, a spheroplast method, a lithium acetate method, and a conjugation method.

[0139] In addition, as the lactic acid bacteria of the present application, as the lactic acid bacteria having the DNA for improving drawability of the present application, examples include the lactic acid bacteria having at least one of the DNAs of (a') to (d') above among the lactic acid bacteria described above as host cells.

[0140] Further, the lactic acid bacteria of the present application having (preferably into which the above-mentioned protein for improving stringiness or the DNA for improving stringiness has been introduced) the above-mentioned protein for improving stringiness or the DNA for improving stringiness can be appropriately confirmed by a known method or a method according to the same, for example, by the detection method of the DNA for improving stringiness described in the evaluation step of the following <Evaluation method of lactic acid bacteria>. Therefore, the "lactic acid bacteria of the present application" also includes lactic acid bacteria evaluated to have the effect of improving the stringiness of fermented milk (including those evaluated to have a high possibility of having the above-mentioned effect) by the following evaluation method of lactic acid bacteria of the present application, and lactic acid bacteria obtained by the following production method of lactic acid bacteria of the present application.

[0141] The DNA possessed by (preferably introduced into) the lactic acid bacteria of the present application can be maintained as the genomic DNA in the lactic acid bacteria, and in addition, if it is a vector, it can be replicated and maintained as an independent body outside the genomic DNA. As the DNA introduced into the lactic acid bacteria, it can be maintained by random insertion into the genomic DNA, or it can be maintained by homologous recombination. In addition, as the lactic acid bacteria of the present application, it can be an artificially mutated strain, a naturally mutated strain, or a genetically recombined strain of the above-mentioned lactic acid bacteria within the range of having the effect of improving the stringiness of fermented milk.

[0142] As the lactic acid bacteria of the present application, it is preferable to have the effect of improving the stringiness of fermented milk. In the present application, the lactic acid bacteria into which the above-mentioned protein for improving stringiness, the above-mentioned DNA for improving stringiness, or the above-mentioned vector has been introduced has the effect of improving the stringiness of fermented milk, which can be confirmed, for example, in such a manner that, when the sticking time of fermented milk obtained using the lactic acid bacteria before the introduction under the same fermentation conditions (for example, conditions under which the lactic acid bacteria before the introduction can be fermented) is taken to be 1, the sticking time of fermented milk obtained using the lactic acid bacteria into which the above-mentioned protein for improving stringiness, the above-mentioned DNA for improving stringiness, or the above-mentioned vector has been introduced is 2 or more, preferably 3 or more, and more preferably 4 or more. The above-mentioned sticking time can be obtained by measuring the time until the fermented milk adhering to the jig is completely disconnected from the fermented milk in the container using the above-mentioned creep meter under the above-mentioned conditions, taking the time immediately after the secondary compression of each fermented milk to be 0 seconds, and raising the jig.

[0143] [Lactic acid bacteria composition]

[0144] The lactic acid bacteria composition of the present application is a composition containing the above-mentioned lactic acid bacteria of the present application. As the lactic acid bacteria composition of the present application, it can further contain other components, and as the above-mentioned other components, although there is no particular limitation, for example, it contains a culture of the culture supernatant, medium components, or the like after the completion of the culture of the above-mentioned lactic acid bacteria; a concentrate, a crude purified product, a purified product, a dilution, a dried product (a spray-dried product, a freeze-dried product, or the like), a frozen product, or the like of the above-mentioned culture; a protective agent, a fermentation accelerator, or the like, and it can be a single one of them, or a combination of two or more of them.

[0145] In addition, the lactic acid bacterial composition of the present application includes a composition containing the lactic acid bacteria of the present application (i.e., the lactic acid bacteria into which at least one selected from the group consisting of the stringiness-improving protein, the stringiness-improving DNA, and the vector of the present application containing the stringiness-improving DNA; the lactic acid bacteria having the stringiness-improving DNA; the lactic acid bacteria evaluated as having the effect of improving the stringiness of fermented milk (including those evaluated as having a high possibility of having the effect) by the evaluation method of the lactic acid bacteria of the present application; and the lactic acid bacteria obtained by the production method of the lactic acid bacteria of the present application), and the fermented milk of the present application described below is also included.

[0146] <Evaluation method of lactic acid bacteria, production method of lactic acid bacteria>

[0147] The evaluation method of the lactic acid bacteria of the present application is a method of evaluating whether or not the lactic acid bacteria have the effect of improving the stringiness of fermented milk with the DNA (i.e., the stringiness-improving DNA of the present application) selected from at least one of the group consisting of the DNA encoding any of the proteins of the present application described above, i.e., the proteins described in (a) to (d) below as an index.

[0148] (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;

[0149] (b) a protein consisting of an amino acid sequence in which one or a plurality of amino acids other than the tyrosine at position 40 in the amino acid sequence represented by SEQ ID NO: 1 are substituted, deleted, inserted, and / or added, and having the effect of improving the stringiness of fermented milk;

[0150] (c) a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1, the amino acid corresponding to position 40 in the amino acid sequence represented by SEQ ID NO: 1 being tyrosine, and having the effect of improving the stringiness of fermented milk; and

[0151] (d) a protein consisting of an amino acid sequence encoded by a DNA that hybridizes to the complementary strand of the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions, the amino acid corresponding to position 40 in the amino acid sequence represented by SEQ ID NO: 1 being tyrosine, and having the effect of improving the stringiness of fermented milk.

[0152] [evaluation steps]

[0153] In the evaluation method of the lactic acid bacteria of the present application, whether or not the lactic acid bacteria have the DNA for improving stringiness of the present application, i.e., whether or not the lactic acid bacteria have the DNA for improving stringiness of the present application is evaluated as an index (evaluation step). When the DNA for improving stringiness described above is present, it can be evaluated that the lactic acid bacteria have the effect of improving stringiness of fermented milk (including those with a high possibility of having the effect). On the other hand, when the DNA described above is not present, the lactic acid bacteria can be evaluated as not having the effect of improving stringiness of fermented milk (including those with a high possibility of not having the effect). Thus, the lactic acid bacteria having the effect of improving stringiness of fermented milk or those with a high possibility of having the effect can be selected. In the evaluation method of the lactic acid bacteria of the present application, the lactic acid bacteria to be evaluated are not particularly limited, and the desired lactic acid bacteria can be appropriately used as the target.

[0154] Whether or not the lactic acid bacteria have the DNA for improving stringiness of the present application can be determined by detecting the DNA. As the method for detecting the DNA for improving stringiness, a publicly known method or a method according to the same can be appropriately used.

[0155] For example, first, genomic DNA is extracted from the lactic acid bacteria to be evaluated. As the method for extracting the genomic DNA, a publicly known method or a method according to the same can be appropriately used, and for example, the PCI method, the GuSCN / Silica method, the SDS phenol method, the CTAB method, and the alkali treatment method can be mentioned. In addition, a commercially available kit can be appropriately used.

[0156] As the method for detecting the DNA for improving stringiness described above, next, the DNA corresponding to the DNA for improving stringiness is isolated, and the nucleotide sequence of the isolated DNA is determined, whereby the method can be implemented. The isolation of the DNA can be performed, for example, by PCR or the like using at least a pair of oligonucleotide primers designed to sandwich the DNA corresponding to the DNA for improving stringiness described above and using the genomic DNA as a template. The determination of the nucleotide sequence of the isolated DNA can be performed by a method known to those skilled in the art, such as the Sanger method and the Maxam-Gilbert method. In addition, the nucleotide sequence of the DNA corresponding to the DNA for improving stringiness described above can be directly determined using a next-generation sequencer or the like from the genomic DNA described above.

