A protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and fermented milk using the same and a method for producing the same.

Abstract

The present invention is at least one protein selected from the group consisting of proteins (a) to (d) below. (a) A protein comprising the amino acid sequence represented by SEQ ID NO: 1; (b) a protein comprising the amino acid sequence represented by SEQ ID NO: 1, in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 are substituted, deleted, inserted and / or added, the protein also having the effect of improving immunostimulatory activity of lactic acid bacteria exopolysaccharide; (c) a protein comprising an amino acid sequence having at least 80% identity with the amino acid sequence represented by SEQ ID NO: 1, in which the amino acids corresponding to positions 334-338 of the amino acid sequence represented by SEQ ID NO: 1 are, from the N terminus, serine, leucine, phenylalanine, serine, and aspartic acid, the protein also having the effect of improving immunostimulatory activity of lactic acid bacteria exopolysaccharide; and (d) a protein comprising an amino acid sequence encoded by DNA hybridizing, under a strict condition, with the complementary strand of DNA comprising the nucleotide sequence represented by SEQ ID NO: 2, in which the amino acids corresponding to positions 334-338 of the amino acid sequence represented by SEQ ID NO: 1 are, from the N terminus, serine, leucine, phenylalanine, serine, and aspartic acid, the protein also having the effect of improving immunostimulatory activity of lactic acid bacteria exopolysaccharide.

Description

[Technical Field]

[0001] The present invention relates to a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and to fermented milk using the same and a method for producing the same. More specifically, the invention relates to a protein, DNA, vector, lactic acid bacteria, and lactic acid bacteria composition that enhance the immunostimulatory activity of extracellular polysaccharides produced when expressed in lactic acid bacteria, as well as fermented milk using these, an immunostimulant, a method for producing the same, a method for improving the immunostimulatory activity of fermented milk, and a method for evaluating lactic acid bacteria. [Background technology]

[0002] Fermented milk is a widely consumed food, and in Japan's "Ministerial Ordinance Concerning Standards for Ingredients of Milk and Dairy Products (Milk and Dairy Products Ordinance)," it is defined as "milk or milk products containing an equivalent or greater amount of non-fat milk solids fermented with lactic acid bacteria or yeast, resulting in a paste-like or liquid form, or frozen versions thereof." Representative examples of such fermented milk include yogurts such as solid-type yogurt (solid fermented milk), soft-type yogurt (paste-like fermented milk), and drinkable yogurt (liquid fermented milk). In recent years, with the growing health consciousness of consumers, there has been a trend towards demanding a variety of functions from fermented milk.

[0003] For example, in the production of fermented milk products such as yogurt, the mainstream method involves inoculating raw milk with lactic acid bacteria and fermenting it. It is known that certain types of exopolysaccharides (EPS) produced by lactic acid bacteria have immunostimulatory activity, such as activating NK cells. For example, Japanese Patent Publication No. 2005-194259 (Patent Document 1) describes an NK cell activator containing acidic polysaccharides derived from lactic acid bacteria as an active ingredient, and International Publication No. 2011 / 065300 (Patent Document 2) describes an antiviral agent containing neutral polysaccharides produced by lactic acid bacteria such as Lactobacillus delbrueckii ssp. bulgaricus (e.g., strain OLL1073R-1) as an active ingredient. Furthermore, Makino et al., 2013, Japanese Journal of Lactic Acid Bacteria, Vol.24, No.1, pp.10-17 (Non-patent Literature 1), describes the immunostimulatory and infection-protective effects of extracellular polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1.

[0004] However, even in the above-mentioned Lctobacillus delbrueckii ssp. bulgaricus OLL1073R-1 and other lactic acid bacteria, it remains unclear which genes or proteins are involved in the production of extracellular polysaccharides with high immunostimulatory activity. As a result, selecting lactic acid bacteria that produce extracellular polysaccharides with superior immunostimulatory activity has required considerable time and effort. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2005-194259 [Patent Document 2] International Publication No. 2011 / 065300 [Non-patent literature]

[0006] [Non-Patent Document 1] Makino et al., 2013, Japanese Journal of Lactic Acid Bacteria, Vol.24, No.1, pp.10-17. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This invention has been made in view of the problems of the prior art described above, and aims to provide a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, as well as fermented milk containing extracellular polysaccharides having excellent immunostimulatory activity, and a method for producing the same. [Means for solving the problem]

[0008] The inventors of this invention have diligently conducted research to achieve the above objective and have identified a protein and the gene encoding it that enhances the immunostimulatory activity of extracellular polysaccharides in lactic acid bacteria. Specifically, as described above, it was known that Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (accession number: FERM BP-10741) (hereinafter referred to as "strain R-1") produces high levels of extracellular polysaccharides with excellent immunostimulatory activity, but it had not been clarified which gene or protein in strain R-1 enhances the immunostimulatory activity of extracellular polysaccharides. Therefore, in order to elucidate a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides, the inventors first demonstrated that the extracellular polysaccharide produced by strain R-1 has higher immunostimulatory activity than the extracellular polysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus 2038 (hereinafter sometimes referred to as "strain 2038").

[0009] Next, the sequences of the EPS gene clusters were compared between the sequences of strain 2038 and strain R-1. It is known that the production of IFN-γ, which is said to be involved in immunostimulatory activity, is higher in acidic extracellular polysaccharides (APS) than in neutral extracellular polysaccharides (NPS) (Makino et al., 2013, Japanese Journal of Lactic Acid Bacteria, Vol.24, No.1, p.10-17, Figure 3). Comparing the structures of NPS and APS, the composition of constituent sugars is almost identical except for the presence of a small amount of phosphorus only in APS (UEMURA et al., "Chemical characterization of exocellular polysaccharide from Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1", Milchwissenschaft 53(8)1998, p.443-446), suggesting that phosphorus is incorporated into the polysaccharide in APS by glycosylation. Therefore, we focused on genes involved in glycosylation within the EPS gene cluster and compared the sequences of both strains. As a result, we found that the R-1 strain and the 2038 strain showed a difference in nucleotide sequence only in the epsF gene, which is involved in glycosylation. We then found that the protein encoded by the epsF gene of the R-1 strain is a protein that enhances the immunostimulatory activity of extracellular polysaccharides.

[0010] Furthermore, on February 5, 2020, a web blast (parameters: default value) was performed on the NCBI nt database using the nucleotide sequence of the R-1 strain's epsF as a query. The top hit sequence was the epsF gene of strain 2038, with Query Coverage of 100% and Per.Identity of 99.9%. Moreover, the differences in the bases were reflected in the differences in the amino acids. Therefore, the inventors also found that the nucleotide sequence of the R-1 strain's epsF gene and its product, the protein, are novel, thus completing the present invention.

[0011] In other words, the present invention relates to a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, as well as fermented milk using the same and a method for producing the same, and more specifically, the present invention is as follows. [1] At least one protein selected from the group consisting of the proteins listed below (a) to (d). (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 1 (b) A protein having an amino acid sequence in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added, and which has an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. (c) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. (d) A protein comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with the complementary strand of DNA consisting of the nucleotide sequence shown in Sequence ID No. 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. [1'] A composition containing at least one protein selected from the group consisting of the proteins (a) to (d) above (preferably a composition for use in improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria). [2] [1] DNA that codes for the protein described. [2'] A composition (preferably, a composition for use in improving the immunostimulatory activity of exopolysaccharides of lactic acid bacteria) comprising at least one DNA selected from the group consisting of DNAs encoding any one of the proteins (a) to (d) above. [3] A vector comprising the DNA of [2]. [3’] A vector comprising at least one DNA selected from the group consisting of DNAs encoding any one of the proteins (a) to (d) above. [4] A composition comprising at least one selected from the group consisting of the protein of [1], the DNA of [2], and the vector of [3]. [5] 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. [5’] A lactic acid bacterium (preferably, a lactic acid bacterium having high immunostimulatory activity of exopolysaccharides) into which the vector of [3’] has been introduced. [6] A lactic acid bacterium having the DNA of [2]. [7] The lactic acid bacterium of [6], which has high immunostimulatory activity of exopolysaccharides. [8] A lactic acid bacterium composition comprising the lactic acid bacterium according to any one of [5] to [7]. [8’] A lactic acid bacterium composition comprising the lactic acid bacterium of [5’] (preferably, a lactic acid bacterium composition for use in improving the immunostimulatory activity of fermented milk). [9] The lactic acid bacterium composition of [8] or [8’], which is fermented milk.

[10] The lactic acid bacterium composition of [8], [8’] or [9], comprising an exopolysaccharide derived from the lactic acid bacterium according to any one of [5] to [7] or [5’].

[11] A method for producing fermented milk, comprising a fermentation step of adding lactic acid bacteria described in any one of [5] to [7] or [5'] or a lactic acid bacteria composition described in any one of [8] to

[10] or [8'] to a milk preparation liquid containing raw milk and fermenting it to obtain a fermented product containing extracellular polysaccharides.

[12] A method for improving the immunostimulatory activity of fermented milk, comprising a fermentation step of adding lactic acid bacteria described in any one of [5] to [7] or [5'] or a lactic acid bacteria composition described in any one of [8] to

[10] or [8'] to a milk preparation liquid containing raw milk and fermenting it to obtain a fermented product containing extracellular polysaccharides.

[13] A method for evaluating lactic acid bacteria, which assesses the immunostimulatory activity of extracellular polysaccharides using at least one DNA selected from the group consisting of DNA encoding one of the proteins (a) to (d) below as an indicator. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 1 (b) A protein having an amino acid sequence in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added, and which has an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. (c) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. (d) A protein comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with the complementary strand of DNA consisting of the nucleotide sequence shown in Sequence ID No. 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria.

[14]

[13] The evaluation method for lactic acid bacteria described above includes an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A step to obtain lactic acid bacteria in which the extracellular polysaccharide is evaluated to have immunostimulatory activity or to have high immunostimulatory activity in the extracellular polysaccharide in the evaluation step, A method for producing lactic acid bacteria, including

[15]

[13] The evaluation method for lactic acid bacteria described above includes an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a formula containing raw milk, and fermenting the mixture, A method for producing fermented milk, including the following:

[16]

[13] The evaluation method for lactic acid bacteria described above includes an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a formula containing raw milk, and fermenting the mixture, A method for improving the immunostimulatory activity of fermented milk, including [specific ingredient / method].

[17] An immunostimulant containing, as an active ingredient, an extracellular polysaccharide derived from any one of the lactic acid bacteria described in [5] to [7] or [5']. [17'] Use of extracellular polysaccharides derived from lactic acid bacteria described in any one of items [5] to [7] or [5'] for immunostimulation. [17''] Use of extracellular polysaccharides derived from lactic acid bacteria described in any one of [5] to [7] or [5'] for the manufacture of immunostimulants. [17'''] An immunostimulatory method comprising administering extracellular polysaccharides derived from lactic acid bacteria as described in any one of items [5] to [7] or [5'].

[18] A method for producing extracellular polysaccharides of lactic acid bacteria, comprising the step of adding lactic acid bacteria described in any one of [5] to [7] or [5'] or a lactic acid bacteria composition described in any one of [8] to

[10] or [8'] to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, fermenting the mixture, and collecting the extracellular polysaccharides contained in the fermented product.

[19]

[13] The evaluation method for lactic acid bacteria described above includes an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A step of adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a culture medium containing glucose and / or sugars having glucose as a constituent sugar, and fermenting the mixture, and collecting the extracellular polysaccharides contained in the fermented product, A method for producing extracellular polysaccharides of lactic acid bacteria, including those contained within the bacterial cell.

