Growth inhibitor for bacteria, staphylococcus aureus or escherichia coli, barrier function improver for vaginal tissue, lactic acid bacteria-producing substance and preparation

Abstract

To obtain a bacterium inhibiting the proliferation of S. aureus and E. coli and participating in the increase of the expression of genes of six factors of CLD1, OCLN, elafin, MUC1, HAS1 and HAS3 relating to the barrier function of vaginal tissues and a preparation, etc., utilizing a culture supernatant or dead cells obtained from the bacterium.SOLUTION: A BG-strain of DL427 belonging to the species Lactobacillusparagasseri deposited under accession number NITEP - 04115, wherein the culture supernatant inhibits the growth of S. aureus and E. coli and increases the expression of the genes for the six factors CLD1, OCLN, Elafin, MUC1, HAS1, HAS3, which are associated with the barrier function of vaginal tissue. A preparation using the culture supernatant or dead bacterial cells of the bacteria can be used as an agent for suppressing proliferation of Staphylococcus aureus or Escherichia coli and as an agent for improving the barrier function of vaginal tissue.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a growth inhibitor for Staphylococcus aureus or Escherichia coli, an agent for improving the barrier function of vaginal tissue, and a lactic acid bacteria-produced substance and preparation, which utilize a bacterium belonging to the species Lactobacillus paragasseri, as well as the culture supernatant or killed cells of said bacterium. [Background technology]

[0002] Even if the birth canal is damaged during childbirth, it is quickly repaired. Therefore, it is thought that the birth canal of a pregnant woman has a unique function during childbirth. The inventors speculate that bacteria present in the vagina may play a role in this function, and have previously provided technologies focusing on bacteria originating from the vagina, such as the creation of an IL10 gene expression enhancer from bacteria originating from the vagina. Technology related to the IL10 gene expression enhancer is described in Japanese Patent No. 7076840 (Patent Document 1).

[0003] Apart from these technologies, the importance of maintaining and improving a healthy intestinal environment has become widely known, and in recent years, the number of people interested in healthy life expectancy has increased, leading to active research into intestinal bacteria that function as so-called beneficial bacteria, such as lactic acid bacteria. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7076840 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the inventors of the present invention have investigated ways to eliminate the causes of vaginal inflammation and odor, considering that it is important to suppress the proliferation of bacteria that cause adverse effects on the vagina during repair of the birth canal. Furthermore, they have investigated ways to enhance the barrier function of the vagina, considering that it is also important to enhance this function.

[0006] On the other hand, in light of the importance of utilizing beneficial bacteria such as lactic acid bacteria derived from the intestines, in the process of examining the effective use of such bacteria, we investigated methods for the effective use of beneficial bacteria, since some intestinal bacteria are difficult to obtain alive from feces. The present invention was born as a result of such research. [Means for solving the problem]

[0007] A first aspect of the present disclosure relates to Lactobacillus paragasseri ( ), deposited under Accession Number: NITE P-04115, which inhibits the growth of Staphylococcus aureus and Escherichia coli and increases gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The bacterium is strain BG-DL427, which belongs to the Bacillus species.

[0008] A first aspect of the present disclosure relates to Lactobacillus paragasseri ( ), deposited under Accession Number: NITE P-04115, which inhibits the growth of Staphylococcus aureus and Escherichia coli and increases gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The bacterium used was the BG-DL427 strain belonging to the Lactobacillus paragasseri species, which can be obtained from the human birth canal. Killed bacteria belonging to the Lactobacillus paragasseri species can suppress the growth of Staphylococcus aureus and Escherichia coli, and increase the gene expression of three factors related to the barrier function of vaginal tissue: CLD1, elafin, and MUC1.

[0009] A second aspect of the present disclosure relates to a culture supernatant of Lactobacillus paragasseri (Deposited under Accession Number: NITE P-04115), which inhibits the growth of Staphylococcus aureus and Escherichia coli and increases gene expression of six factors, CLD1, OCLN, elafin, MUC1, HAS1, and HAS3, that are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The bacterium is strain BG-DL427, which belongs to the Bacillus species.

[0010] A second aspect of the present disclosure relates to a culture supernatant of Lactobacillus paragasseri (Deposited under Accession Number: NITE P-04115), which inhibits the growth of Staphylococcus aureus and Escherichia coli and increases gene expression of six factors, CLD1, OCLN, elafin, MUC1, HAS1, and HAS3, that are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The culture supernatant of the bacterium, BG-DL427 strain, belonging to the Lactobacillus paragasseri species, which can be obtained from the human birth canal, suppresses the growth of Staphylococcus aureus and Escherichia coli and increases the gene expression of six factors related to the barrier function of vaginal tissue: CLD1, OCLN, elafin, MUC1, HAS1, and HAS3.

[0011] A third aspect of the present disclosure relates to Lactobacillus paragasseri (Deposited under Accession Number: NITE P-04115), whose killed cells suppress the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The bacterium is strain BG-DL427, which belongs to the Bacillus species.

[0012] A third aspect of the present disclosure relates to Lactobacillus paragasseri (Deposited under Accession Number: NITE P-04115), whose killed cells suppress the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue. Lactobacillus paragasseri The bacterium used was the BG-DL427 strain belonging to the Lactobacillus paragasseri species, which can be obtained from the human birth canal. Killed bacteria belonging to the Lactobacillus paragasseri species can suppress the growth of Staphylococcus aureus and Escherichia coli, and increase the gene expression of three factors related to the barrier function of vaginal tissue: CLD1, elafin, and MUC1.