[0157] As the DNA corresponding to the DNA for improving stringiness described above, a DNA containing at least a site encoding an amino acid corresponding to the 40th amino acid of R1-EpsC is preferable, and a pair of oligonucleotide primers to be sandwiched can be designed according to the nucleotide sequence of the DNA for improving stringiness described above (e.g., R1-epsC) and a public database (Genbank or the like), respectively. Such oligonucleotides can be designed by a publicly known method or a method according to the same by those skilled in the art.

[0158] As another method for detecting the drawability-improved DNA, for example, there is a PCR-SSP (PCR-Sequence Specific Primer) method. In this method, one of the pair of oligonucleotides constituting the primer has a specific base at the 3' end of the oligonucleotide, for example, when the DNA to be detected is the above (a'), the base type complementary to the site encoding the tyrosine at the 40th position of Rl-EpsC is designed. By PCR using this pair of oligonucleotide primers designed as above, amplification is performed only when the drawability-improved DNA of the present application is used as a template, and amplification is not performed when the genomic DNA encoding the tyrosine at the 40th position is a gene encoding another amino acid. Therefore, the above DNA can be detected using the presence or absence of this amplification as an index.

[0159] In addition, as another method for detecting the drawability-improved DNA, when a restriction fragment polymorphism (RFLP) is designed at the site of the tyrosine at the 40th position of Rl-EpsC or the corresponding position, these RFLP markers can be used as an index, and detection can be performed by, for example, a PCR-RFLP method (or a CAPS [Cleaved Amplified Polymorphic Sequence] method) or the like.

[0160] As another method for detecting the drawability-improved DNA, for example, there is a PCR-SSCP (PCR-Single-Strand Conformation Polymorphism) method. For double-stranded DNA amplified by PCR using a pair of oligonucleotide primers designed to clamp the drawability-improved DNA, after denaturation to single-stranded DNA by heat or alkali or the like, when polyacrylamide gel electrophoresis is performed without a denaturing agent, the single-stranded DNA is folded in the gel due to intramolecular interaction, and a higher structure is formed. The interaction of the folded structure changes depending on the base type, and therefore, by detecting the separated single-stranded DNA using silver staining or a radioisotope, the drawability-improved DNA can be detected using the degree of movement of the single-stranded DNA on the gel as an index.

[0161] As another method for detecting the above-mentioned stretchiness-improved DNA, for example, a method using an intercalator can be given. In this method, first, DNA corresponding to the above-mentioned stretchiness-improved DNA is amplified using the above-mentioned genomic DNA as a template in a reaction system containing an intercalator that emits fluorescence when intercalated between DNA double strands. Then, the temperature of the reaction system is changed, and the change in the intensity of the fluorescence emitted by the intercalator is detected, and the change in the intensity of the fluorescence with the change in the detection temperature is used as an index to detect the above-mentioned stretchiness-improved DNA (particularly, the site encoding the tyrosine at the 40th position of Rl-EpsC or a site corresponding thereto). As such a method, a high-resolution melting curve analysis (HRM) method can be given.

[0162] As another method for detecting the above-mentioned stretchiness-improved DNA, for example, when the DNA to be detected is the above-mentioned (a'), a method using an oligonucleotide probe that hybridizes to a region containing the site encoding the tyrosine at the 40th position of Rl-EpsC can be given. In one mode of this method, first, an oligonucleotide probe that specifically hybridizes to the site encoding the tyrosine at the 40th position and is labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared. Next, the oligonucleotide probe is hybridized to the above-mentioned genomic DNA, and further, DNA containing the site encoding the tyrosine at the 40th position is amplified using the DNA sample in which the oligonucleotide probe is hybridized as a template. Then, the fluorescence emitted by the reporter fluorescent dye as the oligonucleotide probe is decomposed and the inhibition by the quencher is released with the amplification is detected. As such a method, a dual probe method, so-called TaqMan (registered trademark) probe method can be given. As another mode using an oligonucleotide probe labeled with a reporter fluorescent dye and a quencher fluorescent dye, a cycling probe method using a chimera oligonucleotide (chimera of RNA and DNA) that specifically hybridizes to the above-mentioned stretchiness-improved DNA in combination with an enzyme such as RNase H can also be used.

[0163] As another method of detecting the drawability-improving DNA, for example, the LAMP (Loop-Mediated Isothermal Amplification) method can be mentioned. In this method, three regions each on both sides of the target site of the double-stranded DNA are provided, a total of six regions, four kinds of primers (two kinds each on both sides) containing these regions are used, and by performing a reaction in the presence of a strand displacement enzyme, an amplification starting point of a loop structure on both sides of the target site can be generated, and next, a sequence repeat structure complementary to each other is generated on the same strand and the target site is amplified. When the DNA to be detected is (a'), when the target site is set to the site of the tyrosine at the 40th position of R1-EpsC, by determining the nucleotide sequence of the amplification product, detection of the presence or absence of each change can be performed. In addition, when one of the six regions is set to the site of the tyrosine at the 40th position, the target site is not amplified when there is a change, and thus the DNA can be detected using the presence or absence of this amplification as an index.

[0164] The method of detecting the drawability-improving DNA is not limited to the above-described method. For example, other publicly known techniques such as a denaturing gradient gel electrophoresis method (DGGE method), an Invader method, a Pyro-sequencing method, a single nucleotide primer extension (SNuPE) method, an allele-specific oligonucleotide (ASO) hybridization method, a ribonuclease A mismatch cleavage method, a DNA microarray method, a DNA array method, and the like can also be used in the present application.

[0165] Further, as the detection of the drawability-improving DNA, detection of the expression thereof is preferable. As the method of detecting the expression of the drawability-improving DNA, for example, mRNA or protein is extracted from the lactic acid bacteria of the subject according to a conventional method, and the mRNA or protein (i.e., drawability-improving protein) encoded by the drawability-improving DNA is detected by a publicly known method or a method according to the same.

[0166] As the method of detecting the mRNA encoded by the drawability-improving DNA, for example, an RT-PCR method, a Northern blotting method can be mentioned.

[0167] As a method for detecting the protein encoded by the above-described filamentousness-increasing DNA (filamentousness-increasing protein), first, a protein sample is prepared from the subject lactic acid bacterium, and an antigen-antibody reaction is performed using the above-described filamentousness-increasing protein-specific antibody, i.e., an antibody specific to at least the tyrosine at position 40, and the above-described filamentousness-increasing protein is detected. In this antibody-using protein detection method, for example, the above-described filamentousness-increasing protein-specific antibody is added to the above-described protein sample, and an antigen-antibody reaction is performed, and the binding of the above-described antibody to the filamentousness-increasing protein is detected. When the filamentousness-increasing protein-specific antibody is labeled, the filamentousness-increasing protein can be directly detected, and when it is not labeled, it can be further reacted with a labeled molecule (e.g., a secondary antibody or protein A) that recognizes the antibody, and the filamentousness-increasing protein is indirectly detected using the label of the molecule. As such a method, for example, immunohistochemistry (immunostaining), immunoblotting, ELISA, Flow cytometry, Imaging cytometry, Radioimmunoassay, immunoprecipitation, analysis using an antibody array, and the like can be used. As the above-described antibody, a polyclonal antibody or a monoclonal antibody can be used, and the method for preparing these antibodies is known to those skilled in the art.

[0168] [Kit for the evaluation method of the present application]

[0169] As described above, by detecting the filamentousness-increasing DNA of the present application, the filamentousness-increasing effect of the lactic acid bacterium can be evaluated. Therefore, the present application provides a kit for the above-described evaluation method, which contains at least one agent selected from the group consisting of the following (i) to (ii),

[0170] (i) an agent containing an oligonucleotide having a chain length of at least 15 nucleotides that can hybridize to the filamentousness-increasing DNA of the present application, a transcription product thereof, or a complementary nucleotide thereof; and

[0171] (ii) an agent containing an antibody that binds to the filamentousness-increasing protein of the present application.

