Method for screening for lactic acid bacillus, lactic acid bacillus, and cleaning composition, food and drink product, and cosmetic product each containing same

Abstract

[Problem] The present invention provides: a lactic acid bacillus effective for women's health in the fields of gynecology, urology, and the like; a method for screening for the lactic acid bacillus; and a cleaning composition, a food and drink product, and a cosmetic product each containing the lactic acid bacillus. [Solution] A method for screening for a lactic acid bacillus according to the present invention comprises: a culturing step for infecting cells with Lactobacillus crispatus and culturing the infected cells; a measuring step for measuring an HβD-2 production-inducing ability from the cultured cells; and a selecting step for performing the culturing step and the measuring step on a plurality of strains of the Lactobacillus crispatus, comparing HβD-2 production-inducing abilities obtained through the measuring step, and selecting a strain having a high HβD-2 production-inducing ability.

Description

Screening method for lactobacillus, lactobacillus and cleaning composition, food and beverage products, and cosmetic products containing the same

[0001] The present invention relates to a method for screening lactobacilli, lactobacilli, and cleaning compositions, food and beverage products, and cosmetic products containing the same.

[0002] The female hormone estrogen is secreted in increasing amounts from the ovaries during puberty and decreases during menopause. Due to the action of estrogen, glycogen increases in vaginal epithelial cells and vaginal mucus. Lactobacillus bacteria use glycogen or its metabolic products for energy metabolism, producing lactic acid and hydrogen peroxide, which keeps the vagina acidic and inhibits the growth of pathogenic microorganisms.

[0003] It has been reported that one or more species of Lactobacillus, mainly L. crispatus, L. iners, L. gasseri, and L. jensenii, which belong to the genus Lactobacillus, are normally present in the female genus, particularly the vagina and cervix (Non-Patent Document 1).

[0004] It has been reported that when Lactobacillus crispus is predominant in the vagina, the risk of chlamydia (Non-Patent Document 2) and HIV infection (Non-Patent Document 3), bacterial vaginosis (Non-Patent Document 4), premature birth (Non-Patent Document 5), infertility (Non-Patent Document 6), and cervical cancer incidence (Non-Patent Document 7) are significantly lower than when Lactobacillus einerii is predominant. Therefore, it is known that, among lactobacilli, Lactobacillus crispus in particular plays an important role in the health of women in the field of obstetrics and gynecology.

[0005] In addition, it has been reported in recent years that pregnant women with high concentrations of the antibacterial peptide human β-defensin-2 (HβD-2) in their vaginal mucus have a significantly higher rate of normal delivery (normal birth after the 37th week) (Non-patent Document 8).

[0006] Ravel et al.,“Vaginal microbiome of reproductive-age women.”, Proceedings of the National Academy of Sciences of the United States of America., 108 Supplement_1, 4680-4687 (2011)Van Houdt et al.,“Lactobacillus iners-dominated vaginal microbiota is associated with increased susceptibility to Chlamydia trachomatis infection in Dutch women: A case-control study.” Sexual Transmitted Infections, 94(2), 17-123, (2018)Hoang et al., “The cervicovaginal mucus barrier to HIV-1 is diminished in bacterial vaginosis.” PLoS Pathogens, 16(1), e1008236, (2020)Almeida et al., “Lactobacillus crispatus protects against bacterial vaginosis.” Genetics and Molecular Research, 18(4), gmr18475, (2019).Kindinger et al., “The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk. ” Microbiome, 5(1), (2017)Kadogami et al.,“Impact of Lactobacillus in the uterine microbiota on in vitro fertilization outcomes.” Journal of Reproductive Immunology, 160, 104138, (2023) Norenhag et al., “The vaginal microbiota, human papillomavirus and cervical dysplasia: a systematic review and network meta-analysis. ”, An International Journal of Obstetrics & Gynaecology, 127(2), 171-180, (2020) Elovitz et al., “Cervicovaginal microbiota and local immune response modulate the risk of spontaneous preterm delivery.”, Nature Commun., 10(1), 1305 (2019).

[0007] The present invention aims to provide lactobacillus bacteria that are effective for the health of women in the fields of obstetrics and gynecology, urology, etc., a screening method for the same, and cleansing compositions, food and beverage products, and cosmetic products containing the same.

[0008] The present inventors have found that the above-mentioned problems can be solved by the present invention having the following aspects. Aspect 1: A method for screening Lactobacillus, comprising: a culturing step of infecting cells with Lactobacillus crispus and culturing the infected cells; a measuring step of measuring the ability of the cultured cells to induce HβD-2 production; and a selection step of performing the culturing step and the measuring step on multiple strains of Lactobacillus crispus, comparing the HβD-2 production-inducing abilities obtained in the measuring step, and selecting strains with a high ability to induce HβD-2 production. Aspect 2: The method of Aspect 1, wherein the Lactobacillus crispus is collected from the female genitalia. Aspect 3: The method of Aspect 1, wherein the cells are vaginal epithelial cells. Aspect 4: The method of Aspect 1, wherein the culturing step is performed at a multiplicity of infection (MOI) in the range of 17 to 150. Aspect 5: The method of Aspect 1, wherein the measuring step is a step of measuring the level of hβD-2 mRNA expression by quantitative PCR. <Aspect 6> A lactobacillus or killed cells thereof obtained by screening using the method according to any one of Aspects 1 to 5. <Aspect 7> A method for producing killed cells of lactobacillus obtained by screening using the method according to any one of Aspects 1 to 5, the method comprising: a culturing step of culturing lactobacillus in a medium; a sterilization step of sterilizing the lactobacillus in the medium used in the culturing step at 80°C or less for one hour or more to obtain killed cells; and a recovering step of the killed cells. <Aspect 8> The lactobacillus or killed cells thereof according to Aspect 6, which is Lactobacillus crispatus HMS-115 (Accession Number: NITE P-04100) or Lactobacillus crispatus HMS-122 (Accession Number: NITE P-04101). <Aspect 9> A vaginal cleansing composition comprising the lactobacillus or killed cells thereof according to Aspect 6, and water. <Aspect 10> The vaginal cleansing composition according to Aspect 9, further comprising lactic acid and hydrogen peroxide. <Aspect 11> A food or beverage product, a cosmetic product, or an ingredient thereof, comprising the lactobacillus or killed cells thereof according to Aspect 6. <Aspect 12> A method for producing a vaginal cleansing composition, comprising mixing lactobacillus or killed cells thereof with water, wherein the lactobacillus has been screened by the method according to Aspect 1.Aspect 13: A method for producing a vaginal cleansing composition, comprising mixing killed lactobacillus cells with water, wherein the killed lactobacillus cells are produced by the method described in Aspect 7. Aspect 14: A method for producing a food or beverage product or a cosmetic product containing killed lactobacillus cells, wherein the killed lactobacillus cells are produced by the method described in Aspect 7.