[20] A method for producing an immunostimulant, comprising: a fermentation step of adding lactic acid bacteria described in any one of [5] to [7] or [5'] or a lactic acid bacteria composition described in 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 fermenting the mixture to obtain a fermented product containing extracellular polysaccharides; and a step of obtaining an immunostimulant containing the extracellular polysaccharides as an active ingredient. [twenty one]

[13] The evaluation method for lactic acid bacteria described above includes an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a culture medium containing glucose and / or sugars having glucose as a constituent sugar, and fermenting the mixture, A step to obtain an immunostimulant containing the above-mentioned extracellular polysaccharide as an active ingredient, A method for producing an immunostimulant, including the above. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, as well as fermented milk containing extracellular polysaccharides having excellent immunostimulatory activity and a method for producing the same. More specifically, it is possible to provide a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides produced when expressed in lactic acid bacteria, DNA encoding the protein, a vector containing the DNA, lactic acid bacteria and a lactic acid bacteria composition containing the DNA or the vector, and fermented milk, an immunostimulant, and methods for producing the same using these, a method for improving the immunostimulatory activity of fermented milk, and a method for evaluating lactic acid bacteria.

[0013] For example, by introducing the DNA encoding the novel protein of the present invention into various lactic acid bacteria, it becomes possible to easily produce extracellular polysaccharides with excellent immunostimulatory activity, as well as fermented milk and immunostimulants containing them, using these lactic acid bacteria. Furthermore, by using the DNA sequence encoding the novel protein of the present invention as a selection criterion, it becomes possible to easily select lactic acid bacteria capable of producing extracellular polysaccharides with excellent immunostimulatory activity, as well as fermented milk and immunostimulants containing them. [Brief explanation of the drawing]

[0014] [Figure 1] This graph shows the NK cell activity (NK activity (%)) of extracellular polysaccharides derived from strain 2038 (2038 strain EPS) or strain R-1 (R-1 strain EPS) obtained in the <Immunostimulatory Activity Evaluation>. [Modes for carrying out the invention]

[0015] The present invention will be described in detail below with reference to its preferred embodiments.

[0016] <Proteins, DNA, vectors, and compositions containing them> The protein of the present invention is a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and is one of the following proteins (a) to (d): (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 1, (b) A protein having an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid are substituted, deleted, inserted and / or added in the amino acid sequence shown in Sequence ID No. 1, between positions 334 and 338, (c) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and (d) A protein comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with the complementary strand of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. It is at least one protein selected from the group consisting of the following:

[0017] The protein of the present invention is a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria (hereinafter, may be referred to as "immunostimulatory activity-enhancing protein").

[0018] In the present invention, "exopolysaccharide of lactic acid bacteria" refers to exopolysaccharide (EPS; hereafter referred to as "EPS") produced by lactic acid bacteria, and such EPS includes neutral exopolysaccharide (NPS), acidic exopolysaccharide (APS), zwitterionic exopolysaccharide (ZPS), and mixtures thereof.

[0019] Furthermore, in the present invention, "immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria" refers to the effect of activating immunity in a target when extracellular polysaccharides produced by lactic acid bacteria are administered to that target (hereinafter, sometimes simply referred to as "immunostimulatory activity"), and more preferably, the effect of improving (activating) NK cell activity in the said target. Moreover, in the present invention, "effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria" refers to the effect of conferring or improving the immunostimulatory activity to the extracellular polysaccharides produced by lactic acid bacteria when expressed in said lactic acid bacteria (hereinafter, sometimes referred to as "immunostimulatory activity improving effect"). The reason why the protein of the present invention has the immunostimulatory activity improving effect is not clear, but the inventors speculate that it is because the protein of the present invention acts during the biosynthesis of EPS, particularly during the glycosylation process, to generate EPS with a structure that has high immunostimulatory activity.

[0020] In this invention, the immunostimulatory activity of extracellular polysaccharides produced by lactic acid bacteria can be evaluated, for example, by measuring the NK cell activity when the extracellular polysaccharide is administered to a target. In this invention, the NK cell activity can be measured, for example, by the chromium release method according to the method of Takeda et al. (Takeda, K. et al., J.Immunol., 156:3366, 1996). The more high the NK cell activity obtained when administering the extracellular polysaccharide to a target, the higher and superior the immunostimulatory activity can be evaluated.

[0021] The DNA of the present invention is the DNA encoding the immunostimulatory activity-enhancing protein (hereinafter, as may be referred to, "immunostimulatory activity-enhancing DNA"). That is, the DNA of the present invention is the DNA described in (a') to (d') below: (a') DNA encoding a protein consisting of the amino acid sequence shown in Sequence ID No. 1, (b') DNA encoding a protein having an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 1, between positions 334 and 338. (c') DNA encoding a protein having an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and the amino acids have an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, (d') DNA encoding an amino acid sequence that hybridizes under strict conditions with the complementary strand of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and the DNA encoding a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. It is at least one type of DNA selected from the group consisting of the following:

[0022] "(a) The amino acid sequence shown in Sequence ID No. 1" is the amino acid sequence encoded by the epsF gene of Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (accession number: FERM BP-10741) (R-1 strain). "(a') The DNA encoding the amino acid sequence shown in Sequence ID No. 1" is not particularly limited as long as it encodes the amino acid sequence, but it is preferably the nucleotide sequence shown in Sequence ID No. 2. The nucleotide sequence shown in Sequence ID No. 2 is the nucleotide sequence of the epsF gene of the R-1 strain. As described above, the inventors have found that the protein encoded by the epsF gene of the R-1 strain has the above-mentioned immunostimulatory activity-enhancing effect. In the amino acid sequence shown in Sequence ID No. 1, it is particularly important that the amino acids at positions 334-338 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus. When these amino acids are substituted with other amino acids (for example, strain 2038 in the example), even if other sequences are common, the extracellular polysaccharides produced by the lactic acid bacteria do not exhibit superior immunostimulatory activity. Hereinafter, the amino acid sequence shown in SEQ ID NO: 1 will be referred to as "R1-EpsF" and the nucleotide sequence shown in SEQ ID NO: 2 will be referred to as "R1-epsF".

[0023] Furthermore, in nature, mutations in nucleotide sequences can lead to mutations in the amino acid sequence of the protein encoded by that sequence. Moreover, with the current level of technology, a person skilled in the art could, for example, obtain the nucleotide sequence information of the epsF gene of strain R-1 (R1-epsF) or the amino acid sequence information of the protein encoded by it (R1-EpsF), modify that nucleotide sequence, and prepare an immunostimulatory activity-enhancing protein that has a different amino acid sequence from the encoded one, but maintains or further enhances its immunostimulatory activity.

[0024] Therefore, other embodiments of the "immunostimulatory activity-enhancing protein" according to the present invention also include "(b) a protein comprising an amino acid sequence in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334 to 338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added, and which has an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria." Furthermore, other embodiments of the "immunostimulatory activity-enhancing DNA" according to the present invention also include "(b') DNA encoding a protein comprising an amino acid sequence in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334 to 338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added, and which has an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria." Here, "multiple" refers to the number of amino acid modifications in the modified protein (modified product) after substitution, deletion, insertion, and / or addition (hereinafter, these may be collectively referred to as "modifications"), within the range in which the modified protein has an immunostimulatory activity-enhancing effect. This is usually within 100, 1 to 80, preferably 1 to 40, more preferably 1 to 20, and even more preferably 1 to several (for example, 1 to 10, 1 to 8, 1 to 4, or 1 to 2).

[0025] Those skilled in the art can prepare polynucleotides encoding such variants, for example, using known site-directed mutagenesis methods based on the nucleotide sequence information of the epsF gene of the R-1 strain (R1-epsF).

[0026] Furthermore, with the current level of technology, if a person skilled in the art obtains the nucleotide sequence information (R1-epsF) of the epsF gene of the R-1 strain, it is possible to obtain polynucleotides (homologous genes) encoding immunostimulatory activity-enhancing proteins from other microorganisms besides the R-1 strain using hybridization techniques (Southern, EM, J. Mol. Biol., 98:503, 1975) or polymerase chain reaction (PCR) techniques (Saiki, RK, et al. Science, 230:1350-1354, 1985; Saiki, RK et al. Science, 239:487-491, 1988). Therefore, the embodiments of the "immunostimulatory activity-enhancing protein" according to the present invention also include "(d) a protein comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with a complementary strand of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria." Furthermore, other embodiments of the "immunostimulatory activity-enhancing DNA" according to the present invention also include "(d') DNA comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with a complementary strand of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria."In this invention, "amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1" refers to amino acids that, when aligned with the amino acid sequence shown in Sequence ID No. 1 (R1-EpsF) using nucleotide sequence and amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (for example, with parameters set to default values), are aligned with the amino acid sequence shown in Sequence ID No. 1 (R1-EpsF), and are aligned with positions 334-338 of serine, leucine, phenylalanine, serine, and aspartic acid in R1-EpsF.

[0027] To isolate homologous genes, hybridization reactions are typically performed under strict conditions. "Strict conditions" refer to washing the membrane after hybridization in a low-salt solution at high temperature. For example, this might involve washing in a 2×SSC concentration (1×SSC: 15mM trisodium citrate, 150mM sodium chloride), 0.5% SDS solution at 60°C for 20 minutes. Hybridization can be performed according to the instructions provided with, for example, the publicly known ECL Direct DNA / RNA Labeling and Detection System (manufactured by Amersham Pharmacia Biotech). The stricter the hybridization conditions, the higher the expected isolation of DNA with greater identity. However, the above conditions are examples; stringency can be achieved by appropriately combining DNA concentration, DNA length, hybridization reaction time, etc.

[0028] Furthermore, proteins encoded by homologous genes obtained by such methods typically have high homology to the amino acid sequence (R1-EpsF) shown in Sequence ID No. 1. Therefore, the embodiments of the "immunostimulatory activity-enhancing protein" according to the present invention also include "(c) a protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria." Furthermore, the embodiments of the "immunostimulatory activity-enhancing DNA" according to the present invention also include "(c') DNA comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which encodes a protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria."

[0029] The identity of the amino acid sequence can be determined, for example, using BLAST (for example, parameter: default value (i.e., initial setting value)). Furthermore, the identity with the amino acid sequence (R1-EpsF) described in Sequence ID No. 2 is usually sufficient if it is 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).

[0030] The immunostimulatory activity-enhancing protein encoded by the homologous gene may be a protein encoded by a gene isolated from a microorganism other than lactic acid bacteria, but it is preferable that it be isolated from lactic acid bacteria. Examples of the lactic acid bacteria include the families Streptococcuae, Lactobacillaceae, and Leuconostocaceae, and more specifically, the genera Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Liquorilactobacillus, and Limosilactobacillus. Examples include lactic acid bacilli such as Lactobacillus, Levilactobacillus, Lentilactobacillus, and Weissella; lactic acid cocci such as Pediococcus, Leuconostoc, Lactococcus, Streptococcus, and Enterococcus; and Bifidobacterium. Among these, Lactobacillus is preferred, Lactobacillus delbrueckii (including subspecies) is more preferred, and Lactobacillus delbrueckii subspecies bulgaricus is even more preferred.