[0172] As the above-described oligonucleotide, according to the method for detecting the filamentousness-increasing DNA described above, it can be in the form of a primer, or it can be in the form of a probe.

[0173] As the above-described primer, as long as it can hybridize with the fiber drawability-improving DNA of the present application or DNA corresponding to the fiber drawability-improving DNA, or a complementary nucleotide thereof (including cDNA, cRNA), or a transcription product (mRNA) of the fiber drawability-improving DNA, and amplify and detect them, it is not particularly limited. As the above-described primer, it can also be only DNA, and it can also be one in which a part or all thereof is replaced with an artificial nucleic acid such as a crosslinked nucleic acid (modified nucleic acid). As the size of the above-described primer, if it is at least about 15 nucleotides or longer, it is preferable to be 15 to 100 nucleotides, more preferable to be 18 to 50 nucleotides, and further preferable to be 20 to 40 nucleotides. Such a primer can be designed and produced by a person skilled in the art by a known method according to the above-described detection method.

[0174] As the above-described probe, if it can detect the fiber drawability-improving DNA or DNA corresponding to the fiber drawability-improving DNA, or a complementary nucleotide thereof, or a transcription product of the fiber drawability-improving DNA by hybridizing therewith, it is not particularly limited. As the above-described probe, it can be DNA, RNA, an artificial nucleic acid, or a chimera molecule thereof, or the like. As the above-described probe, it can be either single-stranded or double-stranded. As the size of the above-described probe, if it is at least about 15 nucleotides or longer, it is preferable to be 15 to 1000 nucleotides, more preferable to be 20 to 500 nucleotides, and further preferable to be 30 to 300 nucleotides. Such a probe can be designed and produced by a person skilled in the art by a known method. In addition, the above-described probe can be provided in a state of being fixed to a substrate, like a microarray.

[0175] The above-described antibody is not particularly limited as long as it can specifically bind to the fiber drawability-improving protein of the present application. For example, it can be either a polyclonal antibody or a monoclonal antibody, and it can also be a functional fragment (Fab, Fab', scFv, or the like) of an antibody. Such an antibody can be produced by a person skilled in the art by a known method. In addition, as the above-described antibody, in order to be used for ELISA or an antibody array, or the like, it can be provided in a state of being fixed to a substrate such as a plate.

[0176] In addition, the oligonucleotide or antibody included in the above-described kit can be labeled with a labeling substance according to the above-described detection method. As the above-described labeling substance, for example, fluorescent substances such as FITC, FAM, DEAC, R6G, TexRed, Cy5, enzymes such as β-D-glucosidase, luciferase, HRP, and the like can be mentioned. 3 H、 14 C、 32 P、 35 S、 123I radioactive isotopes, biotin, streptavidin, luminol, luciferin, lucigenin, and the like.

[0177] In the evaluation method of the lactic acid bacteria of the present application, a confirmation step of confirming whether or not the above lactic acid bacteria have the effect of improving the stringiness of fermented milk can be further included. As such a confirmation method, although not particularly limited, the fermented milk obtained using the lactic acid bacteria to be evaluated, for example, can be confirmed using the stick time measured using the above creep meter under the above conditions as an index. As the above fermented milk, for example, the above lactic acid bacteria can be inoculated into a 10% skim milk medium to be 1% (wt / wt), and allowed to ferment under anaerobic conditions at 37°C for one night. In addition, for example, the above lactic acid bacteria can be evaluated to have the effect of improving the stringiness of fermented milk for the above stick time of 2 seconds or more, preferably 3 seconds or more, and further preferably 4 seconds or more.

[0178] [Method for producing lactic acid bacteria]

[0179] The method for producing lactic acid bacteria of the present application is a method including the following steps: an evaluation step of evaluating whether or not lactic acid bacteria have the effect of improving the stringiness of fermented milk by the above evaluation method of lactic acid bacteria of the present application; and,

[0180] a step of obtaining lactic acid bacteria evaluated to have the effect of improving the stringiness of fermented milk in the above evaluation step.

[0181] In the method for producing lactic acid bacteria of the present application, as the above evaluation step, the evaluation step of the above <evaluation method of lactic acid bacteria> can be mentioned. By the evaluation step of the method for producing lactic acid bacteria of the present application, lactic acid bacteria having the effect of improving the stringiness of fermented milk or having a high possibility thereof can be selected. By the method for producing lactic acid bacteria of the present application, lactic acid bacteria evaluated to have the effect of improving the stringiness of fermented milk in the above evaluation step (including those evaluated to have a high possibility of the above effect) can be obtained, but, for example, by culturing the selected lactic acid bacteria in a suitable medium, the above lactic acid bacteria can also be obtained in the form of a culture thereof.

[0182] In addition, as the form of the lactic acid bacteria obtained by the method for producing lactic acid bacteria of the present application, the form of a lactic acid bacteria composition such as a culture thereof can be mentioned. Therefore, in the method for producing lactic acid bacteria of the present application, a method for producing a lactic acid bacteria composition including the step of obtaining a lactic acid bacteria composition containing lactic acid bacteria evaluated to have the effect of improving the stringiness of fermented milk by the above evaluation step is also included. As other components other than the above lactic acid bacteria that can be contained in the above lactic acid bacteria composition, those mentioned above can be mentioned.

[0183] [Method for producing fermented milk]

[0184] The method for producing fermented milk of the present application includes a fermentation step in which a lactic acid bacterium or a lactic acid bacterium composition is added to a raw milk-containing prepared milk liquid and allowed to ferment.

[0185] The lactic acid bacterium in the method for producing fermented milk of the present application includes the above-described lactic acid bacterium of the present application (i.e., a lactic acid bacterium into which at least one selected from the group consisting of a stringiness-improving protein, a stringiness-improving DNA, and a vector of the present application containing the stringiness-improving DNA; a lactic acid bacterium having the stringiness-improving DNA; a lactic acid bacterium evaluated as having a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacterium of the present application (including one having a high possibility of having the above-described effect); and a lactic acid bacterium obtained by the production method for lactic acid bacterium of the present application), and one of them alone or two or more in combination can be used. In addition, the lactic acid bacterium composition in the method for producing fermented milk of the present application includes the above-described lactic acid bacterium composition of the present application and a lactic acid bacterium composition obtained by the production method for lactic acid bacterium of the present application, and one of them alone or two or more in combination can be used. By using these lactic acid bacterium or lactic acid bacterium composition, a fermented milk excellent in stringiness can be obtained. Furthermore, when a lactic acid bacterium evaluated as having a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacterium of the present application is used as the above-described lactic acid bacterium, the above-described evaluation step can also be included in the method for producing fermented milk of the present application, but as the evaluation step at this time, only the first time is necessary.

[0186] For the method for producing fermented milk of the present application, other lactic acid bacterium than the above-described lactic acid bacterium of the present application can also be further used in combination. In addition, yeast can also be further added. As the above-described other lactic acid bacterium and yeast, a publicly known lactic acid bacterium or yeast conventionally contained in fermented milk can be mentioned.

[0187] (Prepared milk liquid)

[0188] The prepared milk liquid of the present application contains raw milk. As the above-described raw milk, one containing lactose is preferred, and, for example, raw milk (e.g., milk of cow, buffalo, sheep, goat, etc.), pasteurized milk, whole milk, skim milk, whey, and processed products thereof (e.g., whole milk powder, whole milk concentrate, skim milk powder, skim milk concentrate, condensed milk, whey powder, butter milk, butter oil, cream, cheese, whey protein concentrate (WPC), separated whey protein (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg)) can be mentioned, and one of these or a mixture of two or more can be used.