[0009] According to the present invention, it is possible to provide lactobacillus bacteria that are effective for the health of women in the fields of obstetrics and gynecology, urology, etc., a screening method for the same, and cleansing compositions, food and beverage products, and cosmetic products containing the same.

[0010] Figure 1 shows the types and proportions of bacteria constituting the vaginas of 24 subjects based on the results of amplicon sequencing analysis using a next-generation sequencer. Figure 2 shows the results of measuring the ability of experimental Lactobacillus strains to induce HβD-2 production. Figure 3 shows the results of measuring the ability of 17 strains of Clostridium crispatum collected from subjects to induce HβD-2 production. Figure 4 shows fluorescently stained images of nuclei of vaginal epithelial cells and nuclei of Clostridium crispatum. The white line in the figure indicates 20 μm. Figure 5 shows the difference in hβD-2 mRNA expression levels in vaginal epithelial cells between live and two types of killed bacteria in the DPBS-treated group and HMS-115. Figure 6 shows the difference in the sedimentation properties of live and two types of killed bacteria in HMS-115 in DPBS suspension. Figure 7 shows the difference in mRNA expression levels of the inflammatory cytokine TNF-α between live and killed bacteria in the DPBS-treated group and HMS-115 in vaginal epithelial cells. Figure 8 shows the difference in mRNA expression levels of the inflammatory cytokine IL-8 between live and killed bacteria in the DPBS-treated group and HMS-115 in vaginal epithelial cells. Figure 9 shows the difference in mRNA expression levels of mucus mucin muc1 between live and killed bacteria in the DPBS-treated group and HMS-115 in vaginal epithelial cells. Figure 10 shows the difference in mRNA expression levels of hβD-2 between live and killed bacteria in the DPBS-treated group and HMS-115 and HMS-122 in cervical epithelial cells. Figure 11 shows the difference in mRNA expression levels of hβD-2 between live and killed bacteria in the DPBS-treated group and HMS-115 and HMS-122 in oropharyngeal epithelial cells. Figure 12 shows the difference in hβD-2 mRNA expression levels in skin epithelial cells between the DPBS-treated group and the killed HMS-115 and HMS-122 bacteria. Figure 13 shows the difference in hβD-2 mRNA expression levels in colon epithelial cells between the DPBS-treated group and the killed HMS-115 bacteria.

[0011] The screening method for lactobacillus of the present invention includes a culture step of infecting cells with Lactobacillus crispatus and culturing the infected cells, a measurement step of measuring the ability to induce HβD-2 production from the cultured cells, and a selection step of performing the culture step and the measurement step for multiple strains of Lactobacillus crispatus, comparing the HβD-2 production induction abilities obtained by the measurement step, and selecting strains with high ability to induce HβD-2 production.

[0012] Various factors are known to contribute to women's gynecological health. In particular, the present inventors hypothesized and verified that the recently discovered benefits of HβD-2 on pregnant women are related to vaginal Lactobacillus crispus. As a result, they found that infection of cells with some Lactobacillus crispus enhances HβD-2 production in those cells. While not all Lactobacillus crispus strains are highly capable of inducing HβD-2 production, some strains of Lactobacillus crispus have a very high ability to induce HβD-2 production. These strains, when used as vaginal cleansing compositions or live bacterial preparations, can be maintained as normal flora in the vagina after cleansing, suggesting that such cleansing compositions may be highly effective for women's gynecological health. Furthermore, because the vaginal bacterial flora and the bladder bacterial flora share a high degree of homology, they are also expected to be useful for urological health.

[0013] Furthermore, the hβD-2 mRNA expression enhancing effect of Lactobacillus crispatus obtained by the method of the present invention was also observed in epithelial cells other than vaginal epithelial cells (cervix, oropharynx, skin, colon). Therefore, these lactobacilli can contribute to the maintenance of homeostasis of infection defense and host defense not only through vaginal epithelium but also through epithelial cells throughout the body, such as those of the cervix, oropharynx, skin, and intestinal mucosa, and are therefore expected to be advantageous when used in food and beverage products, cosmetic products, or as raw materials for these products.

[0014] The strain of Lactobacillus crispatus used for screening may be a strain collected from any site in the female genitalia, particularly the vagina, as long as it can exert the ability to induce HβD-2 production in the female genitalia, particularly the vagina. For example, the strain of Lactobacillus crispatus may be collected from the female genitalia.