[0031] In the present invention, the fact that each protein has the immunostimulatory activity-enhancing effect can be confirmed, for example, by administering extracellular polysaccharides produced using lactic acid bacteria having DNA encoding each protein to mice (preferably BALB / c mice), and the NK cell activity is 1.03 or higher, preferably 1.05 or higher, and more preferably 1.10 or higher, compared to the NK cell activity when administering extracellular polysaccharides produced using lactic acid bacteria that do not have any of the DNA encoding any of the proteins (a) to (d), for example, Lactobacillus delbrueckii subspecies bulgaricus strain 2038 (strain 2038)) to the same mice, with the NK cell activity being set to 1. The method for measuring NK cell activity is as described above. Furthermore, the 2038 strain can be isolated by spreading a diluted solution of Meiji Bulgaria Yogurt LB81 (manufactured by Meiji Co., Ltd.) onto BCP agar medium, culturing at 37°C for 48 hours, and then picking up rough-type colonies.

[0032] Furthermore, if the lactic acid bacteria having DNA encoding each of the aforementioned proteins does not have any DNA encoding any of the proteins (a) to (d), that is, if the lactic acid bacteria (transformed organism) is a lactic acid bacterium that does not have any DNA encoding any of the proteins (a') to (d') (for example, at least one lactic acid bacterium selected from the group consisting of Lactobacillus delbrueckii, preferably Lactobacillus delbrueckii subspecies bulgaricus) into which the aforementioned DNA or a vector containing the aforementioned DNA has been introduced in an expressible manner, then the NK cell activity measured in the same manner as above for the extracellular polysaccharide produced using the lactic acid bacteria may be confirmed by setting the NK cell activity measured in the same manner as above for the extracellular polysaccharide produced using the lactic acid bacteria before transformation to 1.03 or higher, preferably 1.05 or higher, and more preferably 1.10 or higher.

[0033] Furthermore, whether or not each lactic acid bacterium possesses any of the DNAs (a') to (d') above can be appropriately confirmed by known methods or similar methods based on the nucleotide sequences of these DNAs. For example, this can be confirmed by the detection method for immunostimulatory activity-enhancing DNA described in the evaluation step of <Evaluation Method for Lactic Acid Bacteria> below. In addition, the method for introducing the DNA or vector encoding the protein into the lactic acid bacterium can be appropriately selected from known methods or similar methods. For example, in the method described in [Immunostimulatory Activity-Enhancing Protein] below, a method using the lactic acid bacterium as the host cell can be cited.

[0034] [Immune-stimulating activity-enhancing protein] The immunostimulatory activity-enhancing protein according to the present invention can be obtained by known methods or similar methods as appropriate. For example, it can be obtained by a production method that includes the step of culturing host cells into which at least one selected from the group consisting of DNA encoding the immunostimulatory activity-enhancing protein and a vector containing the DNA has been introduced, and collecting the protein expressed in the host cells. More specifically, first, the DNA encoding the immunostimulatory activity-enhancing protein (immunostimulatory activity-enhancing DNA) is obtained as isolated DNA by a conventional method from a target microorganism having at least one of the DNAs (a') to (d') above, such as the R-1 strain. The isolated DNA may be chemically synthesized DNA obtained by artificially chemically synthesizing the immunostimulatory activity-enhancing DNA. Next, the DNA (the isolated DNA) or an expression vector containing the same is prepared, and the transformant into which this is introduced is cultured to express the immunostimulatory activity-enhancing protein of the present invention in the transformant, and the protein can be obtained as a recombinant protein from the culture.

[0035] Methods for obtaining the isolated DNA from the target microorganism include, for example, a method of constructing a DNA library or cDNA library by ligating genomic DNA extracted from the microorganism, or cDNA synthesized based on mRNA extracted from the microorganism, with a vector such as a plasmid vector, phage vector, cosmid vector, BAC vector, or PAC vector, and then isolating the desired genomic DNA or cDNA from the library by hybridization using a probe prepared based on the nucleotide sequence of the immunostimulatory activity-enhancing DNA (e.g., R1-epsF); or a method of performing PCR using the genomic DNA or cDNA of the target microorganism as a template with primers prepared based on the nucleotide sequence of the immunostimulatory activity-enhancing DNA (e.g., R1-epsF), and then isolating the desired genomic DNA by ligating the amplified DNA fragment with an appropriate vector as necessary.

[0036] The expression vector is a vector that is replicable within a host cell and contains a protein encoded by its polynucleotide sequence in a state that allows for expression within the host cell. Such an expression vector can be a self-replicating vector, that is, one that exists as an independent extrachromosome and whose replication does not depend on chromosome replication, and can be constructed based on a plasmid, for example. Alternatively, the expression vector may be constructed based on phage DNA that, when introduced into a host cell, is integrated into the genome of that host cell and replicates together with the chromosome into which it is integrated. Examples of such plasmids include plasmids derived from E. coli (pET22, pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, etc.), plasmids derived from yeast (YEp13, YEp24, YCp50, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), and shuttle vectors between E. coli and Lactobacillus (pGMβ1, etc.). Examples of the phage DNA mentioned above include lambda phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.).

[0037] The procedure and method for constructing the expression vector can be any known method or a similar method as appropriate. For example, to insert the immunostimulatory activity-enhancing DNA into the vector, one can first cleave the isolated DNA with a suitable restriction enzyme, insert it into a restriction enzyme site or multi-cloning site on a suitable plasmid, and then ligate it to the plasmid.

[0038] Preferably, the expression vector contains, in addition to the DNA encoding the immunostimulatory activity-enhancing protein of the present invention (immunostimulatory activity-enhancing DNA), a polynucleotide sequence that controls its expression, a polynucleotide sequence that induces the expression of other polynucleotide sequences, and a genetic marker for selecting cells, in order to actually introduce it into host cells and express the immunostimulatory activity-enhancing protein.

[0039] Examples of polynucleotide sequences that control expression include promoters, terminators, and signal peptides, and may be one of these or a combination of two or more. The promoter is not particularly limited as long as it exhibits transcriptional activity in the host cell, and can be a polynucleotide sequence that controls the expression of a gene encoding a protein of the same or different species as the host cell. Examples of polynucleotide sequences that induce expression include, when the host cell is a bacterium, a lactose operon that can induce the expression of a downstream gene by adding isopropyl-β-D-thiogalactopyranoside (IPTG). The gene marker can be appropriately selected depending on the method of selecting the transformant, and for example, a gene encoding drug resistance or a gene complementing nutritional requirements can be used.

[0040] The host cell is not particularly limited, but is preferably a microorganism, such as filamentous fungi, yeast, Escherichia coli, actinomycetes, and lactic acid bacteria. When used in the method for producing the immunostimulatory activity-enhancing protein of the present invention, the host cell is not particularly limited, but when the host cell into which the DNA has been introduced is used as is in the following <Method for producing fermented milk>, <Method for improving the immunostimulatory activity of fermented milk>, <Method for producing extracellular polysaccharides of lactic acid bacteria>, or <Method for producing an immunostimulant>, it is preferably a lactic acid bacterium. The host cell may, if necessary, be one that has already been transformed or a mutant to lack a specific function.

[0041] Methods for introducing the DNA or expression vector into these host cells can be any known or similar methods, such as the heat shock method, electroporation method, spheroplast method, and lithium acetate method. For introducing into lactic acid bacteria, the conjugation method is also an option. Methods for introducing into plant cells include the Agrobacterium method and the particle gun method. Methods for introducing into insect cells include the baculovirus method and the electroporation method. Methods for introducing into animal cells include the calcium phosphate method, lipofection method, and electroporation method.

[0042] The immunostimulatory activity-enhancing protein of the present invention can be collected from a culture (e.g., cultured microbial cells) obtained by culturing a transformant into which the DNA or expression vector has been introduced in a host cell in a suitable medium. Therefore, the present invention can also provide a method for producing the immunostimulatory activity-enhancing protein of the present invention, which includes the steps of culturing the transformant and collecting the immunostimulatory activity-enhancing protein expressed in the transformant.

[0043] As for the culture conditions of the transformants, for example, the culture conditions of the host cells can be applied, and those skilled in the art can appropriately adjust and set the temperature, whether or not air is added, the oxygen concentration, the carbon dioxide concentration, the pH of the medium, the culture temperature, the culture time, the humidity, etc., according to the type of host cell, the culture medium used, etc. Furthermore, as a method for collecting the immunostimulatory activity-enhancing protein from the culture, for example, the immunostimulatory activity-enhancing protein can be expressed in host cells (e.g., E. coli), and after the culture of the transformants is completed, the cultured cells can be recovered by centrifugation or filtration, and the liquid obtained by disrupting the cells can be used as a crude product. Furthermore, this supernatant can be concentrated by ultrafiltration or the like, and a preservative can be added to obtain a concentrated crude product. In addition, the crude product or the concentrated crude product may be purified by using, for example, salting out, organic solvent precipitation, membrane separation, or chromatographic separation, either individually or in combination of two or more methods. Alternatively, the immunostimulatory activity-enhancing protein, to which a purification tag has been attached, may be expressed in host cells (e.g., E. coli), and the crude extract may be passed through a column for purifying tagged proteins, after which the tagged proteins may be eluted for purification.

[0044] The immunostimulatory activity-enhancing protein of the present invention may have other compounds directly or indirectly added to it. Such additions are not particularly limited and may be at the gene level or chemically. There are also no particular limitations on the site of addition; it may be either the amino terminus (also referred to herein as the "N terminus") or the carboxyl terminus (also referred to herein as the "C terminus") of the immunostimulatory activity-enhancing protein of the present invention, or both. Addition at the gene level is achieved by using DNA encoding the immunostimulatory activity-enhancing protein of the present invention (immunostimulatory activity-enhancing DNA) to which DNA encoding another protein has been read and added in the correct reading frame. There are no particular restrictions on the "other proteins" added in this manner. For example, if the purpose is to facilitate the purification of the immunostimulatory activity-enhancing protein of the present invention, purification tag proteins such as polyhistidine (His-) tag proteins, FLAG- tag proteins (registered trademark, Sigma-Aldrich), and glutathione-S-transferase (GST) are preferably used. For example, if the purpose is to facilitate the detection of the immunostimulatory activity-enhancing protein of the present invention, detection tag proteins such as fluorescent proteins such as GFP and chemiluminescent proteins such as luciferase are preferably used. The chemical addition may be covalent or non-covalent. There are no particular restrictions on "covalent bonds," and examples include amide bonds between an amino group and a carboxyl group, alkylamine bonds between an amino group and an alkyl halide group, disulfide bonds between thiols, and thioether bonds between a thiol group and a maleimide group or alkyl halide group. An example of a "non-covalent bond" is the biotin-avidin bond.

[0045] [DNA that improves immunostimulatory activity] The immunostimulatory activity-enhancing DNA of the present invention may be DNA into which mutations have been introduced into natural DNA, DNA consisting of an artificially designed nucleotide sequence, or may be composed of some or all non-natural nucleotides, as long as it encodes the amino acid sequence of the immunostimulatory activity-enhancing protein of the present invention. Furthermore, there are no particular restrictions on its form, and it includes, for example, cDNA, genomic DNA, and chemically synthesized DNA, which were listed as isolated DNA in the above-mentioned [immunostimulatory activity-enhancing protein].

[0046] Furthermore, the immunostimulatory activity-enhancing DNA of the present invention may also take the form of DNA encoding the immunostimulatory activity-enhancing protein of the present invention, with the codons optimized to suit the type of host cell, in order to further improve the expression efficiency of the immunostimulatory activity-enhancing protein it encodes in the host cell.

