Probiotic strain for inhibiting growth of vaginal yeast, immunity against bacteria and HPV, vaginal immunity, intestinal bacterial proliferation, preventing cervical cancer and breast cancer, and mental, psychological, and emotional wellbeing, and composition thereof

Abstract

The present invention provides a Lactobacillus plantarum Probio-87 strain deposited at the Korean Collection for Type Cultures (KCTC) under accession number KCTC15755BP and a composition thereof. The Lactobacillus plantarum Probio-87 strain according to the present invention has beneficial effects for vaginal bacteria, yeast, and virus population control, vaginal immunity enhancement, intestinal bacterial regulation, HPV-mediated cervical cancer and breast cancer prevention, and mental and emotional wellbeing.

Description

Probiotic strains and their compositions for inhibition of vaginal yeast growth, immunity against bacteria and HPV viruses, vaginal immunity, intestinal bacterial proliferation, prevention of cervical and breast cancer, and psychiatric, psychological, and emotional health.

[0001] The present invention relates to probiotic strains and compositions thereof for inhibiting vaginal yeast growth, immunity against bacteria and HPV viruses, vaginal immunity, intestinal bacterial proliferation, prevention of cervical and breast cancer, and mental, psychological, and emotional health. More specifically, the invention relates to probiotic strains and compositions thereof for inhibiting vaginal yeast growth, immunity against bacteria and HPV viruses, vaginal immunity, intestinal bacterial proliferation, prevention of cervical and breast cancer, and mental, psychological, and emotional health, having effects of improving vaginal health, improving vaginal and intestinal microbial imbalances, inhibiting the growth of vaginal pathogenic yeasts and vaginal pathogenic bacteria, reducing vaginal HPV populations, and preventing cervical and breast cancer.

[0002] Probiotics are defined as live microorganisms that provide beneficial effects to the host when consumed in adequate amounts (FAO / WHO, 2006). Probiotic strains have been widely reported to maintain the balance of the gut microbiome and influence immune responses, thereby strengthening the body's systems to fight disease (Hatakka et al., 2001). Over the decades, probiotics have been extensively reported, primarily focusing on enhancing general gut health and immune health (Verna & Lucak, 2010).

[0003] Vaginitis is considered one of the most common gynecological diseases affecting women worldwide, and it is often asymptomatic, primarily caused by an imbalance of the vaginal microbiome. The prevalence of vaginitis has been reported to vary from 5% to 50% by various study groups across major continents such as the United States, Europe, and South Asia (Begum et al., 2011). Bacterial vaginosis (BV) and yeast vaginosis are genitourinary infections that affect approximately one billion women globally annually (Reid et al., 2001). A healthy vaginal microbiome is dominated by Lactobacillus species, but various other bacteria are present in small proportions. Bacterial vaginosis is caused by microbial imbalances caused by other pathogenic bacteria such as Staphylococcus, Gardnerella Vaginalis, and Escherichia coli (Srinivasan and Fredricks, 2008), and yeast vaginosis is caused by pathogenic yeasts such as Candida albicans, Candida glabrata, Candida parasilopsis, Candida krusei, and Candida tropicalis (Turner and Butler, 2014).

[0004] Human Papillomavirus (HPV) refers to a diverse group of viruses that primarily infect epithelial and mucosal tissues (Nunes et al., 2018). These viruses infect epithelial tissues throughout the body, producing benign and malignant lesions, including common and genital warts (Handfield et al., 2018). As a risk factor, HPV accounts for more than 90% of cervical cancer cases and is associated with the etiology of high-risk cancers. High-risk HPV types include V16, V18, V31, V33, V35, V45, V52, V53, V58, V66 / V68, V73, V81, V82, and V84 / V26. It has been reported that HPV testing yields a positive result in up to 100% of patients with cervical cancer. HPV 16 and 18 are the prototypes of high-risk viruses and are the most frequently detected in cervical cancer, accounting for 35.7% and 26%, respectively (Tan et al., 2018).

[0005] The effects of probiotics are mediated through immune modulation, specifically the balance of pro-inflammatory and anti-inflammatory cytokines (Isolauri et al., 2001). Immune responses are initiated by innate immunity following exposure to foreign substances or tissue damage. Innate immunity plays a role in protecting host homeostasis in part by preparing adaptive immunity against persistent invasion and inducing inflammation. However, unbalanced immune responses lead to severe inflammation, uncontrolled tissue damage, and disease. Probiotics have been shown to enhance innate immunity and regulate pathogen-induced inflammation through Toll-Like Receptor (TLR) regulatory signaling pathways (Vanderpool et al., 2008).

[0006] Imbalances in the gut microbiome can occur due to various factors, including disease and diet. The gut microbiome is a metabolically active community of microorganisms residing throughout the gastrointestinal tract and is currently recognized as an important regulator of host homeostasis. It has been hypothesized that regulating the balance of gut microorganisms induces changes in the intestinal metabolic environment, which can affect metabolism, disease development, and immunity.

[0007] Globally, over 20 million new cases of cancer occur annually, with more than 2 million of them being breast cancer. Breast cancer is showing an increasing trend, particularly in developed countries, and ranks first in both incidence and mortality rates among women in most countries worldwide. The gut microbiome plays a pivotal role in various aspects of human health, including nutrient supply, metabolic contribution, defense against pathogens, development of the immune system, and maintenance of epithelial-mucosal equilibrium. In particular, gut microbes play a critical and decisive role in health and pathological conditions that are important for the initiation, progression, and metastasis of cancer through the regulation of inflammation, immune responses, and cellular processes (Ly et al., 2017).

[0008] Vaginitis significantly impacts women's quality of life. These infections can cause a variety of uncomfortable and distressing symptoms, including itching, discomfort, pain, abnormal discharge, foul odor, urinary symptoms, and psychological and sexual effects; consequently, this affects self-esteem, relationships, and overall life. All of these factors have a profound impact on mental and emotional well-being. Probiotics are reported to benefit mental health through the gut-brain axis. The gut-brain axis is a bidirectional communication system between the gastrointestinal tract and the central nervous system. The influence of probiotics or other gut microbiota leads to the production of neurotransmitters and other signaling molecules that affect brain function, having a significant impact on mood, emotions, and mental or emotional health.