[0015] The collected bacteria can be confirmed to be Lactobacillus crispatus by PCR analysis, amplicon sequence analysis, or the like.

[0016] The harvested M. crispatus can be grown in culture before infecting cells. In this case, the culture is grown in a conventional CO ２ The experiment can be carried out at around 37°C using an incubator and an appropriate medium.

[0017] The type of cells to be infected with a strain of C. crispatus is not particularly limited, as long as they are capable of producing HβD-2 in the female genitalia, particularly the vagina, as a result of infection. Examples of such cells include adipocytes, epithelial cells, endothelial cells, epidermal cells, fibroblasts, mesenchymal cells, etc., with vaginal epithelial cells being particularly preferred.

[0018] The cells used can be grown in culture before infection with C. crispatus. In this case, the culture is grown in a conventional CO ２ The experiment can be carried out at around 37°C using an incubator and an appropriate medium.

[0019] When the above-mentioned cells are infected with Bacillus crispatus in the culture step, the multiplicity of infection (MOI) can be set in the range of 17 to 150, or 30 to 70.

[0020] The cells infected with C. crispatus can be further cultured and grown. ２ The experiment can be carried out at around 37°C using an incubator and an appropriate medium.

[0021] After culturing the cells infected with Lactobacillus crispatus in this manner, a measurement step is performed in which the ability of the cells to induce HβD-2 production is measured. The method for measuring the ability to induce HβD-2 production is not particularly limited, and can be measured, for example, by calculating the amount of hβD-2 mRNA expression by quantitative PCR. In this case, specifically, the measurement step can include extracting RNA from the cultured cells, performing a reverse transcription reaction using the RNA as a template to prepare cDNA, and performing quantitative PCR using the cDNA as a template.

[0022] By measuring the HβD-2 production-inducing ability measured in this way in multiple strains of Lactobacillus crispatus, it is possible to select from among them a strain with a high ability to induce HβD-2 production. HβD-2 is a type of antimicrobial peptide produced by host cells, and while it does not exhibit antibacterial activity against Lactobacillus, it does exhibit antibacterial activity against bacteria that cause bacterial vaginosis. Therefore, when a strain with a high ability to induce HβD-2 production is infected into vaginal epithelial cells, a large amount of HβD-2 is produced by the host cells, which can promote women's health in the fields of obstetrics and gynecology and urology.

[0023] The present inventors screened lactobacilli using this method and found that Lactobacillus crispatus HMS-115 (domestic deposit accession number: NITE P-04100; international deposit receiving institution: National Institute of Technology and Evaluation Patent Microorganisms Depositary, date of receipt: April 30, 2025, accession number: NITE ABP-04100) and Lactobacillus crispatus HMS-122 (domestic deposit accession number: NITE P-04101; international deposit receiving institution: National Institute of Technology and Evaluation Patent Microorganisms Depositary, date of receipt: April 30, 2025, accession number: NITE ABP-04101) have extremely high HβD-2 production-inducing ability among Lactobacillus crispatus. ABP-04101) was discovered.

[0024] By contacting a cleansing composition containing the thus screened lactobacillus in water with cells to be infected, the cells become infected with the lactobacillus and the production of HβD-2 can be enhanced. In other words, when this cleansing composition is used for vaginal cleansing, bacterial vaginosis can be prevented, and women's health in the fields of obstetrics and gynecology and urology can be promoted. Furthermore, by further adding an appropriate concentration of lactic acid to such a vaginal cleansing composition, the pH in the vagina can be brought closer to a normal state. The effectiveness of these lactobacillus was strongly confirmed even when they were killed. In other words, it is believed that the bacterial cell structures of these lactobacillus can enhance the production of HβD-2.

[0025] Furthermore, the screened lactobacillus or killed bacteria thereof can contribute to infection defense and maintaining homeostasis of host defense not only through vaginal epithelium but also through epithelial cells throughout the body, such as those in the cervix, oropharynx, skin, and intestinal mucosa. Therefore, food and beverage products, cosmetic products, or ingredients thereof containing lactobacillus or killed bacteria thereof in a form capable of adhering to these cells are also useful. Here, the term "food and beverage products" is interpreted very broadly and includes all forms of food and beverage, and can have any known structure other than the inclusion of lactobacillus or killed bacteria thereof. The term "cosmetic products" is also interpreted very broadly and can take various forms, such as oily compositions, aqueous compositions, emulsions, creams, gels, and pack cosmetics, or cleansing cosmetics such as soaps and toothpastes. Furthermore, these lactobacillus or killed bacteria thereof may be used as ingredients in cosmetic products such as fragrances, perfumes, essential oils, and fragrances.

[0026] In fact, it was found that killed cells obtained by sterilizing the lactobacillus screened in this manner also enhance the production of HβD-2 in the cells. Killed cells have superior preservation properties when formulated compared to live cells, and therefore, killed cells are very effective when formulating a drug product.

[0027] Furthermore, the present inventors have found that lactobacilli killed by a specific sterilization method are particularly advantageous. Specifically, the killed bacteria not only enhance cellular HβD-2 production like live bacteria, but also exert antibacterial and mucus production-enhancing effects without inducing excessive inflammatory responses, demonstrating their usefulness greater than live bacteria.