[0047] 〔vector〕 The immunostimulatory activity-enhancing DNA of the present invention may also take the form of a vector into which the DNA is inserted so that it can be replicated within host cells. Therefore, the present invention also provides a vector containing the immunostimulatory activity-enhancing DNA of the present invention. Examples of vectors of the present invention include the expression vectors listed above in the section on [Immunostimulatory Activity-Enhancing Proteins], including their preferred embodiments.

[0048] [Composition] The present invention provides a composition comprising at least one of the immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and vector described above. The composition of the present invention can be used to improve the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and contains at least one of the immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and vector described above as an active ingredient. For example, by introducing the composition of the present invention into various lactic acid bacteria to obtain the lactic acid bacteria of the present invention described below, and using these to produce extracellular polysaccharides or fermented milk, it becomes possible to obtain extracellular polysaccharides or fermented milk containing them that have superior immunostimulatory activity compared to conventional methods.

[0049] The composition of the present invention may further contain other components. These other components are not particularly limited, but include, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizers, buffers, DNase inhibitors, and preservatives, and may contain only one of these or a combination of two or more.

[0050] <Lactic acid bacteria and lactic acid bacteria compositions> The present invention also provides a transformant obtained by introducing the immunostimulatory activity-enhancing DNA of the present invention, or a vector of the present invention containing the immunostimulatory activity-enhancing DNA, into a host cell. Examples of such transformants include those listed in the above section on [Immunostimulatory Activity-Enhancing Proteins].

[0051] In the present invention, the host cell of the transformant is preferably a lactic acid bacterium, and "the lactic acid bacterium of the present invention" includes lactic acid bacteria into which at least one selected from the group consisting of the immunostimulatory activity-enhancing DNA of the present invention and the vector of the present invention containing the immunostimulatory activity-enhancing DNA has been introduced; and lactic acid bacteria having the immunostimulatory activity-enhancing DNA of the present invention. Furthermore, "the lactic acid bacterium of the present invention" also includes lactic acid bacteria into which the immunostimulatory activity-enhancing protein of the present invention itself has been introduced. As a result, the extracellular polysaccharides produced by the lactic acid bacterium of the present invention can exert immunostimulatory activity.

[0052] Furthermore, the lactic acid bacteria of the present invention may also be in the form of a lactic acid bacteria composition, and the present invention also provides a lactic acid bacteria composition containing at least one of these lactic acid bacteria of the present invention. The lactic acid bacteria composition can be used for the production of the immunostimulatory activity-enhancing protein, as well as for the production of fermented milk, the enhancement of the immunostimulatory activity of fermented milk, the production of extracellular polysaccharides with enhanced immunostimulatory activity, or the production of an immunostimulant.

[0053] [Lactic acid bacteria] The host cells into which the immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, or vector of the present invention are introduced are not particularly limited, but examples include the families Streptococcuaceae, Lactobacillaceae, and Leuconostocaceae, and more specifically, the genera Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, and Liquorilactobacillus. Examples include lactic acid bacilli such as *Lactobacillus tobacillus*, *Limosilactobacillus*, *Levilactobacillus*, *Lentilactobacillus*, and *Weissella*; lactic acid cocci such as *Pediococcus*, *Leuconostoc*, *Lactococcus*, *Streptococcus*, and *Enterococcus*; and *Bifidobacterium*. Among these, *Lactobacillus* is preferred, *Lactobacillus delbrueckii* (including subspecies) is more preferred, and *Lactobacillus delbrueckii subspecies bulgaricus* is even more preferred. The lactic acid bacteria used as the host cell may already possess at least one of the proteins (a) to (d) or at least one of the DNAs (a') to (d'). When such lactic acid bacteria are used as the host cell, further enhancement of immunostimulatory activity can be expected.

[0054] As a method for introducing the immunostimulatory activity-enhancing protein, the immunostimulatory activity-enhancing DNA, or the vector into these lactic acid bacteria, the methods listed above as methods for introducing DNA or expression vectors in the section on [immunostimulatory activity-enhancing protein] can be appropriately employed. For example, it is preferable to introduce the immunostimulatory activity-enhancing DNA or the vector using at least one selected from the group consisting of the heat shock method, electroporation method, spheroplast method, lithium acetate method, and conjugation method.

[0055] Furthermore, as a lactic acid bacterium of the present invention, examples of lactic acid bacteria having the immunostimulatory activity-enhancing DNA of the present invention include, among the lactic acid bacteria listed as the host cell, lactic acid bacteria having at least one of the DNAs (a') to (d') above.

[0056] Furthermore, whether the lactic acid bacteria of the present invention possess (or have introduced) the immunostimulatory activity-enhancing protein or immunostimulatory activity-enhancing DNA of the present invention can be appropriately confirmed by known methods or similar methods, for example, by the detection method for immunostimulatory activity-enhancing DNA described in the evaluation step of <Evaluation Method for Lactic Acid Bacteria> below. Therefore, "lactic acid bacteria of the present invention" also includes lactic acid bacteria whose extracellular polysaccharides have immunostimulatory activity or are evaluated to have high immunostimulatory activity (including those evaluated to have immunostimulatory activity or a high probability of high immunostimulatory activity) according to the evaluation method for lactic acid bacteria of the present invention below, as well as lactic acid bacteria obtained by the production method of lactic acid bacteria of the present invention.

[0057] The DNA possessed by (including introduced into) the lactic acid bacteria of the present invention may be retained within the genomic DNA of the lactic acid bacteria, or, if it is a vector, it may be replicated and retained as an independent entity outside of the genomic DNA. The DNA introduced into the lactic acid bacteria may be retained by random insertion into the genomic DNA, or by homologous recombination. Furthermore, the lactic acid bacteria of the present invention may be an artificial mutant, a natural mutant, or a genetically modified strain of the same strain or its subcultivated strain of the above-mentioned lactic acid bacteria, within the range that has an immunostimulatory activity-enhancing effect.

[0058] The lactic acid bacteria of the present invention preferably produce extracellular polysaccharides with enhanced immunostimulatory activity. In the present invention, the production of extracellular polysaccharides with enhanced immunostimulatory activity by lactic acid bacteria into which the immunostimulatory activity-enhancing protein, the immunostimulatory activity-enhancing DNA, or the vector has been introduced can be confirmed, for example, by measuring the NK cell activity of the extracellular polysaccharides produced using the lactic acid bacteria in the same manner as the method used to confirm that the above-mentioned protein has an immunostimulatory activity-enhancing effect, with the NK cell activity measured for extracellular polysaccharides produced using lactic acid bacteria that do not have any of the above-mentioned DNAs (a') to (d') set to 1, and the NK cell activity being 1.03 or higher, preferably 1.05 or higher, and more preferably 1.10 or higher. Alternatively, for example, the NK cell activity can be confirmed by the NK cell activity being 21% or higher, preferably 22% or higher, and even more preferably 22.5% or higher.

[0059] [Lactic acid bacteria composition] The lactic acid bacteria composition of the present invention is a composition containing the lactic acid bacteria of the present invention described above. The lactic acid bacteria composition of the present invention may further contain other components, and these other components are not particularly limited, but include, for example, a culture product such as the culture supernatant or culture medium components after the completion of culturing the lactic acid bacteria; a concentrate, crude product, purified product, diluted product, dried product (spray dried product, freeze-dried product, etc.), or frozen product of the culture product; a protective agent, a fermentation accelerator, etc., and may contain only one of these or a combination of two or more.

[0060] Furthermore, the lactic acid bacteria composition of the present invention includes any composition containing the lactic acid bacteria of the present invention (i.e., lactic acid bacteria into which at least one selected from the group consisting of an immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and a vector of the present invention containing immunostimulatory activity-enhancing DNA has been introduced; lactic acid bacteria having immunostimulatory activity-enhancing DNA; lactic acid bacteria whose extracellular polysaccharides have immunostimulatory activity or whose extracellular polysaccharides have high immunostimulatory activity according to the evaluation method of lactic acid bacteria of the present invention (including those evaluated as having immunostimulatory activity or being highly likely to have high immunostimulatory activity); lactic acid bacteria obtained by the method for producing lactic acid bacteria of the present invention), and the fermented milk of the present invention described below is also included.

[0061] <Methods for evaluating lactic acid bacteria, methods for producing lactic acid bacteria> The method for evaluating lactic acid bacteria according to the present invention is the immunostimulatory activity-enhancing protein of the present invention, i.e., the proteins (a) to (d) below: (a) A protein consisting of the amino acid sequence shown in Sequence ID No. 1, (b) A protein having an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, in which one or more amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid are substituted, deleted, inserted and / or added in the amino acid sequence shown in Sequence ID No. 1, between positions 334 and 338, (c) A protein having an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has an effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, and (d) A protein comprising an amino acid sequence encoded by DNA that hybridizes under strict conditions with the complementary strand of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in SEQ ID NO: 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. This method evaluates the immunostimulatory activity of extracellular polysaccharides produced by lactic acid bacteria, using at least one DNA selected from the group consisting of DNA encoding any of the following (i.e., the immunostimulatory activity-enhancing DNA of the present invention) as an indicator.

[0062] [Evaluation Process] In the method for evaluating lactic acid bacteria of the present invention, the immunostimulatory activity-enhancing DNA of the present invention is used as an indicator, that is, whether or not the lactic acid bacteria have the immunostimulatory activity-enhancing DNA of the present invention is used as an indicator to evaluate whether or not the extracellular polysaccharides produced by the lactic acid bacteria have immunostimulatory activity, or whether or not the extracellular polysaccharides have high immunostimulatory activity (evaluation step). If the lactic acid bacteria have the immunostimulatory activity-enhancing DNA, the extracellular polysaccharides produced by the lactic acid bacteria are evaluated to have immunostimulatory activity or to have high immunostimulatory activity (including evaluation that there is a high probability that the extracellular polysaccharides have immunostimulatory activity, or to have high immunostimulatory activity). On the other hand, if the lactic acid bacteria do not have the DNA, the extracellular polysaccharides produced by the lactic acid bacteria are evaluated to not have immunostimulatory activity or to have low immunostimulatory activity (including evaluation that there is a high probability that the extracellular polysaccharides do not have immunostimulatory activity, or to have low immunostimulatory activity). This makes it possible to select lactic acid bacteria that produce, or are highly likely to produce, extracellular polysaccharides that have immunostimulatory activity or high immunostimulatory activity. In the method for evaluating lactic acid bacteria of the present invention, there are no particular limitations on the lactic acid bacteria to be evaluated, and any desired lactic acid bacteria can be used as appropriate.

[0063] Whether or not lactic acid bacteria possess the immunostimulatory activity-enhancing DNA of the present invention can be determined by detecting the DNA. A known method or a similar method can be appropriately used for detecting the immunostimulatory activity-enhancing DNA.

[0064] For example, first, genomic DNA is extracted from the lactic acid bacteria to be evaluated. There are no particular restrictions on the method of extracting genomic DNA; known methods or similar methods can be used as appropriate. Examples include the PCI method, GuSCN / Silica method, SDS-phenol method, CTAB method, and alkaline treatment method. Commercially available kits can also be used as appropriate.