[0013] A fourth aspect of the present disclosure is a growth inhibitor for Staphylococcus aureus or Escherichia coli, which contains a culture supernatant of the above bacteria or killed cells of the above bacteria as an active ingredient.

[0014] A fourth aspect of the present disclosure is a growth inhibitor for Staphylococcus aureus or Escherichia coli that contains the culture supernatant of the above bacteria or killed cells of the above bacteria as an active ingredient, and is therefore capable of inhibiting the growth of Staphylococcus aureus or Escherichia coli.

[0015] A fifth aspect of the present disclosure is an agent for improving the barrier function of vaginal tissue, comprising a culture supernatant of the bacterium or killed cells of the bacterium as an active ingredient.

[0016] A fifth aspect of the present disclosure is an agent for improving the barrier function of vaginal tissue, which contains the culture supernatant of the bacteria or killed cells of the bacteria as an active ingredient, and is therefore able to improve the barrier function of vaginal tissue.

[0017] A sixth aspect of the present disclosure is a lactic acid bacteria production substance containing, as an active ingredient, a culture supernatant of the bacterium or killed cells of the bacterium.

[0018] The sixth aspect of the present disclosure provides a lactic acid bacteria production substance containing the culture supernatant of the bacteria or killed cells of the bacteria as an active ingredient, and therefore can contain a production substance derived from the BG-DL427 strain, making it a lactic acid bacteria production substance that is useful for maintaining a good intestinal flora and improving a deteriorated intestinal flora.

[0019] A seventh aspect of the present disclosure is a formulation containing, as an active ingredient, a culture supernatant of the bacterium or killed cells of the bacterium.

[0020] The seventh aspect of the present disclosure is a formulation containing the culture supernatant of the bacteria or killed cells of the bacteria as an active ingredient, and therefore can be in various forms such as liquid, solid, powder, jelly, gel, etc., and can be used as basic cosmetics, cream products such as skin care products, cosmetic liquids, etc. that maintain and improve the barrier function of the epidermis of the face, hands, etc., as well as the delicate areas inside and around the vagina, or as tablets, granules, drinks, etc. that are taken to maintain and improve the intestinal environment. [Effects of the Invention]

[0021] According to one aspect of the present disclosure, the growth of Staphylococcus aureus and Escherichia coli can be inhibited.

[0022] According to one embodiment of the present disclosure, gene expression of six factors associated with the barrier function of vaginal tissue, namely CLD1, OCLN, elafin, MUC1, HAS1, and HAS3, can be increased.

[0023] According to one embodiment of the present disclosure, gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue, can be increased.

[0024] According to one aspect of the present disclosure, the barrier function of vaginal tissue can be improved.

[0025] According to one aspect of the present disclosure, the lactic acid bacteria can be used as a lactic acid bacteria production substance, a preparation, etc. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a scatter plot of the growth inhibition ratio of Staphylococcus aureus and the growth inhibition ratio of Escherichia coli. [Figure 2] This is a heat map showing the degree of increase in gene expression of six barrier function-related factors in DL strains. [Figure 3] 1 is a graph showing the relative expression frequency of the elafin gene in response to treatment with the culture supernatant of each strain. [Figure 4] 1 is a graph showing the relative expression frequency of the CLD1 gene in response to treatment with the culture supernatant of each strain. [Figure 5] 1 is a graph showing the relative expression frequency of the OCLN gene in response to culture supernatant treatment of each strain. [Figure 6] 1 is a graph showing the relative expression frequency of the MUC1 gene in response to treatment with the culture supernatant of each strain. [Figure 7] 1 is a graph showing the relative expression frequency of the elafin gene in response to treatment with killed cells of each strain. [Figure 8] 1 is a graph showing the relative expression frequency of the CLD1 gene in response to treatment with killed cells of each strain. [Figure 9] 1 is a graph showing the relative expression frequency of the OCLN gene in response to treatment with killed cells of each strain. [Figure 10] 1 is a graph showing the relative expression frequency of the MUC1 gene for each strain after treatment with killed cells. DETAILED DESCRIPTION OF THE INVENTION

[0027] One aspect of the present disclosure will be described based on an exemplary embodiment. The following embodiment does not unduly limit the content of the present invention described in the claims. Furthermore, not all of the configurations described in this embodiment are necessarily essential as a solution to the present invention.

[0028] All embodiments and optional embodiments included in this disclosure may be combined with each other to form new embodiments, and all technical features and optional technical features included in this disclosure may be combined with each other to form new technical features.

[0029] The term "or" used in this disclosure is used as an inclusive term. For example, "A or B" means "A, B, or both A and B." "A," "B," and "both A and B" all respectively satisfy "A or B."

[0030] Numerical values ​​or elements modified by the terms "about," "approximately," "nearly," or "substantially" as used in this specification and claims are understood to include the numerical value and any surrounding numerical values, and to include the element and anything that can be considered the same as the element. For example, when describing "about 3," "3" and any subsequent numerical values ​​can be included in "about 3" as long as the technical features and technical significance claimed in this disclosure are not different. Furthermore, when describing "B that is substantially identical to A," B can be completely identical to A, or even if there are differences, they can be included in the scope of "substantially" as long as the technical features and technical significance claimed in this disclosure are the same.

[0031] Unless otherwise specified, the symbol "~" used in this specification and claims is understood to include the lower limit and upper limit, and also to include any value between the lower limit and upper limit. For example, "3 to 5" means "3 to 5," and "2.5 to 6 g" means "2.5 g to 6 g."