[0028] This particular sterilization method involves sterilizing lactobacilli in the culture medium used for culturing them at a relatively low temperature for a long period of time, without washing them after culturing. When sterilization is performed after culturing lactobacilli, it is usually performed after washing the cultured lactobacilli. However, the inventors accidentally discovered that the above-mentioned advantageous effects were exhibited in samples that were mistakenly sterilized without washing. Furthermore, this sterilization method is also usually performed at a high temperature of 90°C or higher, but when sterilization treatment was performed at a low temperature for a long period of time, the above-mentioned advantageous effects were exhibited. When sterilization was performed while lactobacilli were still contained in the culture medium, it is thought that the active substances were protected by the culture medium and were less likely to be inactivated by such a low-temperature, long-term sterilization treatment.

[0029] Therefore, one embodiment of the present invention relates to a method for producing killed lactobacillus cells, which includes a culturing step of culturing lactobacillus such as Lactobacillus crispatus in a medium, a sterilizing step of sterilizing the lactobacillus in the medium used in the culturing step at 80°C or less for 1 hour or more to obtain killed cells, and a recovering step of the killed cells, wherein the recovering step may include a step of washing the killed cells.

[0030] The sterilization step may be performed at a temperature of 75° C. or less, 70° C. or less, or 65° C. or less, or at a temperature of 50° C. or more, 55° C. or more, or 60° C. or more. The temperature may be maintained for 1.5 hours or more, 2.0 hours or more, 2.5 hours or more, or 3.0 hours or more, or for 24 hours or less, 12 hours or less, 8 hours or less, or 6 hours or less.

[0031] The present invention will be explained in more detail in the following examples, but the present invention is not limited thereto.

[0032] Experiment 1: Screening and Evaluation of B. crispatus (Cultivation Process) (Strain Cultivation) Twenty-four premenopausal Japanese women aged 18 years or older who had been diagnosed by a physician as free of genital disease and who had not taken antibiotics or had not taken them within the past month were given informed consent to participate in this study. Vaginal mucus was collected from each woman using a specimen collection swab (regular FLOQ Swab™, Copan Japan Co., Ltd.), immersed in a tube containing specimen preservation solution, and refrigerated. A female genital isolate of B. crispatus (BEI Resources, Virginia, USA) with known subject and genomic information and available for distribution was also obtained.

[0033] These strains were cultured in Lactobacillus MRS broth liquid medium (Becton Dickinson Difco, New Jersey, USA) at 37°C in 5% CO ２ The cells were incubated under static conditions for 24 hours.

[0034] A suspension prepared by mixing equal amounts of the culture medium and 40% glycerol was stored as a glycerol stock at -80°C. Agar powder (Nacalai Tesque, Inc.) was added to each liquid medium to a final concentration of 1.5% by mass to prepare agar plates. The agar plates were cultured anaerobically at 37°C using a sealed pouch (Anelopac, Mitsubishi Gas Chemical Company, Inc.) and a sealed jar (Anelopac Square Jar, Mitsubishi Gas Chemical Company, Inc.).

[0035] <Cell Culture> Vaginal epithelial primary immortalized cells VK2 / E6E7 (VK2, ATCC CRL2616, American Type Culture Collection, Virginia, USA) were obtained.

[0036] The culture was supplemented with 909.1 μg / ml CaCl ２ Keratinocyte-SFM (Thermo Fisher Scientific, Massachusetts, USA) containing 909.1 μg / ml CaCl ２The medium was prepared by mixing equal volumes of EpiLife Medium (Thermo Fisher Scientific, Massachusetts, USA) containing BPE at 50 μg / ml and Recombinant Human EG at 5 ng / ml (hereinafter referred to as "VK2 medium"). An antibiotic for primary cells (Primocin™, Invivogen, California, USA) was added appropriately to a final concentration of 100 μg / ml.

[0037] Culture in VK2 medium at 37°C and 5% CO until confluent. ２ VK2 cells cultured under these conditions were washed with DPBS, and then 0.25% Trypsin / 2.21% EDTA (Trypsin EDTA, Corning, New York, USA) was added and incubated at 37°C in 5% CO ２ The flask was incubated for 5 minutes under these conditions. After confirming that the cells had detached from the bottom of the flask, neutralizing medium (RPMI-1640 medium containing 10% FBS, Sigma-Aldrich, Missouri, USA) was added and mixed. The cell suspension was transferred to a 15 ml tube and centrifuged (2000 rpm, 20°C, 5 minutes). The collected cells were suspended in VK2 medium, diluted appropriately, and used or passaged for experiments.

[0038] Bacterial Identification: A bacterial suspension suspended in specimen preservation solution (eSwab, Copan Japan Co., Ltd.) was serially diluted 10-fold with DPBS. 100 μl of each dilution was smeared onto MRS agar, 1% glucose-containing GAM agar (Shimadzu Diagnostics Co., Ltd.), 5% defibrinated sheep blood-containing tryptic soy agar (Becton Dickinson Difco), and Sabouraud agar plates (Shimadzu Diagnostics Co., Ltd.) using Conlarge. MRS agar, 1% glucose-containing GAM agar, and 5% defibrinated sheep blood-containing tryptic soy agar were cultured anaerobically at 37°C using AnaeroPack and sealed jars, while Sabouraud agar plates were cultured aerobically at 28°C for a set period. Amplicon sequencing analysis using a next-generation sequencer was also outsourced to Techno Suruga Lab.

[0039] The resulting colonies were picked and suspended in 30 μl of sterile purified water. Colony PCR was performed using 1 μl of the suspension as a template with primers and KOD Fx Neo (Toyobo Co., Ltd.) to identify the bacterial species (total 20 μl, 98°C / 10 minutes: 1 cycle, 98°C / 30 seconds, 60°C / 30 seconds, 72°C / 1 minute: 35 cycles, 72°C / 5 minutes: 1 cycle). This identified the colonies as B. crispatus.