[0009] Therefore, it is necessary to provide solutions, particularly those using probiotics, to improve the conditions mentioned above.

[0010] The technical problem of the present invention is to provide a Lactobacillus plantarum Probio-87 strain and a composition thereof having the effects of preventing and improving emotional and psychological health deterioration; improving vaginal health; improving the imbalance between vaginal and intestinal microorganisms; inhibiting the growth of vaginal pathogenic yeasts and vaginal pathogenic bacteria; reducing the vaginal HPV population; preventing HPV-mediated cervical cancer; and preventing breast cancer.

[0011] The present invention relates to the Lactobacillus plantarum Probio-87 strain deposited under accession number KCTC15755BP.

[0012] According to one aspect of the present invention, the strain and composition may alternatively be formulated as a method of probiotic therapy, including administering an effective amount of Lactibactibacillus plantarum as needed. As used herein, “probiotics” means live microorganisms that provide health benefits to a host when administered in appropriate amounts. As used herein, “postbiotics” means preparations of inactivated microorganisms, their components, and metabolites that provide health benefits to a host when administered in appropriate amounts.

[0013] According to another aspect of the present invention, the strain and composition are characterized by regulating an unbalanced vaginal microbiome and preventing vaginal bacterial imbalance by reducing the number of harmful microorganisms and increasing the number of beneficial microorganisms.

[0014] According to another aspect of the present invention, the strain or composition is characterized by inhibiting the proliferation of vaginal pathogenic Candida species, such as Candida albicans, Candida glabrata, Candida parasilopsis, Candida krusei, and Candida tropicalis.

[0015] According to another aspect of the present invention, the strain is characterized by inhibiting vaginal pathogenic bacteria, such as Gardnerella vagininalis, Escherichia coli, and Staphylococcus aureus.

[0016] According to another aspect of the present invention, the strain and composition are characterized by inhibition of HPV populations in the vagina.

[0017] According to another aspect of the present invention, the strain and composition are characterized by anti-inflammatory and immune-modulating protection of the vagina.

[0018] According to another aspect of the present invention, the strain and composition are characterized by reducing the number of harmful microorganisms and increasing the number of beneficial microorganisms to regulate and prevent intestinal microbial imbalance.

[0019] According to another aspect of the present invention, the strain and composition are characterized by the prevention of HPV-mediated cervical cancer.

[0020] According to another aspect of the present invention, the strain and composition are characterized by the prevention of breast cancer.

[0021] According to another aspect of the present invention, the strain and composition are characterized by the enhancement of mental and emotional quality.

[0022] The above strain has all the advantages and medicinal uses mentioned above, but in other cases, one strain must be used per medicinal use.

[0023] An additional element of the present invention comprises the freeze-dried strain, wherein the composition comprising the Lactobacillus plantarum Probio-87 strain (KCTC15755BP) according to the present invention is a freeze-dried living cell, and 10 of the total composition 4 to 10 12 It contains an amount of cfu / g, and the strain is 10 in the composition 4 to 10 12 It exists in amounts in the range of CFU / ml. Here, the composition is 10 4 to 10 12 CFU / ml Having a liquid postbiotic or an equivalent dry form containing an amount of 10 4 to 10 12 It includes postbiotics derived from the above strain present in an amount within the range of CFU / ml, or a composition thereof in a dried form.

[0024] Various medical applications of the above strain or its composition may be expressed as use in the production of food supplements, medicines, infant formulas, edible products, food, or agricultural products to alternatively treat and prevent the mentioned symptoms. Additionally, this may be described as an alternative method of treating and preventing the aforementioned symptoms by administering an effective amount of the above strain or postbiotics to a subject in need. The subject is primarily mammals, particularly humans.

[0025] As used herein with respect to the strain, the term “effective amount” refers to the amount of colony-forming units (CFU) of said strain in the composition. This amount is sufficient to bring about a substantial and positive change in symptoms during treatment, while maintaining a reasonable benefit-risk ratio based on prudent medical judgment to prevent serious side effects.

[0026] Lactiplantibacillus plantarum KCTC 15755BP used in the claims and characteristics of the present invention may simply be referred to as the strain herein.

[0027] The Lactobacillus plantarum Probio-87 strain and its composition according to the present invention have the effects of preventing and improving emotional and psychological health deterioration; improving vaginal health; improving the imbalance between vaginal microbes and intestinal microbes; inhibiting the growth of vaginal pathogenic yeasts and vaginal pathogenic bacteria; reducing the vaginal HPV population; preventing HPV-mediated cervical cancer; and preventing breast cancer.

[0028] Figure 1 is a graph showing the changes in relative gene expression levels in the blood for pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ), anti-inflammatory cytokines (IL-10, IL-4), T cells (CD8, CD117, CD44, FOXP3, CD4), B cells (CD27, CXCR5), NK cells (CD56, CD94, CD34, Nkp30, Nkp46, Nkp44) and mental health / physiological parameters (CREB, BDNF, TPH-2, 5-HT6, IDO, TH, GAD-2, ghrelin, leptin) when a placebo (n=53) or Lactobacillus plantarum Probio-87 strain was administered for 12 weeks.

[0029] Figure 2 is a graph showing alpha diversity for vaginal microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered.

[0030] Figure 3 is another graph showing alpha diversity for vaginal microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered.

[0031] Figure 4 is a graph of principal coordinate analysis (PCoA) measured by PERMANOVA for (A) phylum (PCoA P=0.991), (B) class (PCoA P=0.982), (C) order (PCoA P=0.808), (D) family (PCoA P=0.587), and (E) genus (PCoA P=0.551).

[0032] Figure 5 is a graph of principal coordinate analysis (PCoA) measured by ANOSIM for (A) phylum (PCoA P=0.999), (B) class (PCoA P=0.986), (C) order (PCoA P=0.960), (D) family (PCoA P=0.796), and (E) genus (PCoA P=0.759).