[0065] The method for detecting the immunostimulatory activity-enhancing DNA can then be carried out by isolating the DNA corresponding to the immunostimulatory activity-enhancing DNA and determining the nucleotide sequence of the isolated DNA. The isolation of the DNA can be performed, for example, by PCR using genomic DNA as a template, using a pair of oligonucleotide primers designed to sandwich at least the DNA corresponding to the immunostimulatory activity-enhancing DNA. The nucleotide sequence of the isolated DNA can be determined by methods known to those skilled in the art, such as the Sanger method and the Maxam-Gilbert method. Alternatively, the nucleotide sequence of the DNA corresponding to the immunostimulatory activity-enhancing DNA may be determined directly from the genomic DNA using a next-generation sequencer or the like.

[0066] The DNA corresponding to the immunostimulatory activity-enhancing DNA preferably includes a region encoding amino acids corresponding to positions 334-338 of R1-EpsF, and the pair of oligonucleotide primers flanking this region can be designed based on the nucleotide sequence of the immunostimulatory activity-enhancing DNA (e.g., R1-epsF) and a public database (e.g., Genbank). Such oligonucleotides can be designed by known methods or similar methods by those skilled in the art.

[0067] Another method for detecting the immunostimulatory activity-enhancing DNA is, for example, the PCR-SSP (PCR-sequence-specific primer) method. In this method, the 3' end of one of the pair of oligonucleotides constituting the primer is designed to be a base species complementary to a specific base of the immunostimulatory activity-enhancing DNA. For example, if the DNA to be detected is (a'), the base species is complementary to the sites encoding serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 of R1-EpsF. PCR using this pair of oligonucleotide primers is amplified only when the immunostimulatory activity-enhancing DNA of the present invention is used as a template, and not when genomic DNA is used as a template in which any of the sites encoding serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 encodes another amino acid. Therefore, the presence or absence of such amplification can be used as an indicator to detect the DNA.

[0068] Furthermore, as an alternative method for detecting the immunostimulatory activity-enhancing DNA, if restriction fragment length polymorphisms (RFLPs) can be established at positions 334-338 of R1-EpsF or at the corresponding sites encoding serine, leucine, phenylalanine, serine, and aspartic acid, these RFLP markers can be used as indicators for detection, for example, by PCR-RFLP (or CAPS [Cleaved Amplified Polymorphic Sequence]) method.

[0069] Another method for detecting the immunostimulatory activity-enhancing DNA is, for example, the PCR-SSCP (PCR-Single-Stranded Higher-Order Structure Polymorphism) method. Double-stranded DNA amplified by PCR using a pair of oligonucleotide primers designed to sandwich the immunostimulatory activity-enhancing DNA is denatured by treatment with heat or alkali to obtain single-stranded DNA. When subjected to polyacrylamide gel electrophoresis without a denaturing agent, the single-stranded DNA folds in the gel due to intramolecular interactions, forming a higher-order structure. Since the interactions of the folded structure change depending on the difference in base types, the separated single-stranded DNA can be detected by silver staining or radioisotope detection, and the immunostimulatory activity-enhancing DNA can be detected using the mobility of the single-stranded DNA on the gel as an indicator.

[0070] Another method for detecting the immunostimulatory activity-enhancing DNA is, for example, a method using an intercalator. In this method, first, in a reaction system including an intercalator that emits fluorescence when inserted between DNA double helixes, the DNA corresponding to the immunostimulatory activity-enhancing DNA is amplified using the genomic DNA as a template. Then, the temperature of the reaction system is changed, and the change in the intensity of fluorescence emitted by the intercalator is detected. Using the detected change in fluorescence intensity with respect to the temperature change as an indicator, the immunostimulatory activity-enhancing DNA (particularly positions 334-338 of R1-EpsF or the corresponding sites encoding serine, leucine, phenylalanine, serine, and aspartic acid) can be detected. An example of such a method is high-resolution melting curve analysis (HRM).

[0071] Another method for detecting the immunostimulatory activity-enhancing DNA is, for example, using an oligonucleotide probe that hybridizes to the region of R1-EpsF containing the serine, leucine, phenylalanine, serine, and aspartic acid-coding sites at positions 334-338 when the DNA to be detected is (a') above. In one embodiment of this method, first, an oligonucleotide probe is prepared that specifically hybridizes to the serine, leucine, phenylalanine, serine, and aspartic acid-coding sites at positions 334-338 and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Next, this oligonucleotide probe is hybridized to the genomic DNA, and then the DNA containing the serine, leucine, phenylalanine, serine, and aspartic acid-coding sites at positions 334-338 is amplified using the DNA sample hybridized with the oligonucleotide probe as a template. Then, the fluorescence emitted by the reporter fluorescent dye, whose inhibition by the quencher has been released due to the degradation of the oligonucleotide probe accompanying the amplification, is detected. Such methods include the double-dye probe method, also known as the TaqMan® probe method. Another embodiment using oligonucleotide probes labeled with a reporter fluorescent dye and a quencher fluorescent dye is the cycling probe method, which uses a combination of a chimeric oligonucleotide (a chimera of RNA and DNA) that specifically hybridizes with the immunostimulatory activity-enhancing DNA and an enzyme such as RNase H.

[0072] Another method for detecting the immunostimulatory activity-enhancing DNA is, for example, the LAMP (Loop-Mediated Isothermal Amplification) method. In this method, a total of six regions are set, three on each side of the target site of double-stranded DNA, and these regions are reacted using four types of primers (two on each side) in the presence of a strand-displacing enzyme. This generates loop-structure amplification starting points on both sides of the target site, thereby generating a repeating structure of complementary sequences on the same strand and amplifying the target site. If the DNA to be detected is (a') above, and the target site is the R1-EpsF region at positions 334-338 encoding serine, leucine, phenylalanine, serine, and aspartic acid, the presence or absence of each modification can be detected by determining the nucleotide sequence of the amplified product. Furthermore, if one of the six regions is the site encoding serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338, the target site will not be amplified if there is a modification, and the presence or absence of such amplification can be used as an indicator to detect the DNA.

[0073] The method for detecting the immunostimulatory activity-enhancing DNA is not limited to the above embodiments. For example, other known techniques such as denaturing agent concentration gradient gel electrophoresis (DGGE), Invader method, pyrosequencing, single nucleotide primer extension (SNuPE), allele-specific oligonucleotide (ASO) hybridization, ribonuclease A mismatch cleavage, DNA microarray, and DNA array may also be used in the present invention.

[0074] Furthermore, it is preferable that the detection of the immunostimulatory activity-enhancing DNA involves detecting its expression. As a method for detecting the expression of the stringiness-enhancing DNA, for example, mRNA or protein may be extracted from the target lactic acid bacteria according to a conventional method, and the mRNA or protein encoded by the immunostimulatory activity-enhancing DNA (i.e., immunostimulatory activity-enhancing protein) may be detected by a known method or a similar method.

[0075] Methods for detecting the mRNA encoded by the aforementioned immunostimulatory activity-enhancing DNA include, for example, RT-PCR and Northern blotting.

[0076] As a method for detecting the protein encoded by the aforementioned immunostimulatory activity-enhancing DNA (immunostimulatory activity-enhancing protein), first, a protein sample is prepared from the target lactic acid bacteria, and an antigen-antibody reaction is performed using an antibody specific to the immunostimulatory activity-enhancing protein, that is, an antibody specific to serine, leucine, phenylalanine, serine, and aspartic acid at at least positions 334-338, to detect the immunostimulatory activity-enhancing protein. In such a protein detection method using antibodies, for example, an antibody specific to the immunostimulatory activity-enhancing protein is added to the protein sample to perform an antigen-antibody reaction, and the binding of the antibody to the immunostimulatory activity-enhancing protein is detected. If the antibody specific to the immunostimulatory activity-enhancing protein is labeled, the immunostimulatory activity-enhancing protein can be detected directly. However, if it is not labeled, a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A) is further applied, and the immunostimulatory activity-enhancing protein can be detected indirectly by utilizing the labeling of the molecule. Such methods include, for example, immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and antibody array analysis. The antibody may be either a polyclonal antibody or a monoclonal antibody, and the methods for preparing these antibodies are well known to those skilled in the art.

[0077] [Kit for use in the evaluation method of the present invention] As described above, the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria can be evaluated by detecting the immunostimulatory activity-enhancing DNA of the present invention. Therefore, the present invention provides the following (i) to (ii) agents for use in the evaluation method described above: (i) A drug comprising an oligonucleotide having a chain length of at least 15 nucleotides that hybridizes to the immunostimulatory activity-enhancing DNA of the present invention, its transcript, or its complementary nucleotides, and (ii) A drug comprising an antibody that binds to the immunostimulatory activity-enhancing protein of the present invention, A kit containing at least one drug selected from the group consisting of the above is also provided. The oligonucleotide may be in the form of a primer or a probe, depending on the method for detecting the immunostimulatory activity-enhancing DNA described above.

[0078] The primers are not particularly limited as long as they hybridize to the immunostimulatory activity-enhancing DNA of the present invention or DNA corresponding to the immunostimulatory activity-enhancing DNA, or complementary nucleotides (including cDNA and cRNA), or the transcript (mRNA) of the immunostimulatory activity-enhancing DNA, enabling amplification and detection of these. The primers may consist of DNA alone, or they may be partially or entirely replaced by artificial nucleic acids (modified nucleic acids) such as cross-linked nucleic acids. The size of the primers should be at least about 15 nucleotides in length, preferably 15 to 100 nucleotides, more preferably 18 to 50 nucleotides, and even more preferably 20 to 40 nucleotides. Such primers can be designed and prepared by methods known to those skilled in the art in accordance with the above detection method.

[0079] The probe is not particularly limited as long as it hybridizes to immunostimulatory activity-enhancing DNA, DNA corresponding to immunostimulatory activity-enhancing DNA, its complementary nucleotides, or the transcript of immunostimulatory activity-enhancing DNA, enabling their detection. The probe may be DNA, RNA, artificial nucleic acids, or chimeric molecules thereof. The probe may be single-stranded or double-stranded. The size of the probe should be at least about 15 nucleotides in length, preferably 15 to 1000 nucleotides, more preferably 20 to 500 nucleotides, and even more preferably 30 to 300 nucleotides. Such probes can be designed and fabricated by methods known to those skilled in the art. The probe may also be provided in a form fixed on a substrate, such as a microarray.

[0080] The antibody is not particularly limited as long as it can specifically bind to the immunostimulatory activity-enhancing protein of the present invention. For example, it may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody (Fab, Fab', scFv, etc.). Such antibodies can be produced by methods known to those skilled in the art. The antibody may also be provided in a form immobilized on a substrate such as a plate for use in ELISA or antibody arrays.

[0081] Furthermore, the oligonucleotides or antibodies included in the kit may be labeled with a labeling substance in accordance with the detection method described above. Examples of such labeling substances include fluorescent substances such as FITC, FAM, DEAC, R6G, TexRed, and Cy5, and enzymes such as β-D-glucosidase, luciferase, and HRP. 3 H, 14 C, 32 P, 35 S, 123 Examples include radioactive isotopes such as I, affinity substances such as biotin and streptavidin, and luminescent substances such as luminol, luciferin, and lucigenin.

[0082] The method for evaluating lactic acid bacteria of the present invention may further include a confirmation step to confirm whether the extracellular polysaccharides of the lactic acid bacteria have immunostimulatory activity or have high immunostimulatory activity. There are no particular limitations on such confirmation methods, but for example, if the NK cell activity of the extracellular polysaccharides produced using the lactic acid bacteria to be evaluated is measured in the same manner as the method for confirming that the above-mentioned protein has an immunostimulatory activity-enhancing effect, then if it is 21% or higher, the extracellular polysaccharide can be evaluated as having immunostimulatory activity, preferably 22% or higher, and more preferably 22.5% or higher, then the extracellular polysaccharide can be evaluated as having high immunostimulatory activity.