[0189] As the prepared milk liquid of the present application, only the above-mentioned raw milk can be used, or a water solution, a dilution or a concentrate of the above-mentioned raw milk can be used, or other ingredients can be further added to the above-mentioned raw milk as needed. As such other ingredients, water; soy milk, a sugar such as sugar or a sweetener, a flavoring, a fruit juice, a fruit pulp, a vitamin, a mineral, an oil, a ceramide, collagen, a milk phospholipid, a yeast extract, a polyphenol, and the like food, a food ingredient, a food additive; a stabilizer, a thickening agent, a gelation agent such as pectin, a soy polysaccharide, CMC (carboxymethyl cellulose), agar, gelatin, carrageenan, and the like gum can be used, and one of these or a mixture of two or more of these can be used. The above-mentioned prepared milk liquid can be prepared by mixing the above-mentioned ingredients while being heated and / or homogenized as needed. In addition, the above-mentioned prepared milk liquid can be sterilized by heating.

[0190] (Fermentation)

[0191] As the fermentation step in which the above-mentioned prepared milk liquid is fermented by adding the above-mentioned lactic acid bacteria or lactic acid bacteria composition thereto, a publicly known method or a method according to the same can be appropriately used, and there is no particular limitation, and for example, a method in which the above-mentioned lactic acid bacteria or lactic acid bacteria composition is inoculated into the above-mentioned prepared milk liquid as a starter and is allowed to ferment can be used. As the above-mentioned lactic acid bacteria or lactic acid bacteria composition, the above-mentioned lactic acid bacteria composition can be added to the above-mentioned prepared milk liquid in the form of the above-mentioned lactic acid bacteria composition, preferably in the form of a culture or a concentrate of a culture.

[0192] The amount of the above-mentioned starter to be added can be appropriately set according to the amount to be added in a publicly known method of producing fermented milk, and for example, preferably 1 x 10 7 ~ 5 x 10 9 CFU / mL, and more preferably 1 x 10 8 ~ 2 x 10 9 CFU / mL, with respect to the volume of the above-mentioned prepared milk liquid. In addition, preferably 0.1 to 2% (wt / wt), more preferably 0.5 to 1.5% (wt / wt), and further preferably 0.5 to 1% (wt / wt) with respect to the volume of the above-mentioned prepared milk liquid.

[0193] The inoculation method of the above-mentioned starter is not particularly limited, and a method conventionally used in the method for producing fermented milk can be appropriately used. As the conditions for the above-mentioned fermentation, the conditions can be appropriately selected depending on the growth conditions of the added lactic acid bacteria, the amount of the above-mentioned modulated milk liquid, and the like, and are not particularly limited, and for example, it is preferred that the modulated milk liquid be left to stand or stirred (preferably left to stand) under aerobic or anaerobic conditions at a temperature of 35 to 45°C, more preferably at a temperature of 38 to 43°C, usually for 3 to 24 hours, more preferably for 3 to 8 hours, further preferably for 4 to 6 hours, until the pH of the modulated milk liquid to which the above-mentioned lactic acid bacteria or lactic acid bacteria composition is added is 4.8 or less, more preferably 4.0 to 4.6. In addition, as the above-mentioned anaerobic conditions, for example, fermentation under a nitrogen gas atmosphere can be employed.

[0194] By the above-mentioned fermentation, the fermented milk of the present application can be obtained. The fermented product after the above-mentioned fermentation step (i.e., the modulated milk liquid after the above-mentioned fermentation step and the lactic acid bacteria or lactic acid bacteria composition) can be directly or as needed by concentration, dilution, drying, or freezing or the like, prepared into the fermented milk of the present application. In addition, the lactic acid bacteria in the above-mentioned fermented product can be disrupted or subjected to heat treatment or the like, or as needed, concentrated, diluted, dried, or frozen or the like, to prepare the fermented milk of the present application.

[0195] <fermented milk>

[0196] As the fermented milk of the present application, fermented milk containing at least one lactic acid bacteria selected from the group consisting of the above-mentioned lactic acid bacteria of the present application (i.e., lactic acid bacteria into which at least one selected from the group consisting of a stringiness-improving protein, a stringiness-improving DNA, and a vector of the present application containing a stringiness-improving DNA; lactic acid bacteria having a stringiness-improving DNA; lactic acid bacteria evaluated as having a fermented milk stringiness-improving effect by the evaluation method for lactic acid bacteria of the present application (including those evaluated as having a high possibility of having the above-mentioned effect); and lactic acid bacteria obtained by the production method for lactic acid bacteria of the present application) is provided. As the fermented milk of the present application, it is preferred that the fermented milk contain a stringiness-improving protein and / or an exopolysaccharide from these lactic acid bacteria. In addition, as the fermented milk of the present application, it can further contain other lactic acid bacteria and yeast in addition thereto.

[0197] As the fermented milk of the present application, there is no particular limitation, and for example, it can be any one of fermented milk (more specifically, one having a non-fat milk solid content of 8.0% or more and a lactic acid bacteria count or a yeast count (preferably, a lactic acid bacteria count) of 10 million / mL or more) that meets the standards of "fermented milk" in the regulations (Laws on Milk and Milk Products) of the Ministry of Health, Labour and Welfare of Japan, fermented milk that meets the standards of "milk product lactic acid bacteria beverage" (more specifically, one having a non-fat milk solid content of 3.0% or more and a lactic acid bacteria count or a yeast count (preferably, a lactic acid bacteria count) of 10 million / mL or more), and fermented milk that meets the standards of "lactic acid bacteria beverage" (more specifically, one having a non-fat milk solid content of less than 3.0% and a lactic acid bacteria count or a yeast count (preferably, a lactic acid bacteria count) of 1 million / mL or more). Furthermore, the non-fat milk solid content referred to above means the remaining content (mainly, protein, lactose, minerals, and the like) obtained by subtracting the fat portion from the total milk solid content, and the lactic acid bacteria count and the yeast count are measured by the test methods prescribed in the above-mentioned laws on milk and milk products before sterilization.

[0198] As the fermented milk of the present application, it can be the fermented product after the fermentation step, or it can be a sterilized product of the above-mentioned fermented product, or it can be a concentrated, diluted, dried, or frozen product thereof, and for example, as the fermented milk, it can be a sterilized product of the above-mentioned fermented milk, milk product lactic acid bacteria beverage, or lactic acid bacteria beverage, in which case the lactic acid bacteria count is converted to the number of viable bacteria. In the fermented milk of the present application, in addition to viable bacteria, dead bacteria, and broken and heat-treated products of lactic acid bacteria, concentrated, crude-purified, purified, diluted, dried (spray-dried, freeze-dried, and the like), and frozen products thereof are also contained, and as the lactic acid bacteria contained in the fermented milk of the present application, it is preferable to contain at least viable bacteria.

[0199] As the fermented milk of the present application, it can further contain the above-mentioned other lactic acid bacteria or yeast as lactic acid bacteria, within a range that does not interfere with the effects of the present application. In addition, as the fermented milk of the present application, it can further contain various components that can be contained in a food or drink. As such components, there is no particular limitation, and for example, water, sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, pH adjustors, starches and modified starches, food fibers, fruits and vegetables and processed products thereof, animal and plant crude drug extracts, polymers from nature (collagen, hyaluronic acid, chondroitin, and the like), oils and fats, thickeners, emulsifiers, solvents, surfactants, gelatinizing agents, stabilizers, buffers, suspending agents, viscosity agents, excipients, disintegrants, binders, flow agents, preservatives, colorants, flavorings, taste correctors, sweeteners, and the like can be mentioned, and one of these alone or two or more in combination can be contained.