[0033] Figure 6 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups measured by the Chao1 index for (A) Phylum (W0 P=0.263), (B) Class (W0 P=0.342), (C) Order (W0 P=0.216), (D) Family (W0 P=0.341), and (E) Genus (W0 P=0.827).

[0034] Figure 7 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by observation indices for (A) phylum (W0 P=0.429), (B) class (W0 P=0.212), (C) order (W0 P=0.376), (D) family (W0 P=0.721), and (E) genus (W0 P=0.941).

[0035] Figure 8 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by the ACE index for (A) phylum (W0 P=0.586), (B) class (W0 P=0.435), (C) order (W0 P=0.386), (D) family (W0 P=0.428), and (E) genus (W0 P=0.801).

[0036] Figure 9 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by Fisher's index for (A) phylum (W0 P=0.507), (B) class (W0 P=0.221), (C) order (W0 P=0.370), (D) family (W0 P=0.685), and (E) genus (W0 P=1.000).

[0037] Figure 10 is a graph of principal coordinate analysis (PCoA) measured by PERMANOVA for (A) phylum, (B) class, (C) order, (D) family, and (E) genus.

[0038] Figure 11 is the N metric multidimensional scaling (NMDS) measured by PERMANOVA for (A) phylum, (B) class, (C) order, (D) series, and (E) genus.

[0039] Figure 12 is a graph showing the antimicrobial activity of the Lactobacillus plantarum Probio-87 strain and other probiotic strains against (A) Candida albicans, (B) Candida glabrata, (C) Candida tropicalis, (D) Candida krusei, and (E) Candida parasilopsis.

[0040] FIG. 13 shows (A) living probiotic cells (10 6 Candida albicans cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0041] FIG. 14 shows (A) living probiotic cells (10 6 Candida glabrata cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0042] FIG. 15 shows (A) living probiotic cells (10 6 Candida tropicalis cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0043] FIG. 16 shows (A) living probiotic cells (10 6 Candida krusei cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0044] FIG. 17 shows (A) living probiotic cells (10 6 Candida parasilopsis cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0045] FIG. 18 shows (A) Candida albicans cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0046] FIG. 19 shows (A) Candida glabrata (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0047] FIG. 20 shows (A) Candida tropicalis (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0048] FIG. 21 shows (A) Candida krusei (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0049] FIG. 22 shows (A) Candida parasilopsis (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0050] Figure 23 is a graph showing the antimicrobial activity of CFS derived from Lactobacillus plantarum Probio-87 against (A) Staphylococcus aureus, (B) Escherichia coli, (C) Gardnerella vaginalis, and (D) Lactobacillus iners, and (E) a graph showing the symbiotic activity of Lactobacillus plantarum Probio-87 and Lactobacillus crispatus.

[0051] Figure 24 is a graph showing the cell viability confirmed by MTT assay after treating (A) non-HPV cervical cancer cell line C33A, (B) HPV-16 mediated cervical cancer cell line CaSki, and (C) HPV-18 mediated cervical cancer cell line HeLa with Lactobacillus plantarum (L. plantarum) Probio-87 and other probiotic strains (untreated control group) excluding postbiotics.

[0052] Figure 25 shows a morphological analysis of non-HPV cervical cancer cell line C33A treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope.

[0053] Figure 26 shows a morphological analysis of HPV-16-mediated cervical cancer cells CaSki treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope.

[0054] Figure 27 is a photograph showing the morphological analysis of HPV-18-mediated cervical cancer cells treated with HeLa and Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope.

[0055] Figure 28 is a graph showing the concentrations of angiogenin, angiopoietin-2 (ANG-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) proteins expressed in (A) HeLa, (B) CaSki, and (C) C33A cells with or without CFS treatment with Lactobacillus plantarum (L. plantarum) Probio-87.

[0056] Figure 29 is a graph showing the ratio of changes in transcription factor regulation when (A) HeLa, (B) CaSki, and (C) C33A cells were treated with CFS of Lactobacillus plantarum (L. plantarum) Probio-87.

[0057] Figure 30 is a graph showing the changes in the relative gene expression levels of tumor suppressor factors p21 and ARF in HeLa, CaSki, and C33A cells upon CFS treatment with Lactobacillus plantarum (L. plantarum) Probio-87.

[0058] Figure 31 is a graph showing the cell viability of human breast cancer cell lines MDA-MB-231 (A) and T47D (B) when treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control).

[0059] Figure 32 is a photograph showing the morphological analysis of human breast cancer cells MDA-MB-231 treated with Lactobacillus plantarum Probio-87, other probiotic strains, or postbiotics (untreated control) without postbiotics, performed using a phase-contrast microscope (100 μM).

[0060] Figure 33 is a photograph showing the morphological analysis of human breast cancer cells T47D treated with Lactobacillus plantarum Probio-87, other probiotic strains, or postbiotics (untreated control) without postbiotics, performed using a phase-contrast microscope (100 μM).

[0061] The above-described objects, features, and advantages of the present invention will become more apparent from the detailed description of the drawings and preferred embodiments of the present invention. The following detailed description is not intended to be limiting, and the scope of the present invention is limited only by the appended claims, including all equivalents thereof.

[0062] The present invention will be described in more detail below with reference to the attached drawings and embodiments.

[0063] The Lactobacillus plantarum Probio-87 strain used for the present invention was isolated from the breast milk of a healthy Korean woman. The strain was identified as Lactobacillus plantarum and deposited at the Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Republic of Korea. The strain was deposited by Yong-Ha Park on December 26, 2023, under deposit number KCTC15755BP. The deposited strain is active and retains all characteristics associated with the strain.

[0064] Identification was confirmed through 16S rRNA gene sequence analysis. The list of 16S rRNA sequences is as follows.