[0040] The PCR and qPCR assayed in this example and their primers are described in detail in Kurakawa et al. "Establishment of a sensitive system for analysis of human vaginal microbiota on the basis of rRNA-targeted reverse transcription-quantitative PCR." Journal of microbiological methods, 111: 93-104 (2015) and van Galen et al. "Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion." Cell Stem Cell, 14 (1): 94-106 (2014). These documents can be referenced.

[0041] <Cell infection with C. crispatus> 2.0 x 10 VK2 cells were placed in a 24-well plate. ５ Dispense 1 ml of cells / ml into aliquots and incubate at 37°C in 5% CO ２ After discarding the culture supernatant, the cells were washed once with 1 ml of DPBS, and then 900 μl of VK2 medium was added. 100 μl of a 1 ml suspension of B. crispatus prepared with DPBS to give a multiplicity of infection (MOI) of 50 was added to each well, and the cells were incubated overnight at 37°C in 5% CO ２ The cells were cultured for 6 hours under the conditions described above. A sample containing only DPBS instead of the Lactobacillus crispatus suspension was used as a negative control.

[0042] Quantitative PCR Measurement: The cells cultured as described above were washed twice with 1 ml of DPBS and then used for RNA extraction. RNA extraction was performed using the FastGene RNA Premium kit (Nippon Genetics Co., Ltd.). Using 1 μg of the resulting RNA as a template, reverse transcription was performed using iScript Reverse Transcription Supermix for RT-PCR (Bio-Rad Laboratories, California, USA) (total volume: 20 μl, 25°C / 5 min, 46°C / 20 min, 95°C / 1 min) to prepare cDNA.

[0043] Quantitative PCR (qPCR) was performed using 2.5 μl of the prepared cDNA as a template with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and the primers shown in the table below (10 μl, 40 cycles of 95°C / 5 seconds, 60°C / 1 minute). After obtaining Ct values, the ratio of HβD-2 gene to GAPDH gene in each sample treatment group was calculated, assuming the ratio in the DPBS-only control group was 1.

[0044]

[0045] The results of each test were performed three or more times in total, and the presence or absence of statistically significant differences was determined using Tukey's multiple comparison test or Student's t-test using GraphPad Prism Version 10 (GraphPad Software, California, USA).

[0046] Results: The results of amplicon sequencing are shown in Figure 1. Nine of the 24 subjects had Lactobacillus crispatus (community state type: CST, CST1) as the predominant vaginal bacterium. Einer's disease (CST3) was the most predominant bacterium in eight subjects, Lactobacillus gasseri (CST2) in one, and bacteria other than lactobacillus (CST4) in six.

[0047] Furthermore, by separating the 12 individuals from whom B. crispatus was collected based on colony size, color, shape, growth rate, etc., it was found that a total of 17 strains of B. crispatus were present.

[0048] The mRNA levels of HβD-2 were calculated and compared for the three experimental strains of M. crispatus and the 17 strains of M. crispatus obtained from the test specimens when co-cultured with vaginal epithelial cells.

[0049] First, the amount of HβD-2 mRNA was calculated using three experimental strains of Bacillus crispatus (BEI Resources) (HM-103, HM-637, and HM-638). The results are shown in FIG. 2.

[0050] As shown in Figure 2, mRNA expression levels were 10.8-fold, 50.0-fold, and 3.4-fold higher for HM-103, HM-637, and HM-638, respectively, compared to the DPBS control group, demonstrating significantly increased expression in the HM-637 treatment group compared to the other treatment groups. However, there was no statistically significant difference in HM-638 compared to the control group. Therefore, these results demonstrate that certain strains of Bacillus crispatus significantly increase HβD-2 production in infected cells.

[0051] Next, to select B. crispatus strains that have an effect of enhancing hβD-2 mRNA expression equal to or greater than that of the HM-637-treated group, the hβD-2 mRNA expression levels in vaginal epithelial cells were calculated for the 17 strains selected in this test. The results are shown in Figure 3.

[0052] As a result, the mRNA expression levels of hβD-2 were significantly higher in the HMS-115 (Accession Number: NITE P-04100) and HMS-122 (Accession Number: NITE P-04101) treatment groups, respectively, by 7.07-fold and 4.28-fold compared to the DPBS control group. Furthermore, compared to the HM-637 treatment group, the mRNA expression levels of hβD-2 in the HMS-115 and HMS-122 treatment groups were 2.11-fold and 1.28-fold higher, respectively, and significantly higher in the HMS-115 treatment group. Therefore, it was suggested that the HMS-115 and HMS-122 strains obtained in this study are strains that significantly contribute to the health benefits in the fields of obstetrics and gynecology and urology for women. In addition, the results in Figure 3 show that the expression level of HβD-2 mRNA with HM-637 was about 7 times that of the DPBS control, which is lower than the results in Figure 2. This is because the number of experiments in Figure 3 was increased, resulting in an average increase in the mRNA expression level compared to the DPBS control.

[0053] Experiment 2: Killed Bacteria of Crispertus and Evaluation of the Killed Bacteria Cultivation Process Cultivation of Bacterial Strains The three strains HM-637 (BEI Resources), HMS-115, and HMS-122 were cultured in the same manner as in Experiment 1.