[0200] As such a fermented milk, yogurt, cheese, fermented butter, fermented tallow, etc. are preferable, and yogurt is particularly preferable. As such a yogurt, solid-type yogurt (solid fermented milk), soft-type yogurt (pasty fermented milk), and beverage-type yogurt (liquid fermented milk) such as plain yogurt, etc. can be mentioned, and frozen yogurt using these as a material can also be used. In addition, the fermented milk of the present application can also be used as a material for fermented foods such as cheese, fermented butter, fermented tallow, Kefir, etc.

[0201] The fermented milk of the present application can be obtained by the above-described production method of the fermented milk of the present application, and can be used as a fermented milk having excellent stringiness.

[0202] Method for producing exopolysaccharide

[0203] The present application also provides a method for producing exopolysaccharide of lactic acid bacteria, which comprises the step of: adding the above-described lactic acid bacteria or lactic acid bacteria composition of the present application to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and performing fermentation, and collecting exopolysaccharide contained in the fermentation product.

[0204] In the present application, the "exopolysaccharide of lactic acid bacteria" means exopolysaccharide produced by lactic acid bacteria, and includes neutral exopolysaccharide (NPS), acidic exopolysaccharide (APS), zwitterionic exopolysaccharide (ZPS), and mixtures thereof.

[0205] As such a culture medium, at least one sugar selected from the group consisting of glucose and a sugar having glucose as a constituent sugar must be contained. As a sugar having glucose as a constituent sugar, for example, disaccharides (maltose, sucrose, lactose, etc.), oligosaccharides (galactooligosaccharides, fructooligosaccharides, mannooligosaccharides, etc.), and polysaccharides (starch (amylose, amylopectin), glycogen, etc.) can be mentioned. As the sugar contained in the above-described culture medium, only one of the above-described sugars can be contained, or two or more kinds in combination can be contained, and among these, lactose is preferable. In addition, as the sugar contained in the above-described culture medium, for example, the raw milk contained in the above-described raw milk can be used, and as such a culture medium, the above-described raw milk is preferable, and the above-described prepared milk liquid containing the above-described raw milk is more preferable, and as the above-described raw milk, skim milk powder is preferable.

[0206] As the above-mentioned lactic acid bacteria and lactic acid bacteria composition, and the method of fermentation, including preferred modes thereof, the fermentation step in the above-mentioned method of manufacturing fermented milk is the same except that the above-mentioned culture medium is used as the above-mentioned preparation liquid. As the method of collecting the above-mentioned exocellular polysaccharide from the above-mentioned fermented milk, there is no particular limitation, and a method known in the past or a method based thereon can be appropriately used, and for example, the following methods can be listed: for the fermented milk after fermentation, deproteinization is performed as necessary by adding a protein denaturing agent (trichloroacetic acid or the like) or performing heat treatment, thereby producing a crude purified product, and then, for example, a salting-out method, an organic solvent precipitation method, a membrane separation method, a chromatographic separation method, or a combination of two or more of these methods are used alone to perform purification.

[0207] <Thickener for fermented milk and method of manufacturing the same>

[0208] Further, the present application also provides a thickener for fermented milk containing exocellular polysaccharide as an effective ingredient, the exocellular polysaccharide being derived from at least one lactic acid bacterium selected from the group consisting of the above-mentioned lactic acid bacteria of the present application (i.e., lactic acid bacteria into which at least one selected from the group consisting of a stringiness-improving protein, a stringiness-improving DNA, and a vector of the present application containing a stringiness-improving DNA; lactic acid bacteria having a stringiness-improving DNA; lactic acid bacteria evaluated by the evaluation method for lactic acid bacteria of the present application as having a fermented milk stringiness-improving effect (including those evaluated as having a high possibility of having the above-mentioned effect); and lactic acid bacteria obtained by the method of manufacturing lactic acid bacteria of the present application). The above-mentioned exocellular polysaccharide derived from lactic acid bacteria is: that produced outside the cell when the above-mentioned lactic acid bacteria or lactic acid bacteria composition of the present application is added to the above-mentioned culture medium and subjected to fermentation by the above-mentioned method of manufacturing exocellular polysaccharide, and is contained in the fermented milk after fermentation. The thickener for fermented milk of the present application, for example, can improve the stringiness of fermented milk and thicken the same by being added to the fermented milk.

[0209] The thickener for fermented milk of the present application can be directly the fermented milk after fermentation, can be a concentrate, a crude purified product, a purified product, a paste, a dried product (spray-dried product, freeze-dried product, or the like), a pulverized product, a liquid dispersed in a medium, and a processed product combining two or more of these, and can also be formed only of the exocellular polysaccharide obtained by the above-mentioned method of manufacturing exocellular polysaccharide. Further, within a range that does not interfere with the effects of the present application, other ingredients that can be contained in fermented milk can be contained. As the above-mentioned other ingredients, although there is no particular limitation, for example, various ingredients listed in the above-mentioned <fermented milk> can be listed, and one or a combination of two or more of these can be contained in an appropriate amount.

[0210] The content of the exocellular polysaccharide as the effective ingredient (the total amount if it is a mixture of two or more) in the fermented milk thickener of the present application can be appropriately adjusted, and thus cannot be generalized, but is preferably 0.001% by mass or more, more preferably 0.002% by mass or more, and particularly preferably 0.003% by mass or more, relative to the entire fermented milk thickener. There is no particular limitation on the upper limit of the above content, and for example, it can be 100% by mass or less, and preferably 90% by mass or less.

[0211] <Method for improving stringiness of fermented milk>

[0212] The method for improving stringiness of fermented milk of the present application includes a fermentation step of adding the lactic acid bacteria or lactic acid bacteria composition of the present application to a raw milk-containing modulated milk liquid to ferment it. Thereby, the stringiness of the fermented milk can be improved. As the lactic acid bacteria, lactic acid bacteria composition, and fermentation step, those described above in the method for producing fermented milk of the present application are respectively used. Thereby, the stringiness of the fermented milk can be improved.

[0213] In the present application, that the stringiness of the fermented milk is excellent or improved can be evaluated, for example, by comparing the sticking time of the fermented milk with that of fermented milk obtained using lactic acid bacteria other than the above lactic acid bacteria, i.e., other lactic acid bacteria not having one of the DNAs of (a') to (d'), preferably at least one lactic acid bacteria selected from the group consisting of Lactobacillus delbrueckii and Lactobacillus delbrueckii subsp. bulgaricus, under the same fermentation conditions. For example, when the sticking time of the fermented milk obtained using the above other lactic acid bacteria is set to 1, the fermented milk of the object can be evaluated as having excellent or improved stringiness when the sticking time is 2 or more, preferably 3 or more, and more preferably 4 or more. Or the fermented milk of the object can be evaluated as having excellent or improved stringiness when the sticking time is, for example, 2 seconds or more, preferably 3 seconds or more, and more preferably 4 seconds or more.

[0214] The above sticking time can be measured by using the above creep meter under the above conditions, taking the time immediately after the secondary compression of each fermented milk as 0 seconds, raising the grips, and measuring the time until the fermented milk adhering to the grips is completely disconnected from the fermented milk in the container. In addition, that each lactic acid bacteria does not have one of the DNAs of (a') to (d') can be confirmed, for example, by the detection method of the stringiness-improving DNA described in the evaluation step of the above <Evaluation method of lactic acid bacteria>. As the lactic acid bacteria not having one of the DNAs of (a') to (d'), specifically, for example, Lactobacillus delbrueckii subsp. bulgaricus 2038 strain (2038 strain) is preferable.

[0215] [Examples]

[0216] The present application will be more specifically described below based on examples, but the present application is not limited to the following examples.

[0217] < Lactic acid bacteria >

[0218] The lactic acid bacteria used for the following tests were as follows.