[0065] ctg000000:126255-127818(+)

[0066] TATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCTGCCTAAGGTGGGACAGATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT

[0067] It is evident that, based on the deposited strain, a person skilled in the art can generate additional variants or mutant individuals through conventional mutagenesis or re-isolation techniques. These variants or mutants are designed to retain or enhance the relevant features and benefits described herein for the original strain. Thus, the present invention comprises variants of the described strain. In this specification, the terms “variant” or “mutation” refer to any naturally occurring or intentionally generated strain derived from the deposited strain primarily through mutation while preserving the function of the deposited strain. Functional evaluation can be performed by the methods described in detail in the Examples. For example, the complete genome sequence of the 16S rRNA gene or the “variant” may exhibit sequence identity of approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and 98% as specified herein.

[0068] In certain embodiments, mutations are induced through the application of recombinant DNA technology. In other embodiments, mutations are obtained through random mutagenesis. Consequently, another feature of the present invention includes a method for obtaining variants of the strain. This method includes a process of utilizing a deposited strain as a starting material to apply mutagenesis and ensuring that the resulting variant or mutation maintains or enhances the biological function of the deposited strain.

[0069] In certain embodiments, the strain undergoes fermentation in an artificial medium and subsequent post-fermentation treatment to produce bacterial cells. The produced bacterial cells exist in liquid medium or solid form. Specifically, the post-treatment method is selected from drying, freezing, freeze-drying, fluidized bed drying, spray drying, and refrigeration to obtain cells or postbiotics. The strain is produced through culture (or fermentation) in a suitable artificial medium under appropriate conditions. The term “artificial medium” for microorganisms refers to a medium containing natural substances and, optionally, synthetic chemicals such as polymer polyvinyl alcohol that can mimic some of the functions of blood. Typical artificial media contain essential elements such as a carbon source (e.g., glucose), a nitrogen source (e.g., amino acids and proteins), purified water, and salts required for bacterial growth. The culture medium may be in liquid form or may often be mixed with agar or other gelling agents to create a solid medium.

[0070] The strain may be cultured independently to form a pure culture, cultured in combination with other microorganisms, or cultured separately with other microorganisms and then mixed in a desired ratio. After culture, depending on the intended formulation, the strain may be utilized as a live cell, or the strain culture or cell suspension may be used as is or after appropriate post-treatment for the production of postbiotics. In this specification, the term “biomass” refers to a strain culture obtained after culture (or fermentation, synonymous with culture).

[0071] In this specification, the term “post-processing” refers to any processing performed on biomass for the purpose of obtaining storable bacterial cells or postbiotics. After post-processing, the bacterial cells may exist in solid or liquid form as live bacteria or postbiotics. Bacterial cells stored in solid form may exist in powder or granular form. Regardless of form, solid and liquid states containing bacterial cells or postbiotics do not occur naturally but are produced through artificial post-processing processes.

[0072] The post-treatment process may require the use of one or more elements referred to as so-called “post-treatment agents” in certain embodiments. As specified herein, “post-treatment agent” refers to a compound used to carry out the post-treatment process described herein. Such post-treatment agents may include, without limitation, dehydrating agents, bacteriostatic agents, cryoprotectants, inert fillers (also known as cryoprotectants), carrier materials (also known as core materials), etc., used individually or in combination.

[0073] There are two fundamental methods to reduce the metabolic activity of bacteria, and consequently, two representative approaches for performing post-treatment are available. The first method involves reducing the rate of all chemical reactions by lowering the temperature through refrigeration or freezing using devices such as refrigerators, mechanical freezers, and liquid nitrogen freezers. Alternatively, the rate of all chemical reactions can be reduced by using substances that inhibit bacterial cell growth, particularly bacteriostatic agents. Another method for performing post-treatment involves removing moisture from biomass, which may include sublimating the moisture using a freeze dryer. Suitable techniques for removing moisture from biomass include drying, freeze-drying, spray drying, or fluidized bed drying. Post-treatment into a solid form may include drying, freezing, freeze-drying, fluidized bed drying, or spray drying.

[0074] Freeze-drying is primarily selected as the post-treatment method, which is a process of removing moisture from a frozen bacterial suspension through sublimation under reduced pressure. This method proceeds in three sequential steps: the product is pre-frozen and subjected to primary drying to remove a significant portion of moisture, followed by secondary drying to remove bound water. Given the diversity of industrial processes for manufacturing and isolating freeze-dried cultures, these cultures contain a certain amount of inert filler, also known as cryoprotectants. Cryoprotectants are used to standardize the probiotic content of the product.

[0075] Commercially available freeze-dried cultures often contain inert fillers such as sucrose, sugar, lactose, trehalose, glucose, maltose, maltodextrin, corn starch, inulin, and other pharmaceutically acceptable non-hygroscopic fillers. Additionally, viscous freeze-dried products can be produced by optionally using cryoprotectants such as ascorbic acid. In any case, the resulting powder can be ground to an appropriate size.

[0076] Lactobacillus plantarum Probio-87 (Medical applications of the Lactobacillus plantarum Probio-87 strain)

[0077] The Lactobacillus plantarum Probio-87 strain according to the present invention exhibits distinct advantages in excellent suitability as a probiotic or postbiotic. Probiotic bacteria must meet various criteria related to non-toxicity, viability, adhesion, and beneficial effects. The characteristics of each bacterial strain are unique and cannot be generalized to other strains within the same species.

[0078] Experiments regarding the health-beneficial effects and uses of the strain according to the present invention have been described in detail in the examples.

[0079] 1. The above strain improves the mental, psychological, and emotional health of adult women, as demonstrated by a randomized, double-blind, placebo-controlled study (see Example 1 below). 108 women were recruited under generally healthy conditions. The above strain (1x10 9 As a result of consuming (CFU / day) for 12 weeks, the WHQ survey evaluation showed improvements in depression, physical symptoms, anxiety, sexual symptoms, life expectancy, and menstrual-related symptoms compared to the placebo group as early as week 6. These results indicate that the strain meets the requirements for probiotic strains according to FAO / WHQ recommendations and can be applied as a natural method to improve the mental, psychological, and emotional functions of adult women. Therefore, the strain is characterized by being effective in preventing and improving the decline in mental, psychological, and emotional health.