[0032] The following describes in detail the technology related to bacteria that inhibit the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of six factors related to the barrier function of vaginal tissue: CLD1, OCLN, elafin, MUC1, HAS1, and HAS3. More specifically, the technology relates to bacteria whose culture supernatant inhibits the growth of Staphylococcus aureus and Escherichia coli and increases the gene expression of six factors related to the barrier function of vaginal tissue: CLD1, OCLN, elafin, MUC1, HAS1, and HAS3; bacteria whose killed cells inhibit the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of three factors related to the barrier function of vaginal tissue: CLD1, elafin, and MUC1; culture supernatants of these bacteria; live cells, killed cells, or both live and killed cells of these bacteria; or mixtures containing these bacteria and the culture supernatant; and preparations such as supplements, lactic acid bacteria production substances, and topical skin preparations that use these culture supernatants or mixtures.

[0033] In a preferred embodiment, the bacterium whose culture supernatant inhibits the growth of Staphylococcus aureus and Escherichia coli and increases the gene expression of six factors associated with the barrier function of vaginal tissue, i.e., CLD1, OCLN, elafin, MUC1, HAS1, and HAS3, and whose killed cells inhibit the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of three factors associated with the barrier function of vaginal tissue, i.e., CLD1, elafin, and MUC1, is Lactobacillus paragasseri ( Lactobacillus paragasseri), with strain BG-DL427 (also referred to as "DL427") being particularly preferred. This strain BG-DL427 has been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-04115, date of deposit May 15, 2024, and identification number BG-DL427. The 16S rRNA base sequence of this strain is shown in the Sequence Listing as SEQ ID NO: 1.

[0034] Strain BG-DL427 was identified by a homology search of its 16S rRNA gene sequence against sequences in a database, and was found to belong to the species Lactobacillus paragasseri, with a homology score of 100%. The mycological characteristics of strain BG-DL427 are the same as those of Lactobacillus paragasseri.

[0035] The cells of the BG-DL427 strain can be cultured by the method described below, and killed cells and culture supernatant separated and purified from the culture by means of centrifugation or the like can be used. Furthermore, a culture supernatant prepared from the BG-DL427 strain, a mixture of killed cells, or a mixture of the culture supernatant and killed cells can be used as an active ingredient to prepare a lactic acid bacteria product, which can be in the form of a liquid, gel, jelly, or the like.

[0036] Furthermore, a culture supernatant prepared from BG-DL427 strain cells, a mixture of killed cells, or a mixture of this culture supernatant and killed cells can be appropriately mixed with excipients, disintegrants, binders, stabilizers, wetting agents, etc. to prepare a formulation containing the culture supernatant prepared from BG-DL427 strain cells, a mixture of killed cells, or a mixture of this culture supernatant and killed cells as an active ingredient. This formulation can be in the form of tablets, granules, powders, capsules, liquids, emulsions, creams, gels, jellies, etc.

[0037] Furthermore, a culture supernatant prepared from BG-DL427 strain cells or a mixture of this culture supernatant and killed cells as an active ingredient can be used as a growth inhibitor that suppresses the growth of Staphylococcus aureus and Escherichia coli, and can also be used as an agent for enhancing the gene expression of six factors related to the barrier function of vaginal tissue (CLD1, OCLN, elafin, MUC1, HAS1, and HAS3) or as an agent for improving the barrier function of vaginal tissue.Alternatively, a mixture of killed BG-DL427 strain cells as an active ingredient can be used as a growth inhibitor that suppresses the growth of Staphylococcus aureus and Escherichia coli, and can also be used as an agent for enhancing the gene expression of three factors related to the barrier function of vaginal tissue (CLD1, elafin, and MUC1) or as an agent for improving the barrier function of vaginal tissue.

[0038] These growth inhibitors can inhibit the growth of Staphylococcus aureus and Escherichia coli. Furthermore, these gene expression enhancers or vaginal tissue barrier function improvers can enhance the gene expression of six factors related to the barrier function of vaginal tissue (CLD1, OCLN, elafin, MUC1, HAS1, and HAS3) or three factors related to the barrier function of vaginal tissue (CLD1, elafin, and MUC1), thereby improving the barrier function of vaginal tissue. Furthermore, they can be expected to improve the barrier function of not only vaginal tissue but also epidermis, such as skin. [Example]

[0039] Experiment 1: Obtaining bacteria from the birth canal Fluid collected from the birth canal of healthy pregnant women was anaerobically cultured on BL agar medium (37°C, 2 days), and representative colonies formed were transferred to MRS liquid medium for lactobacilli and further cultured anaerobically (37°C, 2 days), after which they were stored in glycerol at -80°C.

[0040] Next, DNA was extracted from each strain stocked in glycerol and subjected to sequence analysis. Specifically, colonies formed from each strain were scraped with a pipette tip and suspended in 50 μL of dH2O. 50 μL of 100 mM NaOH was added, mixed, and then incubated at 95°C for 15 minutes. Then, 11 μL of 1 M Tris-HCl (pH 7.0) was added, and the mixture was centrifuged. The culture supernatant was collected and used as a DNA solution. The resulting DNA solution was used as a template with a Bacterial 16S rDNA PCR Kit (Takara Bio Inc.) according to the manufacturer's protocol to obtain PCR amplification products for sequence analysis.

[0041] A portion of the PCR amplified product was subjected to agarose gel electrophoresis (100 V, 30 minutes) to confirm whether the desired amplified product had been obtained. The PCR amplified product was then purified from the PCR reaction mixture using a QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's protocol.