[0219] R-1 strain: Lactobacillus delbrueckii subsp. bulgaricus OLL1073 R-1 (Deposit number: FERM BP-10741)

[0220] 2038 strain: Lactobacillus delbrueckii subsp. bulgaricus 2038

[0221] In addition, the 2038 strain is a strain isolated by smearing a diluted solution of Meiji Bulgarian yogurt LB81 (Meiji Co., Ltd.) on a BCP plus agar medium and culturing at 37°C for 48 hours, and picking a rough colony, and the full-length genome of the 2038 strain is registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG), a database that integrates information on genomes, proteins, and compounds in the context of interactions and reactions between molecules, with a relationship network.

[0222] < Stringiness evaluation 1 >

[0223] (1) Preparation of fermented milk

[0224] R-1 strain was inoculated to 1% (wt / wt) in a 10% skim milk medium prepared from skim milk powder 10% (wt / wt), yeast extract 0.1% (wt / wt), and distilled water, and allowed to ferment overnight at 37°C under anaerobic conditions, thereby obtaining fermented milk. In addition, except that 2038 strain was used instead of R-1 strain, fermented milk was obtained under the same conditions.

[0225] (2) Adhesion time measurement

[0226] Each of the fermented milks obtained in the above (1) was evaluated for stringiness using a creep meter (Model: RE2-33005S (Yamato Scientific Co., Ltd.), container: a cylindrical container with a height of 35 mm, clamp: a clamp with a height of 25 mm, speed: 10 mm / sec, return distance: 10 mm, amount of fermented milk: 10 g). That is, for the obtained fermented milk, immediately after allowing the above creep meter to perform secondary compression, the clamp was raised, and the time until the fermented milk adhering to the clamp was completely disconnected from the fermented milk in the container was measured as the adhesion time (sec). The measurement was performed three times for each fermented milk, and the average value was calculated. The longer the adhesion time, the higher the stringiness, and thus it can be evaluated as more excellent. The results are shown in Table 1. Figure 1 Figure 1 ​​As shown, the average of the coagulation time of the fermented milk fermented with R-1 strain was significantly longer than that of the fermented milk fermented with 2038 strain (Tukey-Kramer test, p<0.01), showing high stringiness.

[0227] Comparison of EPS gene cluster regions

[0228] Comparison of EPS gene cluster regions

[0229] A schematic diagram of EPS gene cluster 1 is shown in Fig. 2(a), and a schematic diagram of EPS gene cluster 2 is shown in Fig. 2(b). Figure 2 Figure 2 In Fig. 2, positions at which differences in nucleotide sequences were confirmed between 2038 strain and R-1 strain are indicated by arrows. In the genome of R-1 strain, EPS gene clusters 1 and 2 were also highly conserved. In particular, the entire region (11569 bp) of EPS gene cluster 1 was completely identical to the nucleotide sequence of 2038 strain. On the other hand, as shown in Fig. 2(b), in the case of EPS gene cluster 2, a total of 4 bases were confirmed to be different between the epsC gene and the epsF gene, and between the epsM gene and the transposase gene, among the intergenic regions. Table 1 shows the genes or regions in which differences were confirmed between the genome of 2038 strain and the genome of R-1 strain, the bases, the codons containing the bases, the amino acids encoded by the codons, and the positions of the amino acids in the genes. In addition, in the case of the epsF gene, a frame shift was caused by the difference in bases, and therefore, the codons including the bases in which differences were confirmed between the genome of 2038 strain and the genome of R-1 strain, and the nucleotide sequences downstream of the codons, and the amino acid sequences encoded by the nucleotide sequences are shown in Table 2. In addition, the nucleotide sequence of the genome of 2038 strain described in Table 2 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of the genome of R-1 strain is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. Figure 2 Figure 2 In Fig. 2, positions at which differences in nucleotide sequences were confirmed between 2038 strain and R-1 strain are indicated by arrows. In the genome of R-1 strain, EPS gene clusters 1 and 2 were also highly conserved. In particular, the entire region (11569 bp) of EPS gene cluster 1 was completely identical to the nucleotide sequence of 2038 strain. On the other hand, as shown in Fig. 2(b), in the case of EPS gene cluster 2, a total of 4 bases were confirmed to be different between the epsC gene and the epsF gene, and between the epsM gene and the transposase gene, among the intergenic regions. Table 1 shows the genes or regions in which differences were confirmed between the genome of 2038 strain and the genome of R-1 strain, the bases, the codons containing the bases, the amino acids encoded by the codons, and the positions of the amino acids in the genes. In addition, in the case of the epsF gene, a frame shift was caused by the difference in bases, and therefore, the codons including the bases in which differences were confirmed between the genome of 2038 strain and the genome of R-1 strain, and the nucleotide sequences downstream of the codons, and the amino acid sequences encoded by the nucleotide sequences are shown in Table 2. In addition, the nucleotide sequence of the genome of 2038 strain described in Table 2 is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4. The nucleotide sequence of the genome of R-1 strain is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.​​

[0230] Table 1

[0231]

[0232] Table 2

[0233] Strain Nucleotide sequence (5'-3') Amino acid sequence (N-C) 2038 GG G CTCGCTATTCTCTGA GLAIL R-1 GG-CTCGCTATTCTCTGATTGA GSLFSD

[0234] As shown in Table 1, the first difference is that the adenine (A) at the 118th base of the epsC gene of the genome of the 2038 strain is replaced by thymine (T) in the genome of the R-1 strain. In addition, the amino acid at the 40th position designated by the codon containing this base is asparagine in the genome of the 2038 strain, but is tyrosine in the genome of the R-1 strain. The second difference is that the guanine (G) at the 999th base of the epsF gene of the genome of the 2038 strain is deleted in the genome of the R-1 strain. As a result, a frame shift occurs in the genome of the R-1 strain, the reading frame of the codon shifts, but the amino acid designated by the codon containing this base is glycine (G) at the 333rd position in both the genome of the 2038 strain and the genome of the R-1 strain. However, as shown in Table 2, the amino acid sequence from glycine to the C-terminus differs between the two strains due to the frame shift, which is N-terminus-Gly-Leu-Ala-Ile-Leu-C-terminus in the genome of the 2038 strain, but is N-terminus-Gly-Ser-Leu-Phe-Ser-Asp-C-terminus in the genome of the R-1 strain. The third and fourth differences are the intergenic regions (intergenic 1 and 2) of the epsM gene and the transposase gene. It is generally considered that variations in the intergenic region are not related to either gene, and thus it is presumed that the differences in the epsC gene and the epsF gene and the differences in the amino acid composition caused thereby result in the difference in stretchability between the fermented milk fermented with the R-1 strain and the fermented milk fermented with the 2038 strain.

[0235] <Stretchability Evaluation 2>

[0236] (1) Preparation of 2038-epsC and 2038-epsF strains by transformation of the 2038 strain

[0237] In the transformation of 2038 strains, a shuttle vector pGMβl (obtained from Meiji University) for E. coli and lactic acid bacteria was used. pGMβl was introduced into E. coli DH5 by an electroporation method. After E. coli DH5 retaining pGMβl was selected using an LB medium containing ampicillin (final concentration 50 μg / mL) and erythromycin (final concentration 500 μg / mL), pGMβl was purified by plasmid extraction. In addition, the epsC gene and the epsF gene of R-1 strain were amplified by PCR using the following primers, respectively,

[0238] epsC gene:

[0239] Lb_epsC_rec1_F (nucleotide sequence shown in SEQ ID NO: 7)

[0240] Lb_epsC_rec1_R (nucleotide sequence shown in SEQ ID NO: 8).

[0241] epsF gene:

[0242] Lb_epsF_rec1_F (nucleotide sequence shown in SEQ ID NO: 9)

[0243] Lb_epsF_rec1_R (nucleotide sequence shown in SEQ ID NO: 10).