[0083] [Method for producing lactic acid bacteria] The present invention provides a method for producing lactic acid bacteria, comprising an evaluation step of evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, as described above in the method for evaluating lactic acid bacteria of the present invention, A step to obtain lactic acid bacteria in which the extracellular polysaccharide is evaluated to have immunostimulatory activity or to have high immunostimulatory activity in the extracellular polysaccharide in the evaluation step, This method includes [something].

[0084] In the method for producing lactic acid bacteria of the present invention, the evaluation step is the evaluation step described in the <method for evaluating lactic acid bacteria> above. In the evaluation step of the method for producing lactic acid bacteria of the present invention, lactic acid bacteria that produce or are highly likely to produce extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity are selected. In the method for producing lactic acid bacteria of the present invention, lactic acid bacteria whose extracellular polysaccharides have immunostimulatory activity or are highly evaluated to have high immunostimulatory activity (including those evaluated to have immunostimulatory activity or be highly likely to have high immunostimulatory activity) can be obtained by the evaluation step, but for example, the lactic acid bacteria may be obtained as a culture by culturing the selected lactic acid bacteria in a suitable medium.

[0085] Furthermore, the lactic acid bacteria obtained by the method for producing lactic acid bacteria of the present invention may also be in the form of a lactic acid bacteria composition such as a culture thereof. Therefore, the method for producing lactic acid bacteria of the present invention also includes a method for producing a lactic acid bacteria composition that includes a step of obtaining a lactic acid bacteria composition containing lactic acid bacteria whose extracellular polysaccharides are evaluated in the evaluation step to have immunostimulatory activity or to have high immunostimulatory activity of extracellular polysaccharides. Other components that may be contained in the lactic acid bacteria composition other than the lactic acid bacteria are as described above.

[0086] <Method for producing fermented milk> The present invention provides a method for producing fermented milk, which includes a fermentation step in which lactic acid bacteria or a lactic acid bacteria composition are added to a milk preparation liquid containing raw milk and fermented to obtain a fermented product containing extracellular polysaccharides.

[0087] The lactic acid bacteria used in the method for producing fermented milk of the present invention include the lactic acid bacteria of the present invention described above (i.e., lactic acid bacteria into which at least one selected from the group consisting of an immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and a vector of the present invention containing immunostimulatory activity-enhancing DNA has been introduced; lactic acid bacteria having immunostimulatory activity-enhancing DNA; lactic acid bacteria whose extracellular polysaccharides have immunostimulatory activity or high immunostimulatory activity according to the evaluation method of lactic acid bacteria of the present invention (including those evaluated as having immunostimulatory activity or a high probability of having high immunostimulatory activity); and lactic acid bacteria obtained by the method for producing lactic acid bacteria of the present invention). One of these may be used alone or in combination of two or more. Furthermore, the lactic acid bacteria composition used in the method for producing fermented milk of the present invention includes the lactic acid bacteria composition described above; and the lactic acid bacteria composition obtained by the method for producing lactic acid bacteria of the present invention. One of these may be used alone or in combination of two or more. By using these lactic acid bacteria or lactic acid bacteria compositions, fermented milk containing extracellular polysaccharides that have immunostimulatory activity or high immunostimulatory activity can be obtained. Furthermore, when using lactic acid bacteria that have been evaluated to have immunostimulatory activity or high immunostimulatory activity of extracellular polysaccharides according to the lactic acid bacteria evaluation method of the present invention, the method for producing fermented milk of the present invention may include the evaluation step, but in this case, the evaluation step only needs to be performed once.

[0088] In the method for producing fermented milk according to the present invention, other lactic acid bacteria besides the lactic acid bacteria of the present invention may be used in combination. Yeast may also be added. Examples of other lactic acid bacteria and yeast include lactic acid bacteria and yeast that have been conventionally known to be included in fermented milk.

[0089] (Formula formula) The milk preparation liquid according to the present invention contains raw milk. The raw milk preferably contains lactose, and examples include raw milk (e.g., milk from cows, water buffalo, sheep, goats, 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, buttermilk, butter, cream, cheese, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), β-lactoglobulin (β-Lg)), and may be one of these or a mixture of two or more.

[0090] The formula milk preparation according to the present invention may consist solely of the raw milk, or it may be an aqueous solution, dilution, or concentrate of the raw milk, and may further contain other components as needed in addition to the raw milk. Such other components include water; soy milk, sugars and sweeteners including sucrose, flavorings, fruit juice, fruit pulp, vitamins, minerals, oils and fats, ceramides, collagen, milk phospholipids, yeast extract, polyphenols, and other foods, food components, and food additives; and stabilizers, thickeners, and gelling agents such as pectin, soybean polysaccharides, CMC (carboxymethylcellulose), agar, gelatin, carrageenan, and gums, and may be one of these or a mixture of two or more. The formula milk preparation can be prepared by mixing the components while heating as needed and / or homogenizing as needed. Furthermore, the formula milk preparation may be heat-sterilized.

[0091] (fermentation) As the fermentation step of adding the lactic acid bacterium or the lactic acid bacteria composition to the prepared emulsion and fermenting the same to obtain a fermented product, a known method or a method equivalent thereto can be appropriately employed, and is not particularly limited. Examples thereof include a method of inoculating the prepared emulsion with the lactic acid bacterium or the lactic acid bacteria composition as a fermentation starter and performing fermentation. As the lactic acid bacterium or the lactic acid bacteria composition, it is preferable that the lactic acid bacterium or the lactic acid bacteria composition is added to the prepared emulsion in the form of the lactic acid bacteria composition, more preferably in the form of a culture or a concentrate of a culture.

[0092] The addition amount of the fermentation starter can be appropriately set according to the addition amount employed in known methods for producing fermented milk. For example, in terms of the number of lactic acid bacteria (the total number of bacteria in the case of a combination of two or more types) converted based on the volume of the prepared emulsion, 1×10 7 ~5×10 9 CFU / mL is preferable, and 1×10 8 ~2×10 9 CFU / mL is more preferable. Further, relative to the volume of the prepared emulsion, 0.1 to 2% (wt / wt) is also preferable, 0.5 to 1.5% (wt / wt) is more preferable, and 0.5 to 1% (wt / wt) is even more preferable.

[0093] The method for inoculating the fermentation starter is not particularly limited, and a method conventionally used in methods for producing fermented milk can be appropriately used. The fermentation conditions can be appropriately selected according to the growth conditions of the added lactic acid bacteria, the amount of the prepared emulsion, and the like, and are not particularly limited. For example, at a temperature of 35 to 45°C, more preferably 38 to 43°C, under aerobic or anaerobic conditions, it is preferable to allow standing or stirring (preferably standing) usually for 3 to 24 hours, more preferably 3 to 8 hours, still more preferably 4 to 6 hours, until the pH of the prepared emulsion added with the lactic acid bacterium or the lactic acid bacteria composition becomes 4.8 or less, more preferably 4.0 to 4.6. Further, as the anaerobic condition, for example, fermentation under nitrogen aeration conditions can be employed.

[0094] The fermented milk of the present invention can be obtained by the above fermentation. The fermented product after the fermentation process (i.e., the prepared milk liquid after the fermentation process, and the lactic acid bacteria or lactic acid bacteria composition) can be used as is, or concentrated, diluted, dried, or frozen as necessary to obtain the fermented milk of the present invention. Alternatively, the lactic acid bacteria in the fermented product may be crushed or heat-treated, or concentrated, diluted, dried, or frozen as necessary to obtain the fermented milk of the present invention.

[0095] <Fermented milk> The fermented milk of the present invention is provided, which contains at least one lactic acid bacterium selected from the group consisting of the lactic acid bacteria of the present invention (i.e., lactic acid bacteria into which at least one selected from the group consisting of an immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and a vector of the present invention containing immunostimulatory activity-enhancing DNA has been introduced; lactic acid bacteria having immunostimulatory activity-enhancing DNA; lactic acid bacteria whose extracellular polysaccharides have immunostimulatory activity or whose extracellular polysaccharides have high immunostimulatory activity according to the evaluation method of lactic acid bacteria of the present invention (including those evaluated to have immunostimulatory activity or to have a high probability of having high immunostimulatory activity); lactic acid bacteria obtained by the production method of lactic acid bacteria of the present invention). The fermented milk of the present invention preferably contains immunostimulatory activity-enhancing proteins derived from these lactic acid bacteria, and more preferably contains extracellular polysaccharides derived from these lactic acid bacteria. Furthermore, the fermented milk of the present invention may further contain other lactic acid bacteria and yeast.

[0096] The fermented milk of the present invention is not particularly limited and may be any of the following: fermented milk that meets the standards for "fermented milk" under the Ministerial Ordinance on Standards for Ingredients of Milk and Dairy Products of the Ministry of Health, Labour and Welfare of Japan (more specifically, fermented milk with a non-fat milk solids content of 8.0% or more and a lactic acid bacteria or yeast count (preferably lactic acid bacteria count) of 10 million / mL or more); fermented milk that meets the standards for "dairy product lactic acid bacteria beverage" (more specifically, fermented milk with a non-fat milk solids content of 3.0% or less and a lactic acid bacteria or yeast count (preferably lactic acid bacteria count) of 10 million / mL or more); or fermented milk that meets the standards for "lactic acid bacteria beverage" (more specifically, fermented milk that meets the standards for "dairy product lactic acid bacteria beverage" (more specifically, fermented milk that meets the non-fat milk solids content of less than 3.0% and a lactic acid bacteria or yeast count (preferably lactic acid bacteria count) of 1 million / mL or more). Furthermore, the non-fat milk solids refer to the components remaining after subtracting fat from the total milk solids (mainly protein, lactose, and minerals, etc.), and the number of lactic acid bacteria and yeast is measured before pasteurization using the testing method specified in the aforementioned Ministerial Ordinance on Milk and Dairy Products.

[0097] The fermented milk of the present invention may be the fermented product after the fermentation process, or the fermented product after sterilization, or it may be concentrated, diluted, dried, or frozen. For example, the fermented milk may be the above-mentioned fermented milk, a dairy product containing lactic acid bacteria, or a sterilized lactic acid bacteria beverage. In this case, the number of lactic acid bacteria is calculated on a viable cell count basis. The lactic acid bacteria contained in the fermented milk of the present invention include not only viable bacteria but also dead bacteria, as well as crushed and heat-treated lactic acid bacteria, concentrates, crudely refined products, refined products, diluted products, dried products (spray-dried products, freeze-dried products, etc.), and frozen products thereof. However, it is preferable that the fermented milk of the present invention contains at least viable bacteria.

[0098] The fermented milk of the present invention may further contain other lactic acid bacteria or yeast as lactic acid bacteria, to the extent that it does not inhibit the effects of the present invention. Furthermore, the fermented milk of the present invention may further contain various components that can be included in food and beverages. Such components are not particularly limited and include, for example, water, sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, pH adjusters, starch and modified starch, dietary fiber, fruits, vegetables and their processed products, animal and plant herbal extracts, naturally derived polymers (collagen, hyaluronic acid, chondroitin, etc.), oils and fats, thickeners, emulsifiers, solvents, surfactants, gelling agents, stabilizers, buffers, suspending agents, viscosity modifiers, excipients, disintegrants, binders, fluidizers, preservatives, colorants, flavorings, flavoring agents, sweeteners, etc., and may contain only one of these or two or more in combination.