[0054] Cells Used and Their Culture: Human vaginal epithelial primary immortalized cells VK2 / E6E7 (VK2, ATCC CRL2616) and human oropharyngeal carcinoma cells Detroit 562 (ATCC CCL-138) were obtained from the American Type Culture Collection (ATCC). Human cervical epithelial primary immortalized cells A2EN were obtained from Applied Biological Materials Inc. (British Columbia, Canada). Human endometrial cancer cells Hec-1A (JCRB1117) were obtained from the National Institutes of Biomedical Innovation, Health and Nutrition. Human colon cancer cells HCT116 (RCB2979) were obtained from RIKEN. Human epidermal keratinocytes HaCat were obtained from CLS Cell Lines Service GmbH (Eppelheim, Germany).

[0055] VK2 was cultured in the VK2 medium described above. Detroit 562 was cultured in Eagle's Minimum Essential Medium (EMEM, Thermo Fisher Scientific) containing 10% FBS, 1% non-essential amino acids, 2 mM L-glutamine (Nacalai Tesque, Inc.), 1 mM sodium pyruvate, and 1500 mg / L sodium bicarbonate (hereinafter referred to as "Detroit 562 medium"). A2EN was cultured in a medium containing 50 μg / mL BPE, 5 ng / mL EGF (Human Recombinant Growth Factor), and 909.1 μg / mL CaCl . ２ A medium containing Keratinocyte-SFM containing 10% FBS and 2 mM L-glutamine (Nacalai Tesque, Inc.) was used for culturing Hec-1A cells (hereinafter referred to as "Hec-1A medium"), and a McCoy's 5A medium (Corning) containing 10% FBS and 2 mM L-glutamine (Nacalai Tesque, Inc.) was used for culturing Hec-1A cells (hereinafter referred to as "Hec-1A medium"), and a McCoy's 5A medium containing 10% FBS was used for culturing HCT116 cells (hereinafter referred to as "HCT116 medium").

[0056] As an antibiotic, Primocin (Invivogen) was added to VK2 medium and A2EN medium to a final concentration of 100 μg / ml, and a penicillin-streptomycin mixed solution (Nacalai Tesque) was appropriately added to other cell culture media to a final concentration of 10,000 U / ml and 10,000 μg / ml, respectively.

[0057] Culture in the appropriate cell culture medium until the cells reach confluence in 5% CO at 37°C. ２ After washing with DPBS, 0.25% Trypsin / 2.21% EDTA (Trypsin EDTA, Corning) was added and the cells were incubated at 37°C in 5% CO ２ The flask was incubated for 5 minutes under these conditions. After confirming that the cells had detached from the bottom of the flask, neutralizing medium (RPMI-1640 medium containing 10% FBS, Sigma-Aldrich) was added and mixed. The cell suspension was transferred to a 15 ml tube and centrifuged (2000 rpm, 20°C, 5 minutes). The collected cells were suspended in the appropriate cell culture medium, diluted appropriately, and used or passaged for experiments.

[0058] Killed bacteria preparation method 1: 37°C, 5% CO ２ After 24 hours of static culture under these conditions, 1 ml of the culture medium was transferred to a 1.5 ml tube and, without washing, sterilized at low temperature for a long period of time, i.e., by incubating in a water bath at a constant temperature of 65°C for 4 hours. The culture was then centrifuged twice with 1 ml of DPBS (15,000 rpm, 4°C, 10 minutes), and suspended in the same amount of DPBS as used to prepare live bacteria so that the multiplicity of infection (MOI) was 50, to prepare a suspension of B. crispatus. After smearing this suspension on MRS agar medium and thoroughly culturing it, the absence of bacterial colonies confirmed the death of the bacteria.

[0059] Killed Bacteria Preparation Method 2: 37°C, 5% CO ２ After 24 hours of static culture under these conditions, 1 ml of the culture solution was transferred to a 1.5 ml tube and centrifuged (15,000 rpm, 4°C, 10 minutes) to remove the culture supernatant. The cells were then suspended in 1 ml of DPBS and sterilized at low temperature for a long period of time, i.e., by incubating in a water bath at 65°C for 4 hours. The cells were then centrifuged once with 1 ml of DPBS (15,000 rpm, 4°C, 10 minutes) and suspended in the same amount of DPBS as used to prepare live bacteria so that the multiplicity of infection (MOI) was 50, to prepare a suspension of Bacillus crispatus. After smearing this suspension on MRS agar medium and thoroughly culturing it, the absence of bacterial colonies confirmed the death of the bacteria.

[0060] <Infection of each epithelial cell type with C. crispatus> 2.0 x 10 epithelial cells of each type were placed in a 24-well plate. ５ Dispense 1 ml of cells / ml into aliquots and incubate at 37°C in 5% CO ２ After discarding the culture supernatant, the cells were washed once with 1 ml of DPBS, and then 900 μl of each cell culture medium containing no antibiotics was added. 100 μl of a 1 ml suspension of B. crispatus prepared with DPBS to give an MOI of 50 was added to each well, and the cells were incubated overnight at 37°C in 5% CO ２The cells were cultured for 6 hours under these conditions. After washing twice with 1 ml of DPBS, the cells were used for RNA extraction. A sample containing only DPBS instead of the B. crispatus suspension was used as a negative control.

[0061] Obtaining an image of lactobacilli attached to human vaginal epithelial cells: After placing a round cover glass (Microscope Cover Glasses Φ12 mm) in a 24-well plate, 2.0 × 10 VK2 cells were placed on the well. ５ Dispense 1 ml of cells / ml into aliquots and incubate at 37°C in 5% CO ２ The VK2 cells were cultured overnight under the conditions described above. After washing once with DPBS, 900 μl of antibiotic-free cell culture medium was dispensed into each well. 100 μl of a 1 ml suspension of C. crispatus prepared with DPBS at an MOI of 50 was added to each well.