[0042] The base sequences obtained from the sequence analysis of the PCR amplification products were edited using the sequence editing software ApE, and the base sequences of the 16S rRNA gene of each colony were subjected to a homology search with sequences in the database to identify the species. Two databases were used: NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and DDBJ (http: / / blast.ddbj.nig.ac.jp / top-j.hyml). As a result, Lactobacillus crispatus ( Lactobacillus crispatus ), Lactobacillus jensenii ( Lactobacillus jensenii ), Lactobacillus vaginalis ( Lactobacillus vaginalis) , Lactobacillus faecalis ( Lactobacillus faecalis ), Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus choleohominis ( Lactobacillus coleohominis ), and other unidentified bacteria belonging to the genus Lactobacillus were found.

[0043] Experiment 2: Staphylococcus aureus ( S. aureus ) and E. coli ( E. coli Screening of bacterial strains that exhibit growth inhibitory activity against The glycerol stocks of 133 strains of bacteria identified as belonging to the genus Lactobacillus from Experiment 1 were inoculated into 10 mL of MRS liquid medium to a final concentration of 1% and cultured at 35°C under aerobic conditions for 24 hours. The resulting bacterial solution was centrifuged (14,000 x g, 10 minutes), and the culture supernatant was collected and sterilized by filtration using a 0.22 μm syringe filter and stored at 4°C. This was used as the DL culture supernatant for each of the 133 strains.

[0044] On the other hand, for Staphylococcus aureus, the glycerol stock was inoculated into 10 mL of TS liquid medium to a final concentration of 1% and cultured at 37°C under aerobic conditions for 48 hours. For Escherichia coli, the glycerol stock was inoculated into 10 mL of LB liquid medium to a final concentration of 1% and cultured at 37°C under aerobic conditions for 24 hours. The resulting bacterial suspensions were cultured at OD of 1000 in their respective liquid media. 620 The solution was diluted to a value of approximately 0.20 to 0.25 and dispensed in 200 μL portions into a 96-well plate.

[0045] Each DL culture supernatant was added to the 96-well plate to a final concentration of 10%, and after gentle stirring, culture was carried out at 37°C under aerobic conditions for 24 hours. OD was measured using a plate reader. 620 The values ​​were measured and the relative values ​​were calculated based on the untreated condition. This experiment was repeated three times, and the average of the relative values ​​obtained from the three experiments was calculated. This value was used to evaluate the growth inhibitory effect on Staphylococcus aureus and Escherichia coli.

[0046] A scatter plot was created with the growth inhibition ratio against Staphylococcus aureus on the y-axis and the growth inhibition ratio against E. coli on the x-axis, as shown in Figure 1. The strength of the growth inhibition ratio was represented by the density of the plot for Staphylococcus aureus and by the size of the plot for E. coli, and DL culture supernatants with high growth inhibition effects against Staphylococcus aureus and E. coli were searched for.

[0047] From this scatter plot, 16 strains were found out of 133 strains that had small plots (high Escherichia coli growth inhibition) and light colors (high Staphylococcus aureus growth inhibition). These DL culture supernatants may inhibit the growth of both Staphylococcus aureus and Escherichia coli and prevent vaginal inflammation and odor. The 16 strains found were named strains DL56, DL57, DL166, DL181, DL184, DL200, DL201, DL286, DL325, DL352, DL376, DL423, DL424, BG-DL427 (DL427), DL428, and DL430. These strains are collectively referred to, or each individual strain is referred to as a "DL strain".

[0048] Since Staphylococcus aureus and Escherichia coli are bacteria that cause vaginal inflammation and odor, the above 16 strains are considered to be strains that function beneficially to make it difficult to cause vaginal inflammation and odor and maintain it well. Also, all of the above DL strains are bacteria belonging to the genus Lactobacillus, and there is an expectation for their use as alternative bacteria such as lactic acid bacteria belonging to the genus Lactobacillus derived from the intestine. That is, the above DL bacteria are cells that can be obtained as live bacteria, and since the vagina has a different environment from the intestine, there is a possibility of obtaining bacteria with some different characteristics from intestinal bacteria.

[0049] Experiment 3: <Identification of the species of 16 strains that are DL strains> For the 16 strains obtained through the above Experiment 1 and Experiment 2, the species were identified by the following method. Glycerol stocks of the DL16 strain (DL56, DL57, DL166, DL181, DL184, DL200, DL201, DL286, DL325, DL352, DL376, DL423, DL424, BG-DL427 (DL427), DL428, and DL430) were inoculated into 4 mL of MRS liquid medium to a final concentration of 1% and cultured at 35°C under aerobic conditions for 24 hours. A 1.0 mL aliquot was recovered from the resulting bacterial suspension and centrifuged (9,000 × g, 10 minutes, 4°C). The culture supernatant was discarded to obtain a bacterial cell pellet. The bacterial cell pellet was dissolved in 50 μL of dH2O, mixed with 50 μL of 100 mM NaOH, and then incubated at 95°C for 15 minutes. Thereafter, 11 μL of 1 M Tris-HCl (pH 8.5) was added, gently mixed, and then centrifuged (11,000×g, 3 minutes, 4° C.), and the culture supernatant was collected to prepare a DNA solution.