[0099] Preferred fermented milk products include yogurt, cheese, fermented cream, and fermented butter, with yogurt being particularly preferred. Specifically, examples of yogurt include solid-type yogurts (such as plain yogurt), soft-type yogurts (paste-like fermented milk), and drinkable yogurts (liquid fermented milk). Frozen yogurt using these as ingredients may also be used. Furthermore, the fermented milk of the present invention can also be used as an ingredient in fermented foods such as cheese, fermented cream, fermented butter, and kefir.

[0100] The fermented milk of the present invention can be obtained by the method for producing the fermented milk of the present invention described above, and because it contains extracellular polysaccharides having or having high immunostimulatory activity, it can be made into fermented milk with improved immunostimulatory activity.

[0101] <Production method of exopolysaccharide> The present invention also provides a method for producing extracellular polysaccharides of lactic acid bacteria, comprising the steps of adding the lactic acid bacteria or lactic acid bacteria composition of the present invention to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, fermenting the mixture, and collecting the extracellular polysaccharides contained in the fermented product.

[0102] The culture medium must contain at least one sugar selected from glucose and sugars that contain glucose as a constituent sugar. Examples of sugars that contain glucose as a constituent sugar include disaccharides (maltose, sucrose, lactose, etc.), oligosaccharides (galactooligosaccharides, fructooligosaccharides, mannanoligosaccharides, etc.), and polysaccharides (starch (amylose, amylopectin), glycogen, etc.). The culture medium may contain only one of the above sugars or a combination of two or more, but it is preferable that it contains lactose. Furthermore, the sugar contained in the culture medium may be, for example, one contained in the raw milk, and it is preferable that the culture medium contains the raw milk, and more preferably that it is a prepared milk solution containing the raw milk, and skim milk powder is preferred as the raw milk.

[0103] The lactic acid bacteria and lactic acid bacteria composition, as well as the fermentation method, are the same as in the fermentation step in the above-described method for producing fermented milk, except that the culture medium may be used as the milk preparation liquid, including preferred embodiments thereof. The method for collecting the extracellular polysaccharide from the fermented milk is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, the fermented product may be purified by adding a protein denaturant (such as trichloroacetic acid) or removing proteins by heat treatment as needed to obtain a crude product, and then purifying it by using, for example, a salting-out method, an organic solvent precipitation method, a membrane separation method, or a chromatographic separation method, either alone or in combination of two or more.

[0104] <Immunostimulant and Method for Producing the Same> Furthermore, the present invention also provides an immunostimulant containing, as an active ingredient, an extracellular polysaccharide derived from at least one lactic acid bacterium selected from the group consisting of the lactic acid bacterium of the present invention (i.e., lactic acid bacterium into which at least one selected from the group consisting of an immunostimulatory activity-enhancing protein, immunostimulatory activity-enhancing DNA, and a vector of the present invention containing immunostimulatory activity-enhancing DNA has been introduced; lactic acid bacterium having immunostimulatory activity-enhancing DNA; lactic acid bacterium whose extracellular polysaccharide has immunostimulatory activity or high immunostimulatory activity of the extracellular polysaccharide by the evaluation method of the lactic acid bacterium of the present invention (including those evaluated to have immunostimulatory activity or a high probability of high immunostimulatory activity); lactic acid bacterium obtained by the method for producing the lactic acid bacterium of the present invention). The extracellular polysaccharide derived from the lactic acid bacterium is produced extracellularly when the lactic acid bacterium or lactic acid bacterium composition of the present invention is added to the culture medium and fermented by the method for producing the extracellular polysaccharide, and is contained in the fermented product after fermentation.

[0105] The immunostimulant of the present invention can be administered to a target, such as a human or a non-human animal (preferably a mammal), by either oral or parenteral administration. This primarily improves the NK cell activity of the target, thereby contributing to the prevention of infections such as influenza, and the prevention or inhibition of cancer progression. Therefore, the present invention also provides an immunostimulatory method, more preferably a method for improving NK cell activity, which includes the step of administering the extracellular polysaccharide to a target. In this invention, oral administration includes the ingestion of food and beverage compositions or feed compositions.

[0106] The immunostimulant of the present invention may be the fermented product after fermentation, or it may be a concentrated, crudely purified, purified, pasteurized, dried (spray-dried, freeze-dried, etc.) product of the fermented product, a granulated product, a pulverized product, a liquid dispersed in a medium, or a processed product combining two or more of these, or it may consist only of extracellular polysaccharides obtained by the method for producing extracellular polysaccharides. Furthermore, depending on the purpose, target, method, and dosage of administration, it can be, for example, a pharmaceutical composition, a quasi-drug composition, a food and beverage composition, a feed composition, etc.

[0107] The aforementioned pharmaceutical composition and quasi-drug composition can be, for example, a preparation, and its form is not particularly limited, but examples include solid preparations such as tablets, pills, granules, powders, and capsules; liquid preparations such as general liquids, suspensions, emulsions, and syrups; jelly preparations; injections and intravenous preparations; enteral and nasogastric preparations; and suppositories. The aforementioned preparation can be manufactured, for example, by adding one or more preparation aids such as solvents, dispersants, emulsifiers, thickeners, gelling agents, surfactants, buffers, stabilizers, preservatives, excipients, binders, disintegrants, solubilizers, lubricants, colorants, flavoring agents, sweeteners, coating agents, and fragrances to the above-mentioned extracellular polysaccharide, according to known methods or similar methods.

[0108] The form of the food and beverage composition is not particularly limited, and examples include solid forms such as bars, liquid forms such as beverages and liquid foods, paste forms, semi-liquid forms, gel forms (jelly forms), gel-like fats and oils (semi-solid fats and oils), and powder forms. Examples of such food and beverage compositions are not particularly limited, but examples include the fermented milk of the present invention (including lactic acid bacteria beverages, yogurt, etc.), beverages (teas, carbonated drinks, cocoa, coffee, soy milk beverages, fruit and vegetable juice beverages, soft drinks, nutritional drinks, alcoholic beverages, etc.), processed foods (chocolate, gum, gummies, jelly, baked goods (bread, cakes, cookies, biscuits, etc.), candy, etc.), dairy products (prepared milk powder, processed milk, milk beverages, ice cream, margarine, condensed milk, etc.), seasonings (sauces, soups, dressings, mayonnaise, mayonnaise-type seasonings, cream, etc.), supplements, edible oils, functional edible fats and oils, etc. Such food and beverage compositions can be produced, for example, by the above-mentioned method for producing fermented milk in the case of the fermented milk of the present invention; by a method of incorporating the above-mentioned fermented product or extracellular polysaccharide into an existing food or beverage; or by a method of adding the above-mentioned fermented product or extracellular polysaccharide during the manufacturing process of the food or beverage.

[0109] The aforementioned food and beverage composition may further contain various components that can be included in food and beverages, as long as they do not hinder the effects of the present invention. Such components are not particularly limited and include, for example, the various components listed in the above-mentioned <fermented milk>, the formulation aids listed in the above-mentioned pharmaceutical composition and quasi-drug composition, and one or more of these may be included in appropriate amounts in combination.

[0110] Examples of the aforementioned feed composition include those obtained by appropriately modifying the above-mentioned food and beverage composition depending on the purpose, target, method, and dosage of administering the feed composition.

[0111] In the immunostimulant of the present invention, the content of extracellular polysaccharides, which are the active ingredients (the total amount if it is a mixture of two or more), is determined appropriately according to the dosage form, dosage, etc., and therefore cannot be stated in general terms. However, it is preferably 0.001 to 90% by mass, more preferably 0.002 to 50% by mass, even more preferably 0.003 to 10% by mass, even more preferably 0.01 to 5% by mass, and also preferably 0.1 to 1% by mass or 0.003 to 1% by mass, relative to the entire immunostimulant.

[0112] Furthermore, the dosage of the immunostimulant of the present invention cannot be generalized as it is determined appropriately on a case-by-case basis, taking into consideration the species, age, weight, sex, and therapeutic purpose of the target organism. However, the lower limit of the amount of extracellular polysaccharides (the total amount if it is a mixture of two or more types) per adult day can be, for example, 0.01 mg / kg, preferably 0.02 mg / kg, and more preferably 0.05 mg / kg. There is no particular upper limit for the dosage, but it can be, for example, 1 g / kg per adult day.

[0113] <Methods for improving the immune-boosting activity of fermented milk> The present invention provides a method for improving the immunostimulatory activity of fermented milk, which includes a fermentation step in which the lactic acid bacteria or lactic acid bacteria composition of the present invention is added to a milk preparation liquid containing raw milk and fermented to obtain a fermented product containing extracellular polysaccharides. This makes it possible to obtain fermented milk containing extracellular polysaccharides with improved immunostimulatory activity, thereby improving the immunostimulatory activity of the fermented milk. The lactic acid bacteria, lactic acid bacteria composition, and fermentation step are as described in the above-described method for producing fermented milk of the present invention.

[0114] In the present invention, superior immunostimulatory activity of fermented milk can be determined, for example, by measuring the NK cell activity of extracellular polysaccharides purified from the fermented milk under evaluation by a standard method, in the same manner as the method used to confirm that the above-mentioned protein has an immunostimulatory activity-enhancing effect, and finding that the NK cell activity is 21% or higher, preferably 22% or higher, and more preferably 22.5% or higher, then the fermented milk can be considered to have superior immunostimulatory activity. Furthermore, improved immunostimulatory activity of fermented milk can be confirmed, for example, by measuring the NK cell activity of extracellular polysaccharides purified from the fermented milk under evaluation by a standard method, in the same manner as the method used to confirm that the above-mentioned protein has an immunostimulatory activity-enhancing effect, and finding that the NK cell activity is 1.03 or higher, preferably 1.05 or higher, and more preferably 1.10 or higher, with the NK cell activity measured for extracellular polysaccharides purified from fermented milk obtained in the same manner using lactic acid bacteria that do not have any of the above-mentioned DNAs (a') to (d') set to 1. [Examples]

[0115] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0116] <Lactic acid bacteria> The lactic acid bacteria used in the following tests are as follows: R-1 strain: Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (Accession number: FERM BP-10741) 2038 strain: Lactobacillus delbrueckii subsp. bulgaricus 2038 Strain 2038 was isolated by spreading a diluted solution of Meiji Bulgaria Yogurt LB81 (manufactured by Meiji Co., Ltd.) onto BCP agar medium, culturing at 37°C for 48 hours, and then picking up rough-type colonies. The full-length genome of strain 2038 is registered as entry number T01957 in the Kyoto Encyclopedia of Genes and Genomes (KEGG), an integrated database of biological systems information that integrates genome, protein, and compound information through intermolecular interactions, reactions, and relational networks.

[0117] <Immunostimulatory Activity Evaluation> (1) Preparation of extracellular polysaccharides (Preparation of fermented milk) Strain R-1 was seeded at a concentration of 1% (wt / wt) in a 10% skim milk powder medium prepared with 10% (wt / wt) skim milk powder, 0.1% (wt / wt) yeast extract, and distilled water. Fermentation was carried out overnight at 37°C under anaerobic conditions to obtain fermented milk. Fermented milk was also obtained under the same conditions, except that strain 2038 was used instead of strain R-1.