[0080] 2. The above strain can improve vaginal health by enhancing immune and inflammatory parameters, as shown in a randomized, double-blind, placebo-controlled study (see Example 2 below). The above strain (1x10) was studied in 108 women. 9 CFU / day) was administered for 12 weeks in a randomized, double-blind, placebo-controlled clinical trial. The group administered the strain upregulated the blood immunomodulatory genes IL-10, CD44, CD8, CD56, CD94, Nkp30, Nkp46, and Nkp44 compared to the placebo group. These genes are primarily associated with NK cells, which implies that inflammation was prevented and greater defense against antigen invasion was initiated in the group administered the strain compared to the placebo group. Therefore, the strain is characterized by its effectiveness in improving vaginal health by influencing anti-inflammatory and immunomodulatory functions.

[0081] 3. The above strain has a regulatory effect on vaginal microbial imbalance (see Example 3 below). 108 women were recruited based on general health conditions. The above strain (1x10 9As a result of consuming (CFU / day) for 12 weeks, compared to the placebo group, the increase in ecological diversity of vaginal bacteria within the group across various taxa was prevented, as indicated by the alpha diversity index. The beta diversity index indicated that the strain prevented changes in microbial community composition that occurred in the placebo group over time. Analysis of vaginal microbial composition revealed that the strain prevented the overgrowth of specific bacterial groups associated with bacterial vaginosis compared to the placebo group. The Nugent score also showed a decrease in the group administered the strain compared to the placebo group. Therefore, the strain is characterized by the regulation of vaginal microbial imbalance and postbiotic concentrations.

[0082] 4. The above strain is characterized by being effective in regulating intestinal microbiota and postbiotic imbalances (see Example 4 below). 108 women were recruited based on general health conditions. Healthy women [tested] the above strain (1x10⁻⁶⁻⁶ for 12 weeks 9 As a result of consuming CFU / day, alpha diversity analysis showed that loss of within-group ecological diversity across relative abundances of other taxa, which was not observed in the placebo group, was prevented. The beta diversity index indicated that the strain prevented changes in microbial community composition that occurred in the placebo group over time. Analysis of gut microbial composition revealed that after 12 weeks, the strain showed an increase in the proportion of some beneficial gut microorganisms compared to the placebo group, while the proportion of some harmful gut bacteria decreased. Therefore, the strain is characterized by improving gut microbial imbalance.

[0083] 5. The strain is characterized by inhibiting the growth of vaginal pathogenic Candida through growth inhibition and cell aggregation (see Example 5 below). The postbiotics of the strain inhibit the growth of vaginal pathogenic Candida species, such as Candida albicans, Candida glabrata, Candida krusalis, Candida parasilopsis, and Candida tropicalis. Furthermore, the postbiotics of the strain inhibit high yeast cell aggregation, thereby preventing the proliferation of pathogens and their attachment to host cells. Additionally, the living cells of the strain inhibit the cell cavity aggregation of yeast cells, thereby inhibiting growth and proliferation. Through this, it can be explained that the strain has an antimicrobial effect against vaginal pathogenic yeast species. Therefore, the strain or its postbiotics are characterized by the inhibition of the growth of vaginal pathogenic yeast species.

[0084] 6. The strain above has activity that inhibits the growth of vaginal pathogenic bacteria (see Example 6 below). The postbiotics of the strain above inhibit the proliferation of vaginal pathogenic bacteria such as Gardnerella vagninalis, Escherichia coli, and Staphylococcus aureus. The postbiotics of the strain above also inhibit the proliferation of Lactobacillus iners, a lactic acid bacterium known to be abundant in the vagina of women with bacterial vaginosis, and grow symbiotically with Lactobacillus crispatus, known as a beneficial bacterium. Based on this, the strain above was found to have antibacterial activity against vaginal pathogenic bacteria. Therefore, the strain above is characterized by the inhibition of the growth of vaginal pathogenic bacteria.

[0085] 7. The strain above suppresses the HPV population in the vagina of adult women who are positive for HPV (Human Papillon virus), as demonstrated in a randomized, double-blind, placebo-controlled study (see Example 7 below). Eighty-seven sexually active women aged 26 years or older who were confirmed to be positive for the L1 strain were recruited. As a result of quantifying the L1 gene, which indicates HPV abundance in the vagina of HPV-positive women, the strain above (1 x 10⁻⁶ 9 In the group administered the strain (CFU / day) for 12 weeks, vaginal HPV abundance decreased compared to HPV-positive women. It was also confirmed that the Nugent score in the group administered the strain decreased compared to the placebo group. Vaginal health and sexual function also improved over time compared to the placebo group as a result of administering the strain for 12 weeks. Regarding immunity measured by blood gene expression, inflammation and immune cell indicators were found to improve compared to the placebo group when the strain was consumed for 12 weeks. Correlation analysis revealed that changes in the vaginal microbiome were associated with immunity, vaginal health, and sexual function parameters. Therefore, the strain is characterized by a reduction in the vaginal HPV population.

[0086] 8. The strain above prevents HPV-mediated cervical cancer by inhibiting the growth of HPV-mediated cervical cancer cells (see Example 8 below). The postbiotics of the strain above inhibit the growth of CaSki cell lines, which are HPV-16-mediated cervical cancer cells, and HeLa cell lines, which are HPV-18-mediated cervical cancer cells. Phase-contrast microscopy analysis showed that the postbiotics of the strain above reduced the number of cancer cells and altered their morphology, such as through cell shrinkage and surface exfoliation. In angiogenesis analysis, the postbiotics of the strain above were found to downregulate angiogenesis promoters compared to the carrier control group, specifically VEGR, ANG-2, and angiogenin. In apoptosis analysis, the postbiotics of the strain above were found to promote apoptosis in cancer cells by upregulating genes such as p53, TGFβ, NFκB, Myc / Max, MAPK / ERK, Notch, and ARF. Therefore, the strain above and its postbiotics are characterized by the prevention of HPV-mediated cervical cancer.