[0050] The resulting DNA solution was used as a template, and the primer set 27F / 1492R was used. PCR was performed using KAPA HiFi HS ReadyMix (KAPABIOSYSTEMS) as the PCR enzyme. The reaction mixture consisted of 12.5 μL of KAPA HiFi HS ReadyMix, 0.75 μL of 27F primer (10 μM), 0.75 μL of 1492R primer (10 μM), 1.0 μL of template DNA, and 11 μL of dH2O. The reaction conditions were an initial denaturation step at 95 °C for 3 minutes, followed by 30 cycles of denaturation at 98 °C for 20 seconds, annealing at 60 °C for 15 seconds, and extension at 72 °C for 30 seconds. The reaction was then repeated for 30 cycles, followed by a final extension step at 72 °C for 90 seconds.

[0051] The resulting PCR amplification products were purified using the FastGene™ GeL / PCR Extraction Kit (Nippon Genetics Co., Ltd.) according to the manufacturer's protocol and subjected to sequence analysis. The sequence analysis was outsourced to FASMAC Co., Ltd. using the primer set 518F / 800R. The sequence information of the primers used in this experiment is shown in Table 1 below.

[0052] [Table 1]

[0053] The sequence and wavelength information obtained from the sequence analysis was edited using the sequence editing software ApE, and the species was identified by performing a homology search of the 16S rRNA gene sequence of each DL strain with the sequences registered in NCBI. The species of each of the 16 strains identified through homology searches are shown in Table 2. As a result, Lactobacillus gasseri ( Lactobacillus gasseri ), Lactobacillus paragasseri ( Lactobacillus paragasseri ), Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus murielis ( Lactobacillus mulieris ) Four species of bacteria belonging to the genus Lactobacillus were found.

[0054] [Table 2]

[0055] Experiment 4: <Search for DL ​​strains that express barrier function-related genes> Using human cervical epithelial NCE16IIA cells, we analyzed the expression of the following six barrier function-related genes when the DL culture supernatant of the DL16 strain obtained in Experiment 2 was added: CLD1 and OCLN, which are related factors of cell adhesion molecules; elafin, an antimicrobial peptide; MUC1, a humoral component; and HAS1 and HAS3, which are hyaluronan synthases.

[0056] Elafin is an elastase inhibitor and antimicrobial peptide found in mucosal tissues such as the intestine and vagina. It is a multifaceted mediator that inhibits inflammatory cell infiltration and activation, promotes wound healing, and promotes intercellular adhesion. OCLN and CLD1 are essential proteins for the formation of tight junctions in epidermal cells and are required for normal skin barrier function. MUC1, a type of mucin, functions as an adhesion protein while also protecting epithelial cells from bacteria and enzymes. HAS1 and HAS3 are enzymes essential for hyaluronan synthesis, a major component of the extracellular matrix, and regulate cell adhesion, migration, and differentiation. Therefore, by focusing on these six factors, we can investigate their direct or indirect involvement in biological barrier function in a broad sense.

[0057] Human cervical epithelial NCE16IIA cells (National Institutes of Biomedical Innovation, Health and Nutrition; NIBIOHN) were cultured in MCDB153 medium (Functional Peptide Institute) supplemented with 5 μg / mL insulin, 0.5 μg / mL hydrocortisone, 10 μg / mL transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng / mL epidermal growth factor (EGF), and 50 μg / mL bovine pituitary extract (BPE). Then, 1 × 10 cells were cultured per well. 5 The cells were seeded onto a 24-well plate and cultured until they reached 90% confluency. After replacing the culture medium with fresh one, the DL culture supernatant was added to a final concentration of 10% (v / v) and exposed for 24 hours. After that, the culture medium was removed and RNA was extracted.

[0058] RNA was extracted from cells treated with the DL culture supernatant using ISOGEN II (Nippon Gene) according to the manufacturer's protocol. The extracted RNA was dissolved in dH2O, and genomic DNA was removed using DNase I (Sigma). cDNA was then synthesized by reverse transcription using ReverTra™ Ace qPCR Master Mix (Toyobo).

[0059] Quantitative gene expression analysis was performed using THUNDERBIRD SYBR Next qPCR Mix (TOYOBO) with the CFX Connect™ Real-Time Detection System (Bio-Rad) according to the manufacturer's protocol. Primer sequences for CLD1, elafin, HAS1, HAS3, MUC1, OCLN, and the reference gene β-actin are shown in Table 3.

[0060] [Table 3]

[0061] Gene expression levels were evaluated using the ΔΔCt method, with untreated conditions as the control. This was repeated three times, and the average relative values ​​obtained from the three experiments was calculated. The resulting data was analyzed using R version 4.2.2, and the expression levels of each gene were scaled (z-scored) to have a mean of 0 and a variance of 1. Cluster analysis and heat maps using the 'ComplexHeatmap' package were then used to examine the effects on the expression of barrier function-related genes between bacterial species and strains. The results are shown in Figure 2 (note that in Figure 2, "BG-DL427" is referred to as "DL427").

[0062] As shown in Figure 2, although the same bacterial species tend to exhibit similar behavior, the expression levels of each of the six barrier function-related genes varied depending on the strain. Among these, it was found that the Lactobacillus paragasseri BG-DL427 strain significantly increased the expression levels of all six barrier function-related genes. In addition to the expression of intercellular adhesion molecules such as CLD1 and OCLN, the BG-DL427 strain also induced a dramatic increase in the expression of the antimicrobial peptide elastase inhibitor elafin, suggesting that it may be effective against vaginal inflammation, pathogen invasion, bacterial vaginosis, etc. Furthermore, given that the culture supernatant of the BG-DL427 strain inhibits the growth of Staphylococcus aureus and Escherichia coli, the culture supernatants of both strains may be applicable to products that solve or prevent vaginal problems.