[0118] (Purification of extracellular polysaccharides) Trichloroacetic acid was added to each of the fermented milk products obtained above to a final concentration of 10% by mass, and the resulting denatured proteins were removed to obtain a crude product. An equal volume of cold ethanol was added to the obtained crude product, and it was allowed to stand at 4°C for 16 hours to perform ethanol precipitation, obtaining precipitate 1 containing extracellular polysaccharides (EPS). Precipitate 1 was dialyzed against MilliQ water using a dialysis membrane (molecular weight cutoff: 6-8 kDa) to enzymatically decompose nucleic acids and residual proteins, and then ethanol precipitation was performed again to obtain precipitate 2. Precipitate 2 was dissolved in MilliQ water, dialyzed again, and then freeze-dried to obtain each purified extracellular polysaccharide. The purified extracellular polysaccharide obtained from fermented milk using strain R-1 was designated "Strain R-1 EPS," and the purified extracellular polysaccharide obtained from fermented milk using strain 2038 was designated "Strain 2038 EPS."

[0119] (2) Evaluation of NK cell activity For each purified exopolysaccharide obtained in (1) above, NK cell activity was measured as an evaluation of immunostimulatory activity. Specifically, first, a total of 20 female BALB / c mice (7 weeks old, purchased from CLEA Japan, Inc.) were divided into two groups: one group was the R-1 strain EPS-administered mouse group (n=10), and the other was the 2038 strain EPS-administered mouse group (n=10). In both groups, each purified exopolysaccharide was orally administered at a dose of 100 μg / mouse / day for 3 weeks. After completion of the administration period, the spleen was extracted from each mouse to obtain spleen cells.

[0120] Subsequently, for each preparation of spleen cells, NK cell activity was measured by the chromium release method according to the method of Takeda et al. (Takeda, K. et al., J. Immunol., 156: 3366, 1996). Specifically, the effector cells were each preparation of spleen cells, and the target cells were 51 51Cr-labeled YAC-1 cells (mouse lymphoma cells). After culturing for 4 hours at an E / T ratio (number of effector cells / number of target cells) of 200:1, the radioactivity of the culture supernatant and the whole culture medium was measured. The ratio of the radioactivity in the supernatant to the radioactivity of the whole culture medium was defined as NK cell activity (NK activity (%)). The results are shown in Figure 1. As shown in Figure 1, significantly higher NK cell activity was observed in mice administered with R-1 strain EPS compared with mice administered with 2038 strain EPS, confirming that R-1 strain EPS has higher immunostimulatory activity than 2038 strain EPS.

[0121] <Comparison of EPS gene cluster regions> From the nucleotide sequences of 2038 strain genomes registered in KEGG, the -100bp to +100bp regions of two EPS gene cluster regions: LBU1598-LBU1588 (EPS gene cluster 1) and LBU1630-LBU1618 (EPS gene cluster 2) were extracted. The full-length genome of strain R-1 was obtained using the next-generation sequencer MiSeq (Illumina). Using Homology / Local BLASTN in Genetyx Ver.13 (Genetics Corporation), nucleotide sequences homologous to the two EPS gene cluster regions mentioned above were extracted from the genome of strain R-1 (E-value threshold=0.00001, word size=11).

[0122] In both the 2038 and R-1 genomes, EPS gene clusters 1 and 2 were highly conserved. In particular, the nucleotide sequence of the 2038 strain and the R-1 strain were completely identical across the entire region (11,569 bp) of EPS gene cluster 1. On the other hand, in EPS gene cluster 2, a total of 4 base pairs were found in the epsC and epsF genes, as well as in two intergeneric regions between the epsM gene and the transposase gene, out of a total of 15,769 bp. Since the epsC gene is not involved in glycosylation, and the mutations in the intergeneric regions are not thought to be related to any of the genes, it can be said that the base pair difference in the epsF gene, which is involved in glycosylation, and the resulting difference in amino acid composition, are involved in the difference in immunostimulatory activity between the extracellular polysaccharide produced by the R-1 strain and the extracellular polysaccharide produced by the 2038 strain. Table 1 shows the nucleotides in the epsF gene that differed between the 2038 strain genome and the R-1 strain genome, the codon containing those nucleotides, the amino acid encoded by that codon, and the position of that amino acid within the gene. Furthermore, because a frameshift occurred in the epsF gene due to the difference in these nucleotides, Table 2 below shows the nucleotide sequences below the codon containing the nucleotides that differed between the 2038 strain genome and the R-1 strain genome, and the amino acid sequences that they encode. Additionally, the nucleotide sequence of the 2038 strain genome is shown as SEQ ID NO: 3, and the amino acid sequence as SEQ ID NO: 4; the nucleotide sequence of the R-1 strain genome is shown as SEQ ID NO: 5, and the amino acid sequence as SEQ ID NO: 6; respectively.

[0123] [Table 1]

[0124] [Table 2]

[0125] As shown in Table 1, the difference between the 2038 strain genome and the R-1 strain genome was that guanine (G) at position 999 of the epsF gene in the 2038 strain genome was deleted (del) in the R-1 strain genome. This caused a frameshift in the R-1 strain genome, shifting the codon reading frame. However, the amino acid specified by the codon containing this base was glycine (G) at position 333 in both the 2038 and R-1 strain genomes. However, as shown in Table 2, the frameshift resulted in different amino acid sequences from glycine to the C-terminus between the two strains. In the 2038 strain genome, the sequence was N-terminal -Gly-Leu-Ala-Ile-Leu-C-terminal, while in the R-1 strain genome, it was N-terminal -Gly-Ser-Leu-Phe-Ser-Asp-C-terminal. These results suggest that serine, leucine, phenylalanine, serine, and aspartic acid at positions 334-338 of the protein encoded by the epsF gene of the R-1 strain are important for enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria. [Industrial applicability]

[0126] As described above, the present invention makes it possible to provide a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, as well as fermented milk containing extracellular polysaccharides having excellent immunostimulatory activity and a method for producing the same. More specifically, it makes it possible to provide a novel protein that enhances the immunostimulatory activity of extracellular polysaccharides produced when expressed in lactic acid bacteria, DNA encoding the protein, a vector containing the DNA, lactic acid bacteria and a lactic acid bacteria composition containing the DNA or the vector, and fermented milk, an immunostimulant, and methods for producing the same using these, a method for improving the immunostimulatory activity of fermented milk, and a method for evaluating lactic acid bacteria.

[0127] For example, by introducing the DNA encoding the novel protein of the present invention into various lactic acid bacteria, it becomes possible to easily produce extracellular polysaccharides with excellent immunostimulatory activity, as well as fermented milk and immunostimulants containing them, using these lactic acid bacteria. Furthermore, by using the DNA sequence encoding the novel protein of the present invention as a selection criterion, it becomes possible to easily select lactic acid bacteria capable of producing extracellular polysaccharides with excellent immunostimulatory activity, as well as fermented milk and immunostimulants containing them.

Claims

1. At least one protein selected from the group consisting of the proteins listed below (a) to (c). (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 1 (b) A protein having an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, comprising an amino acid sequence in which 1 to 20 amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334 to 338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added. (c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria.

2. DNA encoding the protein described in claim 1.

3. A vector comprising the DNA described in claim 2.

4. A transformant bacterium into which the vector described in Claim 3 has been introduced, wherein the NK cell activity of extracellular polysaccharides is improved compared to before transformation.

5. A lactic acid bacteria composition containing the lactic acid bacteria described in claim 4.

6. The lactic acid bacteria composition according to claim 5, which is fermented milk.

7. A lactic acid bacteria composition according to claim 5 or 6, comprising an extracellular polysaccharide derived from the lactic acid bacteria described in claim 4.

8. A method for producing fermented milk, comprising a fermentation step of adding the lactic acid bacteria described in claim 4 or the lactic acid bacteria composition described in any one of claims 5 to 7 to a milk preparation liquid containing raw milk and fermenting it to obtain a fermented product containing extracellular polysaccharides.

9. A method for improving the immunostimulatory activity of fermented milk, comprising a fermentation step of adding the lactic acid bacteria described in claim 4 or the lactic acid bacteria composition described in any one of claims 5 to 7 to a milk preparation liquid containing raw milk and fermenting it to obtain a fermented product containing extracellular polysaccharides.

10. A method for evaluating lactic acid bacteria, comprising the step of detecting at least one DNA selected from the group consisting of DNA encoding any of the proteins (a) to (c) below from lactic acid bacteria, and if detected, evaluating that the extracellular polysaccharide produced by the lactic acid bacteria has immunostimulatory activity or has high immunostimulatory activity, wherein the immunostimulatory activity of the extracellular polysaccharide is evaluated using the DNA as an indicator. (a) Protein consisting of the amino acid sequence shown in Sequence ID No. 1 (b) A protein having an effect of enhancing the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, comprising an amino acid sequence in which 1 to 20 amino acids other than serine, leucine, phenylalanine, serine, and aspartic acid at positions 334 to 338 in the amino acid sequence shown in Sequence ID No. 1 are substituted, deleted, inserted, and / or added. (c) A protein having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in Sequence ID No. 1, wherein the amino acids corresponding to positions 334-338 of the amino acid sequence shown in Sequence ID No. 1 are serine, leucine, phenylalanine, serine, and aspartic acid from the N-terminus, and which has the effect of improving the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria.

11. The method for evaluating lactic acid bacteria according to claim 10, comprising an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A step to obtain lactic acid bacteria in which the extracellular polysaccharide is evaluated to have immunostimulatory activity or to have high immunostimulatory activity in the extracellular polysaccharide in the evaluation step, A method for producing lactic acid bacteria, including

12. The method for evaluating lactic acid bacteria according to claim 10, comprising an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a formula containing raw milk, and fermenting the mixture, A method for producing fermented milk, including the following:

13. The method for evaluating lactic acid bacteria according to claim 10, comprising an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a formula containing raw milk, and fermenting the mixture, A method for improving the immunostimulatory activity of fermented milk, including [specific ingredient / method].

14. A method for producing extracellular polysaccharides of lactic acid bacteria, comprising the steps of adding the lactic acid bacteria described in claim 4 or the lactic acid bacteria composition described in any one of claims 5 to 7 to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, fermenting the mixture, and collecting the extracellular polysaccharides contained in the fermented product.

15. The method for evaluating lactic acid bacteria according to claim 10, comprising an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A step of adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a culture medium containing glucose and / or sugars having glucose as a constituent sugar, and fermenting the mixture, and collecting the extracellular polysaccharides contained in the fermented product, A method for producing extracellular polysaccharides of lactic acid bacteria, including those contained within the bacterial cell.

16. A method for producing an immunostimulant, comprising: a fermentation step of adding the lactic acid bacteria described in claim 4 or the lactic acid bacteria composition described in any one of claims 5 to 7 to a culture medium containing glucose and / or a sugar having glucose as a constituent sugar, and fermenting the mixture to obtain a fermented product containing extracellular polysaccharides; and a step of obtaining an immunostimulant containing the extracellular polysaccharides as an active ingredient.

17. The method for evaluating lactic acid bacteria according to claim 10, comprising an evaluation step for evaluating the immunostimulatory activity of extracellular polysaccharides of lactic acid bacteria, A fermentation step to obtain a fermented product containing extracellular polysaccharides by adding lactic acid bacteria, which were evaluated in the evaluation step to have extracellular polysaccharides that have immunostimulatory activity or have high immunostimulatory activity, to a culture medium containing glucose and / or sugars having glucose as a constituent sugar, and fermenting the mixture. A step to obtain an immunostimulant containing the above-mentioned extracellular polysaccharide as an active ingredient, A method for producing an immunostimulant, including the above.

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