[0087] 9. The strain above prevents HPV-mediated breast cancer by inhibiting the growth of breast cancer cells (see Example 9 below). The postbiotics of the strain above inhibit the growth of metastatic (MDA-MB-231) and non-metastatic (T47D) human breast cancer cell lines. Phase-contrast microscopy analysis showed that the KCTC 15755BP postbiotics reduced the number of cancer cells and decreased morphological changes in cells, such as shrinkage and exfoliation of the cell surface. In the case of MDA-MB-231 cells, which exhibit an invasive phenotype through star-shaped protrusions connecting cells, treatment with the postbiotics of the strain above also reduced the formation and / or dissolution of connections. Therefore, the strain above and its postbiotics are characterized by the prevention of breast cancer.

[0088] Product form

[0089] According to one aspect of the present invention, the strain and its composition are 10 per gram as a freeze-dried composition 4 to 10 12 The present invention comprises a composition containing a strain present in an amount within the CFU range. Additionally, the present invention comprises 10 postbiotics derived from said strain. 4 to 10 12 It includes a composition in an amount in the range of CFU / ml or an equivalent dry form.

[0090] Determining the effective amount of bacterial cells depends on several factors, including the intended goal, the patient's age and physical condition, the severity of underlying diseases, and the final formulation. When administered orally, the above strain is 10 in accordance with current laws 7 to 10 12 CFU range, preferably 10 9 to 10 11 It is included in the composition in an amount that provides an effective daily dose in the CFU range. The term "colony-forming unit" ("CFU") refers to the number of bacterial cells determined by microbiological counting on an agar plate. When included in the composition, the strain is preferably present in a 1:1 concentration ratio.

[0091] The primary use of the strain according to the present invention is in the form of viable cells, but this application extends to postbiotics such as inactivated cultures or cell lysates (achieved through methods such as altered pH, sonication, radiation, temperature, or pressure exposure). In addition, physiologically active factors and postbiotics produced by or generated from the strain are also included.

[0092] In certain embodiments, the composition may be selected in various forms, such as food supplements, pharmaceuticals, infant formula, edible products, food, or agricultural products. Additionally, in other embodiments, the composition is prepared as a tablet, capsule, pill, or liquid for oral administration.

[0093] The composition of the present invention may be prepared in various forms that do not impair the viability or bioactivity of bacterial cells. Considering the intended purpose of the composition, the selection of excipients and appropriate formulation methods fall within the competence of skilled individuals in the fields of pharmaceutical and food technology.

[0094] The compositions described in the present invention may be formulated in various forms in which bacterial cells act as a single active agent or in combination with one or more additional active agents. Additionally, in the case of food, pharmaceutically acceptable excipients, suitable additives, and ingredients may be mixed and used. In specific embodiments, the composition may contain one or more additional active agents, and other probiotic strains that do not exhibit an antagonistic effect against bacterial cells in the compositions of the present invention are preferred.

[0095] Depending on the formulation, bacterial cells may be included as purified bacteria, as part of a bacterial culture, as a bacterial culture post-treated with postbiotics alone, or together with a suitable carrier or ingredient. Prebiotics may also be included.

[0096] This composition may take the form of a pharmaceutical product, wherein the term “pharmaceutical product” includes all compositions containing an active ingredient (in this case, a bacterial cell) together with pharmaceutically acceptable excipients. The term “medicine” is not limited to medicines. The term “pharmaceutically acceptable” means a compound, substance, composition, and / or dosage form suitable for a reasonable benefit-risk ratio within the scope of medical judgment, which does not cause excessive toxicity, irritation, allergic reaction, or other problems upon contact with the subject’s tissues. All carriers, adjuvants, etc., must be “acceptable” in terms of compatibility with other compositional components, and appropriate options must be found in standard medicine texts.

[0097] This medicine may have various forms or names depending on the product approval channel and national regulations. For example, medicines and medical foods are considered distinct types of medicines in this context. The term "medical food" refers to food specifically manufactured and intended for the dietary management of diseases with nutritional needs that cannot be met by a standard diet alone. In some countries, this category is defined by regulations such as the US FDA Orphan Drugs Act of 1988 amendment and the European Commission Directive 1999 / 21 / EC.

[0098] Probiotic or postbiotic compositions such as those presented herein are often classified as food supplements. Food supplements, also known as dietary supplements or nutritional supplements, are another specific type of medicine intended to supplement the diet and provide nutrients or beneficial components that are generally not consumed in a standard diet or in insufficient amounts. Generally, food supplements are considered foods, but they may sometimes be defined as drugs, natural health products, or functional foods. In the context of the present invention, food supplements also include functional foods and are generally available as over-the-counter medicines without a prescription. If a food supplement is in the form of a pill or capsule, it may contain excipients similar to those used in medicines. However, food supplements may also appear as fortified foods, such as infant formula. Therefore, in certain embodiments, the compositions described in the present invention may be considered food supplements.

[0099] The composition of the present invention is versatile when administered directly or mixed with a suitable edible liquid or solid. It may be lyophilized or generally used in unit doses in various forms such as tablets, pills, capsules, slowly dissolving or dissolving solids, granules, powders, suspensions, sachets, and syrups. Additionally, it may take the form of a single dose of lyophilized composition provided with a separate liquid container to be mixed before administration, or as a standalone liquid formulation.

[0100] Furthermore, the above composition applies to various foods or edible items, including but not limited to infant dairy products. The term "edible product" may be broadly interpreted to encompass all ingestible products that meet sensory criteria. Meanwhile, "food" refers to an edible item that provides additional nutritional support to the body. Foods of particular note include food supplements and infant formula. Ideally, the food incorporates carrier materials such as oatmeal porridge, lactic acid fermented foods, resistant starch, dietary fiber, carbohydrates, proteins, and glycosylated proteins. In certain embodiments, the bacterial cells of the present invention are homogenized with other ingredients, such as cereal or powder, to form infant formula.

[0101] Another feature of the present invention relates to a solid composition characterized by an antifreeze agent, freeze-dried biomass containing the strain of the present invention, and a pharmaceutically acceptable carrier. The carrier may be selected from options such as emulsion, gel, paste, granule, powder, and gum.