[0063] Experiment 5: Comparison of vaginal barrier function-related gene expression between intestinal Lactobacillus paragasseri type strains and vaginal Lactobacillus paragasseri isolates In Experiment 4, we compared the expression of barrier-related genes in vaginal cells using the BG-DL427 strain and two JCM strains of human intestinal Lactobacillus paragasseri, JCM1130 and JCM5343, which were found to significantly increase the expression of barrier-related genes in vaginal cells.

[0064] Culture supernatants for the JCM2 strain were obtained in the same manner as the DL culture supernatants obtained in Experiment 2. Specifically, glycerol stocks of each JCM2 strain were inoculated into 10 mL of MRS liquid medium to a final concentration of 1%, and cultured at 35°C under aerobic conditions for 24 hours. The resulting bacterial solution was centrifuged (14,000 × g, 10 minutes), and the culture supernatant was collected, sterilized by filtration using a 0.22 μm syringe filter, and stored at 4°C. This was used as the culture supernatant for each JCM2 strain.

[0065] Human cervical epithelial NCE16IIA cells (gift from NIBIOHN) were cultured in the same manner as in Experiment 4, and then JCM2 cell culture supernatant was added to a final concentration of 10% (v / v), just as in Experiment 4, and the cells were exposed to the culture supernatant for 24 hours. After that, the culture medium was removed and RNA was extracted. RNA extraction, cDNA synthesis, and quantitative gene expression analysis from cells treated with the DL and JCM culture supernatants were also performed in the same manner as in Experiment 4. The gene expression levels were evaluated by the ΔΔCt method using untreated conditions as a control, and significant differences were determined by multiple comparison testing using Tukey's test (n=6).

[0066] The results of this gene expression analysis are shown in Figures 3 to 6 (note that "BG-DL427" is abbreviated as "DL427" in Figures 3 to 6). While the intestinal-derived JCM5343 strain was effective in increasing the gene expression of MUC1, it was less effective for elafin, CLD1, and OCLN, and the JCM2 strain, including the JCM1130 strain, did not significantly increase all six genes related to barrier function.

[0067] The culture supernatant of the BG-DL427 strain inhibits the growth of Staphylococcus aureus and Escherichia coli, thereby suppressing vaginal inflammation and odor under normal conditions. In addition, it induces a dramatic increase in the expression of the antibacterial peptide elastase inhibitor elafin, which attacks and kills pathogens even in cases of inflammation and vaginitis. It also increases the gene expression of intercellular adhesion molecules such as CLD1 and OCLN, thereby exerting an excellent barrier function to prevent pathogens from entering the body. Furthermore, increased gene expression of MUC1, HAS1, and HAS3 is expected to function in mucosal protection. Therefore, culture supernatants that increase the gene expression of these factors could be used in products that prevent and maintain the normal state of tissues, treat disease conditions, and protect after recovery, not only in the vagina but also externally, such as on the skin surface, and internally, such as in the intestine.

[0068] Experiment 6: Comparison of barrier function-related gene expression by treatment with killed bacteria When killed cells of the above-mentioned human intestinal-derived JCM2 strain and the above-mentioned BG-DL427 strain were applied to human cervical epithelial NCE16IIA cells, the gene expression of cell adhesion molecule-related factors CLD1 and OCLN, the antimicrobial peptide elafin, and the humoral component MUC1 was examined.

[0069] Glycerol stocks of the JCM2 strain and the BG-DL427 strain were inoculated into 10 mL of MRS liquid medium to a final concentration of 1% and cultured at 35°C under aerobic conditions for 24 hours. A 1.0 mL aliquot was collected from the resulting bacterial solution and centrifuged (9,000 × g, 10 minutes, 4°C). The culture supernatant was discarded to obtain a bacterial cell pellet. The bacterial cell pellet was dissolved in PBS(-), gently mixed, centrifuged (9,000 × g, 10 minutes, 4°C), the culture supernatant was discarded, and the pellet was redissolved in PBS(-). This was heat-treated at 95°C for 30 minutes, centrifuged (9,000 × g, 10 minutes, 4°C), and the culture supernatant was discarded to prepare killed bacterial cells of each strain. The killed bacterial cells were stored at -80°C.

[0070] Human cervical epithelial NCE16IIA cells (gift from NIBIOHN) were cultured in MCDB153 medium (Functional Peptide Institute) supplemented with 5 μg / mL insulin, 0.5 μg / mL hydrocortisone, 10 μg / mL transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng / mL EGF, and 50 μg / mL bovine pituitary extract (BPE). 2 × 10 cells per well were cultured. 5 The cells were seeded in a 24-well plate and cultured until they reached 90% confluency. After replacing the culture medium with new medium, 1 × 10 5 The killed bacteria were added to the culture medium at a concentration of 1000 cells / mL and exposed for 24 hours. After that, the culture medium was removed and RNA was extracted.

[0071] RNA extraction from cells treated with the killed bacteria, cDNA synthesis, and quantitative gene expression analysis were performed as in Experiment 4. Gene expression levels were evaluated using the ΔΔCt method with untreated conditions as the control, and significant differences were determined by multiple comparison testing using Tukey's test (n = 3).