[0244] Next, the purified pGMβl was treated with Sac I or Sal I and dephosphorylated. Separately, the amplified product of the epsC gene of the R-1 strain was treated with Sac I, and the amplified product of the epsF gene was treated with Sal I, and they were each ligated with the above pGMβl, to obtain plasmids "pGMβl-epsC" and "pGMβl-epsF", respectively. The pGMβl-epsC or pGMβl-epsF was introduced into E. coli DH5 by electroporation. After selecting E. coli DH5 harboring the pGMβl-epsC or pGMβl-epsF using LB medium containing ampicillin (final concentration 50 μg / mL) and erythromycin (final concentration 500 μg / mL), the pGMβl-epsC and pGMβl-epsF were each purified by plasmid extraction. Next, the pGMβl-epsC or pGMβl-epsF was introduced into L. lactis IL 1403 (L. lactis IL 1403) by electroporation. Next, the culture solution of L. lactis IL 1403 harboring the pGMβl-epsC or pGMβl-epsF and the culture solution of the 2038 strain were aspirated through a 0.45 μm membrane filter provided in a suction bottle, and the pGMβl-epsC or pGMβl-epsF was introduced into the 2038 strain by conjugative transfer. The 2038 strain harboring the pGMβl-epsC or pGMβl-epsF was selected by culturing in MRS medium containing erythromycin (final concentration 25 μg / mL) at 40°C. Thereafter, after repeating subculture in MRS medium, the 2038 strain into which the epsC gene of the R-1 strain was introduced (Example 1: designated as "2038-epsC strain") and the 2038 strain into which the epsF gene of the R-1 strain was introduced (Comparative Example 1: designated as "2038-epsF strain") were each obtained.

[0245] (2) Preparation of fermented milk

[0246] Each fermented milk was obtained by inoculating the 2038-epsC strain and the 2038-epsF strain into a 10% skim milk medium prepared from skim milk powder 10% (wt / wt), yeast extract 0.1% (wt / wt) and distilled water to 1% (wt / wt) and fermenting under anaerobic conditions at 37°C overnight. Also, each fermented milk was similarly obtained for the R-1 strain and the 2038 strain. The appearance of the fermented milk obtained using each strain is shown in Figure 3 As shown in Figure 3 In the fermented milk obtained by fermentation with the R-1 strain or the 2038-epsC strain, separation of whey was hardly observed, compared with the fermented milk obtained by fermentation with the 2038 strain or the 2038-epsF strain.

[0247] (3) Coagulation time measurement

[0248] For each fermented milk obtained in (2) of <Stretchability Evaluation 2> above, the adhesion time was measured in the same manner as in (2) of <Stretchability Evaluation 1>, and the stretchability was evaluated. The adhesion time was measured three times for each fermented milk, and the average value was calculated. The results are as follows: Figure 4 As shown. Figure 4 As shown, compared with the average adhesion time of fermented milk obtained from fermentation using strain 2038 or 2038-epsF, the average adhesion time of fermented milk obtained from fermentation using strain 2038-epsC was significantly longer (Tukey-Kramer test, p<0.01), indicating high stringiness. As shown above, it was confirmed that introducing the DNA of the epsC gene from strain R-1 into lactic acid bacteria can improve the stringiness of the resulting fermented milk. Furthermore, it was confirmed that the epsC gene of strain R-1, which achieves high stringiness, differs from the epsC gene of strain 2038, which has poor stringiness, only at amino acid position 40. This indicates that the tyrosine residue at position 40 of the protein encoded by the epsC gene is crucial for improving the stringiness of fermented milk.

[0249] Industrial availability

[0250] As explained above, the present invention can provide novel proteins that improve the stringiness of fermented milk, as well as fermented milk with excellent stringiness and a method for manufacturing the same. More specifically, it can provide novel proteins that improve the stringiness of fermented milk, DNA encoding the aforementioned protein, a vector containing the aforementioned DNA, lactic acid bacteria containing the aforementioned DNA or the aforementioned vector, and compositions of the aforementioned lactic acid bacteria, as well as fermented milk with excellent stringiness using the above-mentioned materials, fermented milk thickeners, and methods for manufacturing the same, methods for improving the stringiness of fermented milk, and methods for evaluating lactic acid bacteria.

[0251] For example, by introducing DNA encoding the novel protein of this invention into various lactic acid bacteria, it is easier than before to produce fermented milk with high stringiness and a thick consistency. Furthermore, by using the sequence of DNA encoding the novel protein of this invention as a selection criterion, it is easier than before to select lactic acid bacteria capable of producing fermented milk with high stringiness and a thick consistency. Moreover, by improving the stringiness of fermented milk with the novel protein of this invention, the amount of free whey can be reduced, and the appearance of the fermented milk can also be improved. sequence list <110> Meiji Co., Ltd. <120> Proteins that improve the stringiness of fermented milk, fermented milk using these proteins, and methods for their production. <130> IBPF21-534WO <150> JP2020-172077 <151> 12-OCT-2020 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 255 <212> PRT <213> Lactobacillus delbrueckii <400> 1 Met Ala Phe Gly Arg Lys Lys His Leu Asn Asn Asp Thr Met Lys Asn 1 5 10 15 Gly Val Lys Leu Ile Thr Leu Ala Asn Pro Gln Ser Val Ile Ser Glu 20 25 30 Gln Phe Arg Asn Ile Arg Thr Tyr Ile Asn Phe Met Asn Val Asp Arg 35 40 45 Glu Val Lys Thr Ile Val Phe Thr Ser Ala Met Ala Ser Ala Gly Lys 50 55 60 Ser Thr Val Ser Ala Asn Val Ala Ile Thr Met Ala Gln Ala Gly Lys 65 70 75 80 Lys Thr Ile Leu Val Asp Ala Asp Leu Arg Arg Pro Thr Met His Ser 85 90 95 Thr Phe Asn Val Ser Asn Ser Asn Gly Leu Thr Thr Leu Leu Thr Ser 100 105 110 Arg Ser Met Glu Met Asp Ala Asn Ser Val Ile Arg Glu Ser Gly Val 115 120 125 Glu Asn Leu Ser lie Leu Thr Ala Gly Pro lie Pro Pro Asn Pro Ser 130 135 140 Glu Leu Leu Ser Ser Lys His Met Leu Asp Leu lie Glu Asp Leu Lys 145 150 155 160 Gln Glu Tyr Asp Met Val Val Leu Asp Leu Ala Pro lie Leu Asp Ala 165 170 175 Gly Glu Thr Gin Gin Leu Thr Ser Ser Leu Asp Gly Thr lie Leu Val 180 185 190 Val Arg Gin Ala His Ser Gin Lys Ser Ala Val Lys Arg Ala Val Glu 195 200 205 Leu Leu Lys Leu Thr Lys Ser Pro lie Leu Gly Tyr Val Met Asn Asp 210 215 220 Val Asp Ala Asp Gly Asp Asp Gly Tyr Gly Tyr Gly Tyr Gly Tyr Gly 225 230 235 240 Tyr Gly Glu Glu Asp Glu Lys Lys Gly Leu Phe Gly Arg Lys Lys 245 250 255 <210> 2 <211> 768 <212> DNA <213> Lactobacillus delbrueckii <400> 2 atggcattcg gtcgaaagaa gcatttaaac aacgatacga tgaagaacgg ggtcaagctg 60 attactttgg ccaacccgca aagcgttatt tcagaacagt tcagaaacat ccgaacctat 120 atcaatttta tgaacgttga tagggaagta aagacgattg tctttacttc agccatggcc 180 agcgccggca agtcgactgt cagtgctaat gtagccatta ccatggcgca agccggcaag 240 aagacgatct tggtcgatgc ggacttgcgc cggccaacta tgcactctac ttttaacgta 300 tctaatagca atggcttgac aactttgttg actagccggt ctatggaaat ggacgcaaat 360 agcgtgatcc gggaaagtgg tgtagagaac ctgtcaatct tgacggcagg tccgattccg 420 ccaaacccat cagaacttct gtcttccaag catatgttgg atttgattga agatttaaag 480 caagaatatg atatggtcgt gcttgactta gcaccgatct tggacgcggg cgaaacccag 540 caactgacca gttctttgga cgggacgatc ttggttgtgc gccaggcaca ttcacagaag 600 tcagcagtta agcgggcagt tgagctgctt aagctgacta agtcaccaat cttgggttat 660 gttatgaacg atgttgatgc cgatggggat gacggctatg gatatggtta tggctacggt 720 TATGGCGAAG AAGATGAGAA GAAGGGGCTC TTTGGGAGAA AGAAGTAG 768 <210> 3 <211> 18 <212> DNA <213> Lactobacillus delbrueckii <400> 3 GGGCTCGCTA TTCTCTGA 18 <210> 4 <211> 5 <212> PRT <213> Lactobacillus delbrueckii <400> 4 GLY LEU ALA ILE LEU 1 5 <210> 5 <211> 21 <212> DNA <213> Lactobacillus delbrueckii <400> 5 GGCTCGCTAT TCTCTGATTG A 21 <210> 6 <211> 6 <212> PRT <213> Lactobacillus delbrueckii <400> 6 GLY SER LEU PHE SER ASP 1 5 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Lb_epsC_rec1_F <400> 7 CAGGAGCTCA GGTGAAATCG CACATATCAC GA 32 <210> 8 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Lb_epsC_rec1_R <400> 8 gacgagctct caactaccgg catgagctac t 31 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Lb_epsF_rec1_F <400> 9 ccggtcgaca caatcggtgt tatcttcc 28 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Lb_epsF_rec1_R <400> 10 cgggtcgacg atcataggct ttagacact 29