[0102] Additional aspects may be extended to oral care products, pharmaceutical compositions, edible products, dietary supplements, and cosmetic compositions, all of which contain an effective amount of the previously defined composition. In certain embodiments, the oral care product may be chewing gum, toothpaste, mouth spray, candy, or an orally dispersible tablet. Similarly, in certain embodiments, the pharmaceutical composition, edible product, or dietary supplement may take the form of a lozenge or an orally dispersible tablet.

[0103] The term “comprising” and variations thereof as used in the detailed description and claims according to the present invention are not intended to exclude the inclusion of other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention may be identified by those skilled in the art by a close examination of the description or obtained through the actual application of the present invention. Additionally, the present invention includes all possible combinations of the specific and preferred embodiments described herein. The following embodiments and examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0104] Examples

[0105] Bacterial strains and cultures

[0106] The Lactobacillus plantarum Probio-87 strain according to the present invention was isolated from the breast milk of a generally healthy woman in South Korea. All stock cultures were stored in 20% glycerol (-20°C) and activated three consecutive times in sterile de Mann, Rogosa, Sharper (MRS) liquid medium (Hi-Media, Mumbai, India) using a 10% (v / v) inoculum. They were cultured at 37°C for 24 hours prior to use.

[0107] In vitro experiment

[0108] manufacturing of postbiotics

[0109] The Lactobacillus plantarum Probio-87 strain was obtained from Probiotic Co., Ltd. (South Korea). Commercial controls were isolated from their respective commercial products. Lactobacillus crispatus VP and Lacticaseibacillus rhamnosus VP were isolated from the Jarrow Women's Fem Dophilus® vaginal probiotic product. Lactobacillus rhamnosus FD was isolated from the Jarrow Women's Fem Dophilus® product, and Lactiplantibacillus plantarum GC was isolated from the Bayer Gyno-Canesflor® product. Species identification was performed on all strains via 16S rRNA gene sequencing.

[0110] All stock cultures of probiotic strains preserved in 20% glycerol (-20℃) were activated three consecutive times using a 10% (v / v) inoculum in sterile de Mann, Rogosa, Sharpe (MRS) liquid medium prior to use and incubated at 37℃ for 24 hours. The activated cultures were 10 9 CFU / ml It was diluted and autoclaved at 121°C for 15 minutes at 15 psi, and stored as a postbiotic under sterile conditions at -80°C for subsequent analysis.

[0111] CFS preparation

[0112] The activated strain from the above-mentioned preserved strain was diluted to an optical density (OD) of 1.0 at 600 nm and centrifuged at 12,000×g for 5 minutes at 4°C to collect the supernatant. The supernatant was neutralized to pH 7.0 and the cell solution was stored. The CFS was sterilized using a filter and stored under sterile conditions at -80°C for subsequent analysis.

[0113] Inhibition of Candida growth by postbiotics

[0114] The postbiotic antimicrobial activity of probiotic strains against pathogenic yeasts Candida albicans, Candida glabrata, Candida parasilopsis, Candida krusei, and Candida tropicalis was evaluated in 96-well plates. Pathogenic yeasts were obtained as clinical isolates from the University Hospital of Malaysia (Kelantan, Malaysia). All yeast cultures were stored at -20°C in 20% glycerol (v / v) and activated in sterile YEPG medium (Hi-media) containing 10% (v / v) inoculum at 37°C for 24 hours prior to use. The activated yeast cultures had an optical density (600 nm) of 0.60 (10 6 It was standardized to CFU / mL. A total of 100 mL of yeast cell suspension and 100 mL of probiotic postbiotics were added to a 96-well microplate and incubated at 37°C for 72 hours. Turbidity was measured by absorbance at 600 nm. A negative control was prepared using MRS liquid medium to replace the postbiotics. A positive control was prepared using clotrimazole (50 μM and 200 μM) to replace the postbiotics.

[0115] Candida co-aggregation by living probiotic cells

[0116] Activated yeast culture 10 in phosphate-buffered saline (pH 7.4) 6 Standardized to CFU / mL, live probiotics (10 6The cells were mixed with (CFU / mL) at a 1:1 (v:v) ratio and cultured in a culture shaker at 0.4 xg at 37°C for 8 hours. Cell morphology and aggregate formation were examined using Gram staining and visualized using an optical microscope equipped with an a×100 magnification lens. Aggregation was obtained from each microscope image by converting the images to black and white using Image J software version 1.39 (National Institutes of Health, Bethesda, Md). Scores were calculated by Image J based on aggregated sections converted to black. Yeast cells were grown exclusively in YEPG medium without postbiotics and were used as a control.

[0117] Candida co-aggregation by postbiotics

[0118] Activated yeast culture 10 in phosphate-buffered saline (pH 7.4) 6 The cells were standardized to CFU / mL, mixed with postbiotics at a 1:1 (v:v) ratio, and cultured in a culture shaker at 0.4 xg at 37°C for 8 hours. Cell morphology and aggregate formation were examined using Gram staining and visualized using an optical microscope equipped with an a×100 magnification lens. Aggregation was obtained from each microscope image by converting the images to black and white using Image J software version 1.39 (National Institutes of Health, Bethesda, Md). Scores were calculated by Image J based on aggregated sections converted to black. Yeast cells were grown exclusively in YEPG medium without postbiotics and used as a control.

[0119] Antimicrobial activity against pathogenic bacteria

[0120] Pathogenic and symbiotic cultures were obtained from the University of Industrial Technology, Universiti Sains Malaysia (Penang, Malaysia). However, Gardnerella vaginalis was purchased from the Korean Collection for Type Cultures using accession number KCTC 5096. Escherichia coli and Staphylococcus epidermidis were cultured and maintained in trypticase soy liquid medium (Hi-media), Lactobacillus iners and Lactobacillus crispatus in MRS liquid medium (Hi-media), and Gardnerella vaginalis in vaginalis solid medium containing human blood. All growth analyses were performed at an optical density of 600 nm. However, Garnerella vaginalis was determined by the injection plate method after incubation at 37°C for 18 hours.