[0072] The results of gene expression analysis are shown in Figs. 7 to 10 (in Figs. 7 to 10, "BG-DL427" is denoted as "DL427"). In the BG-DL427 strain, no fluctuation was observed in the gene expression of OCLN, but an increase in the expression of the genes of CLD1, elafin, and MUC1 was observed. However, no significant difference was recognized in the gene expression between the BG-DL427 strain and the JCM strain. From these results, it was found that the behavior was different from the case of treatment with the culture supernatant. In addition, since an increase in the expression of the CLD1, elafin, and MUC1 genes was recognized in the killed cells of the BG-DL427 strain, it is thought that even if it is not as effective as the culture supernatant, the killed cells may also be beneficially effective against inflammation in the vagina, invasion of pathogens, bacterial vaginitis, etc., and could be applied to products for solving and preventing vaginal troubles.

[0073] Experiment 7: <Production of the original solution of the BG-DL427 culture supernatant> The bacterial solution obtained by thawing the cryopreserved BG-DL427 strain was inoculated into a soymilk-like medium (glucose 0.6%, yeast extract 0.5%, skim milk powder 2.8%, soybean powder 7.7%, antifoaming agent 0.01%, the balance water (each weight%)) so that the inoculated weight was 0.1 wt% of the medium weight, and cultured for 72 hours to adjust the pH to 4.5 to 5.5. Then, the obtained culture solution was centrifuged, the culture supernatant was recovered, clarified, and further ultrafiltered to produce a 5-fold concentrated solution, which was used as the original solution of the BG-DL427 culture supernatant for the production of various preparations thereafter.

[0074] Experiment 8: <Production of various preparations from the original solution of the BG-DL427 culture supernatant> 1-1. Production of a liquid preparation or a lotion preparation (1) The above-mentioned original solution of the BG-DL427 culture supernatant was added to a diluent prepared by blending a fragrance, an emulsifier, and a preservative in 80 wt% of distilled water, 10 wt% of alcohol, and several wt% of glycerin so that the original solution of the BG-DL427 culture supernatant was 4 wt% of the total amount, to obtain a lotion preparation. Also, a liquid preparation was obtained by substituting the glycerin component with distilled water. This lotion preparation or liquid preparation can be applied as a care preparation for the skin and around delicate zones by using it as it is as a lotion or a lotion.

[0075] Manufacture of liquid preparation or lotion preparation (2) To a diluted solution prepared by adding a sweetener, such as sugar, to 95 wt% of soy milk, the above-mentioned BG-DL427 culture supernatant stock solution was added to obtain a drink such that the BG-DL427 culture supernatant stock solution accounted for 4 wt% of the total amount. This drink can be applied as a beverage to maintain a healthy intestinal environment or as a health supplement drink to create a healthy intestinal environment.

[0076] Manufacture of cream preparation To a cream prepared by blending petrolatum, olive oil, ethanol, a pigment with a fragrance, an emulsifier, and a preservative, the above-mentioned BG-DL427 culture supernatant stock solution was added to obtain a cream-like preparation such that the BG-DL427 culture supernatant stock solution accounted for 4 wt% of the total amount. This cream-like preparation can be directly applied as a cream product or as a care preparation for the skin and around delicate zones. It can also be applied as an anti-inflammatory agent to be applied to inflamed areas.

[0077] Manufacture of tablets The above-mentioned BG-DL427 culture supernatant stock solution was added to starch and starch paste, and molded to obtain tablets such that the BG-DL427 culture supernatant stock solution accounted for 4 wt% of the total amount. These tablets can be applied as oral preparations, as care preparations for skin diseases such as atopy, and as anti-inflammatory preparations for injuries and inside the digestive organs.

[0078] Manufacture of capsule preparations The above-mentioned liquid preparation, lotion preparation, and cream preparation can be filled into capsules and encapsulated, and can be applied as an insert for vaginal insertion for daily care or as a vaginal care preparation when the vagina becomes inflamed. It can also be applied as an oral preparation.

[0079] Experiment 9: <Manufacture of BG-DL427 cell-containing liquid> The bacterial solution obtained by thawing the cryopreserved BG-DL427 strain was added to MRS liquid medium so that the inoculated weight was 0.1 wt% of the medium weight, and cultured for 72 hours to obtain an OD 600 value of 4.5 - 5.0. The obtained culture solution was centrifuged, the culture supernatant was discarded, and a cell pellet was obtained. Then, this cell pellet was diluted with PBS(-) to obtain a BG-DL427 cell-containing solution for producing various preparations later. The BG-DL427 cell-containing solution contains viable cells of the BG-DL427 strain.

[0080] Experiment 10: Production of Various Preparations from the BG-DL427 Cell-Containing Solution 1-1. Production of Liquid Preparation or Lotion Preparation (1) To a dilution prepared by mixing 80 wt% distilled water, 10 wt% alcohol, and several wt% glycerin with a fragrance, an emulsifier, and a preservative, the above-mentioned BG-DL427 cell-containing solution was added such that among a total of 1 g, the number of cells of the BG-DL427 strain was 1×10 8 ~5×10 9 to obtain a lotion preparation. Also, by substituting the content of glycerin with distilled water, a liquid preparation was obtained.

[0081] This lotion preparation or liquid preparation can be directly used as a lotion or a lotion, and is applicable as a care preparation for the skin and around delicate areas.

[0082] 1-2. Production of Liquid Preparation or Lotion Preparation (2) To a dilution prepared by mixing 95 wt% soy milk with a sweetener such as sugar, the above-mentioned BG-DL427 culture supernatant stock solution was added to obtain a drink such that the BG-DL427 culture supernatant stock solution was 4 wt% of the total amount. This drink can be applied as a beverage or a health supplement drink for maintaining a healthy intestinal flora or achieving a healthy intestinal flora.