[0062] After centrifugation (1,000 rpm, 30 seconds), the mixture was incubated at 37°C in 5% CO ２ The cells were co-cultured for 4 hours under the conditions described above. After co-culture, the cells were washed twice with DPBS and then fixed overnight at 4°C with 1 ml of 4% paraformaldehyde phosphate buffer (4% paraformaldehyde powder (Nacalai Tesque), 3x DPBS, pH 7.4) added. After washing twice with DPBS, the cells were air-dried. Subsequently, the cells were treated with 95% ethanol for 10 minutes and air-dried again. A fluorescent mounting medium (VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, California, USA)) was dropped onto the slide, which was then covered with a cover glass and left to stand in the dark. Fluorescent staining images of the samples were observed and photographed using a confocal laser scanning microscope (LSM980, Carl Zeiss, Oberkochen, Germany).

[0063] <Quantitative PCR Measurement> In the same manner as in Experiment 1, RNA was extracted from the cells after the addition of the Lactobacillus crispatus suspension, cDNA was prepared, and quantitative PCR (qPCR) was performed using the primers shown in Table 1 and the following Table 2. After obtaining the Ct value, the ratio of each sample treatment group was calculated when the ratio of the hβd-2, TNF-α, il-8, or muc1 gene to the gapdh gene in the DPBS control group was set to 1. Statistical processing was also performed in the same manner as in Experiment 1.

[0064]

[0065] <<Results>> <Evaluation of the ability of Lactobacillus crispatus to adhere to vaginal epithelial cells>> Figure 4 shows fluorescent staining images of the nuclei of vaginal epithelial cells and the nuclei of Lactobacillus crispatus. The upper row is an image of a bacterium with a high ability to induce hβD-2 expression, and the lower row is an image of a bacterium with a low ability to induce hβD-2 expression. The lower row shows essentially only the nuclei of vaginal epithelial cells, whereas the upper row shows the presence of a large number of Lactobacillus crispatus around the cell nuclei.

[0066] Therefore, in the HM-637, HMS-115, and HMS-122 treatment groups, in which the effect of enhancing hβD-2 mRNA expression in vaginal epithelial cells was observed, many L. crispatus bacteria adhered to the vaginal epithelial cells. In contrast, in the HMS-103, HMS-110, and HMS-119 treatment groups, in which the effect of enhancing expression was not observed, almost no L. crispatus bacteria adhered to the vaginal epithelial cells. This suggests that the ability of L. crispatus to enhance hβD-2 mRNA expression in vaginal epithelial cells is related to its ability to adhere to epithelial cells.

[0067] <Evaluation of the effect of live or dead Lactobacillus crispots on enhancing hβD-2 mRNA expression> Since it was suggested that the ability of Lactobacillus crispots to enhance hβD-2 mRNA expression in vaginal epithelial cells is related to its ability to adhere to epithelial cells, we used HMS-115 to confirm whether the same activity is observed in dead rather than live bacteria.

[0068] In this study, we confirmed the difference in the ability to enhance hβD-2 mRNA expression in vaginal epithelial cells between the killed bacteria prepared by Killed Bacterial Preparation Method 1 and the killed bacteria prepared by Killed Bacterial Preparation Method 2. Since HMS-115 was killed by heating for 4 hours, killed bacteria after heating for 4 hours were used.

[0069] As a result, as shown in Figure 5, the group (Dead1) treated with HMS-115 killed by killed bacteria preparation method 1 showed the same level of ability to enhance hβD-2 mRNA expression as the live bacteria treatment group, and showed a significant increase in hβD-2 mRNA expression compared to the DPBS-treated group. On the other hand, the group (Dead2) treated with killed bacteria preparation method 2 showed the same level of ability to enhance hβD-2 mRNA expression as the DPBS-treated group, with no significant difference observed. This suggests that the use of killed bacteria maintained the ability to enhance hβD-2 mRNA expression in vaginal epithelial cells, and that the ability to enhance hβD-2 mRNA expression varied greatly depending on the killed bacteria preparation method.

[0070] After preparing the live or killed cell suspension, the cells were vortexed and allowed to stand at room temperature for 24 hours. As shown in Figure 6, live cells and cells killed by killed cell preparation method 1 (Dead1) settled, whereas most cells of cells killed by killed cell preparation method 2 (Dead2) remained floating rather than settling. The sedimentation tendency of the cells may be due to the cell surface structure, and it is thought that changes in the cell surface structure due to differences in the killed cell preparation methods affected the ability of hβD-2 to enhance mRNA expression.

[0071] From the above, it was thought that the effect of Lactobacillus crispatus in enhancing HβD-2 production in vaginal epithelial cells was not due to metabolic products of Lactobacillus crispatus, but rather to bacterial constituents related to its ability to adhere to vaginal epithelial cells.

[0072] <Evaluation of effects other than the hβD-2 mRNA expression enhancement effect of dead and alive Lactobacillus crispus cells> Since dead Lactobacillus crispus cells maintained the ability to enhance hβD-2 mRNA expression in vaginal epithelial cells, the effects on other factors were examined.

[0073] The results showed that the mRNA expression levels of the inflammatory cytokines TNF-α and IL-8 were significantly elevated in the live bacteria group compared to the DPBS-treated group, whereas the expression levels were similar to those in the DPBS-treated group and no elevated expression was observed in the killed bacteria group (Figures 7 and 8). Furthermore, the expression levels of muc1, a mucus component found in vaginal epithelial cells, were similar to those in the DPBS-treated group and no significant difference was observed, whereas the expression levels were significantly elevated in the killed bacteria group compared to the DPBS-treated group (Figure 9).