Claims

1. A protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

2. A DNA encoding the protein of claim 1.

3. A vector containing the DNA of claim 2.

4. A composition containing at least one selected from the group consisting of the protein of claim 1, the DNA of claim 2, and the vector of claim 3.

5. A lactic acid bacterium into which at least one selected from the group consisting of the DNA of claim 2 and the vector of claim 3 is introduced.

6. A lactic acid bacterium having the DNA according to claim 2, wherein, The lactic acid bacteria is a lactic acid bacteria other than Lactobacillus delbrueckii subsp. bulgaricus (OLL1073R-1). Lactobacillus delbrueckii subsp. bulgaricus ) OLL1073R-1.

7. The lactic acid bacterium according to claim 6, which has a fermentation milk stringiness-improving effect.

8. A lactic acid bacterium composition containing the lactic acid bacterium according to any one of claims 5 to 7.

9. The lactic acid bacterium composition according to claim 8, which is fermented milk.

10. The lactic acid bacterium composition according to claim 8 or 9, which contains exopolysaccharide from the lactic acid bacterium according to any one of claims 5 to 7.

11. A method of manufacturing a fermented milk, comprising: a fermentation step of adding the lactic acid bacterium according to any one of claims 5 to 7, or the lactic acid bacterium composition according to any one of claims 8 to 10 to a prepared milk liquid containing raw milk and allowing it to ferment.

12. A method of improving the stretchability of a fermented milk, comprising: a fermentation step of adding the lactic acid bacterium according to any one of claims 5 to 7, or the lactic acid bacterium composition according to any one of claims 8 to 10 to a prepared milk liquid containing raw milk and allowing it to ferment.

13. A method for evaluating a lactic acid bacterium, which evaluates whether or not it has a fermentation milk stringiness-improving effect, using a DNA encoding a protein of (a) below as an index, (a) a protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

14. A fermented milk containing a lactic acid bacterium evaluated as having an effect of improving the stringiness of fermented milk by the evaluation method for a lactic acid bacterium according to claim 13, wherein, The lactic acid bacteria is a lactic acid bacteria other than Lactobacillus delbrueckii subsp. bulgaricus (OLL1073R-1). Lactobacillus delbrueckii subsp. bulgaricus ) OLL1073R-1.

15. A method for producing a lactic acid bacterium, comprising: an evaluation step of evaluating whether or not a lactic acid bacterium has a fermentation milk stringiness-improving effect, by the method for evaluating a lactic acid bacterium according to claim 13; and a step of obtaining a lactic acid bacterium evaluated as having a fermentation milk stringiness-improving effect in the evaluation step.

16. A method for producing fermented milk, comprising: an evaluation step of evaluating whether or not a lactic acid bacterium has a fermentation milk stringiness-improving effect, by the method for evaluating a lactic acid bacterium according to claim 13; and a fermentation step of adding a lactic acid bacterium evaluated as having a fermentation milk stringiness-improving effect in the evaluation step to a prepared milk liquid containing raw milk and allowing it to ferment.

17. A method for improving the stringiness of fermented milk, comprising: an evaluation step of evaluating whether or not a lactic acid bacterium has a fermentation milk stringiness-improving effect, by the method for evaluating a lactic acid bacterium according to claim 13; and a fermentation step of adding a lactic acid bacterium evaluated as having a fermentation milk stringiness-improving effect in the evaluation step to a prepared milk liquid containing raw milk and allowing it to ferment.

18. A fermented milk thickening agent containing exopolysaccharide from the lactic acid bacterium according to any one of claims 5 to 7 as an effective ingredient. ​ ​ ​ 19. A method for producing exopolysaccharide of lactic acid bacteria, comprising the steps of: adding the lactic acid bacteria according to any one of claims 5 to 7 or the lactic acid bacteria composition according to any one of claims 8 to 10 to a medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, and collecting exopolysaccharide contained in the fermentation product; the sugar having glucose as a constituent sugar is at least one of maltose, sucrose, lactose, galactooligosaccharide, fructooligosaccharide, mannanoligosaccharide, starch, and glycogen.

20. A method for producing exopolysaccharide of lactic acid bacteria, comprising: an evaluation step of evaluating whether or not the lactic acid bacteria have a fermentation milk stringiness-improving effect by the evaluation method for lactic acid bacteria according to claim 13; and a step of adding the lactic acid bacteria evaluated as having a fermentation milk stringiness-improving effect in the evaluation step to a medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, and collecting exopolysaccharide contained in the fermentation product; the sugar having glucose as a constituent sugar is at least one of maltose, sucrose, lactose, galactooligosaccharide, fructooligosaccharide, mannanoligosaccharide, starch, and glycogen.

21. A method for producing a fermented milk thickener, comprising: a fermentation step of adding the lactic acid bacteria according to any one of claims 5 to 7 or the lactic acid bacteria composition according to any one of claims 8 to 10 to a medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, to obtain a fermentation product containing exopolysaccharide; and a step of obtaining a fermented milk thickener containing the exopolysaccharide as an effective ingredient; the sugar having glucose as a constituent sugar is at least one of maltose, sucrose, lactose, galactooligosaccharide, fructooligosaccharide, mannanoligosaccharide, starch, and glycogen.

22. A method for producing a fermented milk thickener, comprising: an evaluation step of evaluating whether or not the lactic acid bacteria have a fermentation milk stringiness-improving effect by the evaluation method for lactic acid bacteria according to claim 13; a fermentation step of adding the lactic acid bacteria evaluated as having a fermentation milk stringiness-improving effect in the evaluation step to a medium containing glucose and / or a sugar having glucose as a constituent sugar, and allowing it to ferment, to obtain a fermentation product containing exopolysaccharide; and a step of obtaining a fermented milk thickener containing the exopolysaccharide as an effective ingredient; the sugar having glucose as a constituent sugar is at least one of maltose, sucrose, lactose, galactooligosaccharide, fructooligosaccharide, mannanoligosaccharide, starch, and glycogen.

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