[0083] 2. Production of Cream Preparation To a cream prepared by mixing petrolatum, olive oil, ethanol, and a pigment with a fragrance, an emulsifier, and a preservative, the above-mentioned BG-DL427 cell-containing solution was added such that among a total of 1 g, the number of cells of the BG-DL427 strain was 1×10 8 ~5×10 9A cream preparation was obtained so as to contain [quantity]. This cream preparation can be applied as it is as a cream product or as a care preparation for the skin and around delicate areas. It can also be applied as an anti-inflammatory agent to be applied to the inflamed area.

[0084] 3. Production of tablets Starch and starch paste were added with the above BG-DL427 cell-containing liquid, and among a total amount of 1 g thereof, the cells of the BG-DL427 strain were 1×10 8 ~5×10 9 Tablets were obtained by molding so as to contain [quantity]. This tablet can be applied as an oral preparation, as a care preparation for skin diseases such as atopy, and as an anti-inflammatory preparation for trauma and inside the digestive organs.

[0085] 4. Production of capsules The above liquid preparation, lotion preparation, and cream preparation can be filled into capsules and encapsulated, and can be applied as an insert for vaginal insertion for daily care or as a vaginal care preparation when the vagina becomes inflamed. It can also be applied as an oral preparation.

[0086] Experiment 11: <Production of various preparations from the BG-DL427 culture supernatant stock solution and the BG-DL427 cell-containing liquid> The above BG-DL427 culture supernatant stock solution and the above BG-DL427 cell-containing liquid were mixed in an appropriate amount, and liquid preparations, lotion preparations, tablets, and capsules were produced in the same manner as the production methods of the above various preparations except that they were made to contain the above effective amount. More specifically, the contribution ratios of the BG-DL427 culture supernatant stock solution and the above BG-DL427 cell-containing liquid were determined, and the respective amounts of active ingredients were determined and formulated according to the contribution ratios.

[0087] Experiment 12: <Production of lactic acid bacteria-produced substances> Multiple species of common lactic acid bacteria belonging to the genus Lactobacillus, the genus Lactococcus, and the genus Enterococcus, and each of the BG-DL427 strains were inoculated into MRS medium and cultured alone at 34°C in an aerobic environment, OD 660Lactic acid bacteria were isolated and cultured until a β-glucan value of approximately 1.3 was achieved. From these, groups were created by combining lactic acid bacteria known to work well together. Lactic acid bacteria from each group were added to soy milk medium, mixed, and cultured at 32-36°C for approximately 1 day under an aerobic environment. The BG-DL427 strain was included in the same group as other Lactobacillus lactic acid bacteria. All cultures were then mixed and cultured in soy milk medium for an additional 25-30 hours. The resulting culture was pulverized using a homogenizer to obtain a uniform suspension. This suspension was then filtered through a cloth filter to obtain a filtrate and a fermentation residue. The filtrate was then filtered to obtain a lactic acid bacteria product. This lactic acid bacteria product contained bacterial components derived from killed cells of the lactic acid bacteria and BG-DL427 strain, products produced by the lactic acid bacteria and BG-DL427 strain, and liquid components derived from the soybeans and soy milk used as the culture medium ingredients.

[0088] The above-mentioned lactic acid bacteria produced substances contain a large amount of acetic acid components, and therefore, are expected to have effects such as inhibiting fat synthesis, reducing visceral fat, preventing high blood pressure, and inhibiting postprandial increases in blood sugar levels. Furthermore, since the active ingredient contains culture supernatant or killed cells of vaginal-derived Lactobacillus bacteria, it is expected to have a different effect from that of intestinal bacteria, improving the barrier function of tissues.

Claims

1. A bacterium, the BG-DL427 strain belonging to the species Lactobacillus paragasseri, deposited under Accession Number: NITE P-04115, which inhibits the growth of Staphylococcus aureus and Escherichia coli and increases the gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue.

2. The bacterium is the BG-DL427 strain belonging to the species Lactobacillus paragasseri deposited under Accession Number: NITE P-04115, and the culture supernatant thereof suppresses the growth of Staphylococcus aureus and Escherichia coli and increases the gene expression of six factors, namely CLD1, OCLN, elafin, MUC1, HAS1, and HAS3, which are associated with the barrier function of vaginal tissue.

3. The bacterium is the BG-DL427 strain belonging to the species Lactobacillus paragasseri deposited under Accession Number: NITE P-04115, and its killed cells suppress the growth of Staphylococcus aureus and Escherichia coli and increase the gene expression of three factors, CLD1, elafin, and MUC1, which are associated with the barrier function of vaginal tissue.

4. A growth inhibitor for Staphylococcus aureus or Escherichia coli, comprising a culture supernatant of the bacterium according to claim 1 or 2 or killed cells of the bacterium according to claim 1 or 3 as an active ingredient.

5. 10. An agent for improving the barrier function of vaginal tissue, comprising a culture supernatant of the bacterium according to claim 1 or claim 2 or killed cells of the bacterium according to claim 1 or claim 3 as an active ingredient.

6. A lactic acid bacterium product comprising a culture supernatant of the bacterium according to claim 1 or claim 2 or killed cells of the bacterium according to claim 1 or claim 3 as an active ingredient.

7. A preparation comprising a culture supernatant of the bacterium according to claim 1 or claim 2 or killed cells of the bacterium according to claim 1 or claim 3 as an active ingredient.

Images