[0074] These results suggest that killed B. crispatus bacteria induce the expression of mucus mucin without the expression of inflammatory cytokines, while maintaining the ability to enhance hβD-2 mRNA expression in vaginal epithelial cells. In other words, killed B. crispatus bacteria are more useful than live bacteria in that they exert antibacterial and mucus-enhancing effects without inducing excessive inflammatory responses.

[0075] <Evaluation of the relationship with the ability of Lactobacillus crispatus to enhance hβD-2 mRNA expression in various epithelial cells> Although we have focused on the vaginal mucosa so far, we calculated and compared the amount of hβD-2 mRNA when Lactobacillus crispatus was co-cultured with epithelial cells from sources other than the vagina (cervix, oropharynx, skin, and colon). Since the lactic acid produced by Lactobacillus crispatus has a significant effect on cells in culture media for oropharynx, skin, and colon epithelial cells, we analyzed the presence or absence of activity in these cells using the above-mentioned killed bacteria.

[0076] First, we calculated the amount of hβD-2 mRNA in cervical epithelial cells using live B. crispatus HMS-115 and HMS-122 cells. Significantly elevated expression was observed only in the live or killed HM-115 treatment groups compared to the DPBS control group (Figure 10). That is, the hβD-2 mRNA expression enhancement effect of B. crispatus was observed in cervical epithelial cells, both in live and killed cells, as in vaginal epithelial cells.

[0077] Similar experiments were then performed on oropharyngeal, skin, and colonic epithelial cells, and the hβD-2 mRNA expression in each cell group was significantly enhanced in the HMS-115 and HMS-122 killed bacteria treatment groups compared to the DPBS control group (Figures 11 to 13). In particular, hβD-2 mRNA expression was extremely high in skin epithelial cells, as shown in Figure 12, strongly suggesting the usefulness of the Lactobacillus crispatus of the present invention for skin epithelial cells.

[0078] HβD-2 has been reported to exhibit high antibacterial activity against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, and fungi such as Candida albicans. Although HβD-2 has low antibacterial activity against Gram-positive bacteria, it has been reported to enhance the bactericidal activity against the Gram-positive bacterium S. aureus, which causes suppurative diseases and food poisoning, by acting synergistically with a protease secreted by Staphylococcus epidermidis, a normal skin bacterium. It has also been reported to have antiviral activity against viruses such as HIV, respiratory syncytial virus, which causes respiratory infections, and varicella zoster virus, which causes chickenpox and shingles. Furthermore, it functions as a regulator of host immune responses, such as by enhancing the expression of cytokines and chemokines.

[0079] In this study, the effect of Lactobacillus crispatus in enhancing hβD-2 mRNA expression was observed in epithelial cells other than vaginal epithelial cells (cervix, oropharynx, skin, and colon), suggesting that HMS-115 and HMS-122 of Lactobacillus crispatus may contribute to infection defense and maintaining homeostasis of host defense through epithelial cells throughout the body, including not only the vaginal epithelium but also the cervix, oropharynx, skin, and intestinal mucosa.

[0080] [Rule 26, amended 22.05.2025]

Claims

1. A method for screening lactobacillus, comprising: a culturing step of infecting cells with Lactobacillus crispatus and culturing the infected cells; a measuring step of measuring the ability of the cultured cells to induce HβD-2 production; and a selection step of carrying out the culturing step and the measuring step for multiple strains of Lactobacillus crispatus, comparing the HβD-2 production induction abilities obtained by the measuring step, and selecting strains with a high ability to induce HβD-2 production.

2. The method of claim 1, wherein the Lactobacillus crispatus is collected from the female genitalia.

3. The method of claim 1, wherein the cells are vaginal epithelial cells.

4. The method according to claim 1, wherein the culturing step is carried out at a multiplicity of infection (MOI) in the range of 17 to 150.

5. The method according to claim 1, wherein the measuring step is a step of measuring the expression level of hβD-2 mRNA by quantitative PCR.

6. Lactobacillus or killed cells thereof obtained by screening using the method according to any one of claims 1 to 5.

7. A method for producing killed lactobacillus bacteria obtained by screening using the method according to any one of claims 1 to 5, comprising a culturing step of culturing lactobacillus bacteria in a culture medium, a sterilizing step of sterilizing the lactobacillus bacteria in the culture medium used in the culturing step at 80°C or below for at least one hour to obtain killed bacteria, and a recovering step of the killed bacteria.

8. The lactic acid bacterium or killed cells thereof according to claim 6, which is Lactobacillus crispatus HMS-115 (Accession No.: NITE P-04100) or Lactobacillus crispatus HMS-122 (Accession No.: NITE P-04101).

9. A vaginal cleansing composition comprising the lactobacillus or killed lactobacillus thereof according to claim 6 and water.

10. The vaginal cleansing composition of claim 9, further comprising lactic acid and hydrogen peroxide.

11. A food or beverage product, a cosmetic product, or an ingredient thereof, comprising the lactobacillus or killed cells thereof according to aspect 6.

12. A method for producing a vaginal cleansing composition, comprising mixing lactobacillus or killed lactobacillus bacteria with water, wherein the lactobacillus has been screened by the method of claim 1.

13. A method for producing a vaginal douche composition, comprising mixing killed lactobacillus bacteria with water, wherein the killed lactobacillus bacteria are produced by the method of claim 7.

14. A method for producing a food or beverage product or a cosmetic product containing killed lactobacillus cells, wherein the killed lactobacillus cells are produced by the method according to aspect 7.

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