[0121] Cancer cell lines, origin, and culture

[0122] HPV-mediated cervical cancer cells (HeLa and CaSki) were cultured in DMEM medium (Nacalai Tesque, Japan) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. HPV-non-mediated cervical cancer cells C33A were cultured in DMEM F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cell lines were cultured in T75 tissue culture flasks with ventilation caps in a 5% CO2 incubator at 37°C.

[0123] Human breast cancer cell lines T47D and MDA-MB-231 were cultured in DMEM high-glucose medium supplemented with 20% fetal bovine serum. All cell lines were cultured in T75 tissue culture flasks with ventilation caps in a 5% CO2 incubator at 37°C.

[0124] MTT

[0125] MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assays were performed in 96-well plates according to the manufacturer's standard protocol. Hela and Caski cells were seeded at 2,500 cells per well, while C33A cells were seeded overnight in 96-well plates at 3,000 cells per well. T47D and MDA-MB-231 cells were seeded overnight in 96-well plates at 1.2 x 10⁶ cells. 5 cell / ml It was vaccinated.

[0126] Inoculated cells were treated with postbiotics of probiotic strains at a ratio of 30% postbiotics to 70% medium. After 48 hours, 10 μL of MTT (5 mg / mL) was added per well. The corresponding plates were covered with aluminum foil and incubated at 37°C for 4 additional hours. The medium was removed, and the formazan crystals were dissolved in 100 ml of dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm (measure) and 630 nm (reference). All experiments were performed in technical and biological triples. Cell viability was measured using the formula (control absorbance - sample absorbance) / control absorbance x 100%.

[0127] Morphological evaluation using phase-contrast microscopy

[0128] Cells were seeded overnight in 12-well plates at a density of 25,000 cells per well and treated with a postbiotic of a probiotic strain at a ratio of 30% postbiotic and 70% medium. Cell morphological changes were observed and imaged using an inverse phase-contrast microscope at 100X magnification. At least 1,000 cells were imaged per treatment condition.

[0129] Angiogenesis Array

[0130] Angiogenesis analysis was performed according to the specifications provided by the manufacturer. In the preliminary stage, a population of 10,000 HeLa / Caski / C33A cells was seeded into each well of a 96-well plate, with 100 μl of complete culture medium in each well. These cells were cultured under standard conditions for 24 hours to facilitate attachment. After cell attachment, the treatment regimen was applied as follows: a mixture of 30% conditioned fetal serum / vehicle control (CFS / VC) and 70% culture medium for Hela and Caski cell lines, and a mixture of 10% CFS / VC and 90% culture medium for C33A cell lines. This treatment was continued for 48 hours. Subsequently, the adjusted medium was collected for further analysis.

[0131] Sandwich enzyme-linked immunosorbent assays (ELISA) were performed on collected samples in compliance with the protocols specified in the analysis kit. The analysis procedure was based on fluorescence detection of the analysis microarrays. For data extraction, array-specific Gene Array List (GAL) files were utilized with microarray analysis software platforms such as GenePix, ScanArray Express, ArrayVision, and MicroVigene. These GAL files are accessible at www.RayBiotech.com / Gal-Files.html. Additionally, data were analyzed and results were generated using a specialized QAnalyzer tool designed for these specific arrays.

[0132] Cancer pathway and Dualuciferase test

[0133] The effects of KCTC15755BP postbiotics on signaling pathways were evaluated using the Cignal Finder Cancer 10-Pathway Reporter Array (tube format) according to the manufacturer's instructions. The study procedure began by combining 1 μL of DNA construct (concentration: 100 ng) with 48.4 μL of Opti-MEM® I reduced serum medium (fetal bovine serum [FBS] and antibiotic-free) in each well of a white opaque microplate. This mixture was allowed to equilibrate at 25°C for 5 minutes. Subsequently, 0.6 μL of FuGENE® 4K Transfection Reagent was introduced for every 100 ng of DNA construct. The resulting transfection complex, with a total volume of 50 μL, underwent an additional 30-minute incubation period at 25°C. Afterward, 2.5 x 10⁶ prepared in Dulbecco's phosphate-buffered saline (DPBS) 4 Cell volumes were added to the transfection complex. These cells were resuspended in 50 μL of Opti-MEM® I reduced serum medium supplemented with 10% FBS and 1% Gibco® MEM non-essential amino acids (100X concentration). After adjusting the final volume of each well of the microplate to 100 μL, an incubation period of 48 hours was observed.

[0134] After transfection, the medium containing the transfection reagent was replaced with the treatment medium. This treatment medium consisted of 75 μL of complete Dulbecco's Modified Eagle Medium (DMEM) or F12 DMEM for the C33A cell line, to which 30% CFS of the probiotic or vehicle control (pure MRS liquid medium) and 10% CFS for C33A cells were added. Subsequently, the cells were cultured for an additional 24 hours.

[0135] Luciferase analysis was performed using the Dual-Glo® Luciferase Analysis System according to the manufacturer's protocol. 75 μL of Dual-Glo® Reagent was added to each well, and the samples were incubated at 25°C for 30 minutes in a light-shielded environment. The luminescence of Firefly luciferase was quantified using a BMG labtech CLARIOstar microplate reader. 75 μL of Dual-Glo® Stop & Glo® reagent was added to each well, and after incubation under similar conditions for 15 minutes, Renilla luciferase luminescence was measured. The relative fold change in activity was calculated by dividing the Firefly / Renilla ratio of the treatment group by the Firefly / Renilla ratio of the control group, where a value greater than 1 indicates upregulation, while a value less than 1 indicates downregulation of the target transcription factor.

[0136] Human clinical research

[0137] Probiotics and placebo products

[0138] The probiotic strain Lactobacillus plantarum Probio-87 complies with the characteristic criteria for probiotics, such as resistance to acid and bile conditions in the upper gastrointestinal tract, does not possess antibiotic resistance in accordance with the requirements of the European Food Safety Authority (EFSA), and possesses the ability to perform the effects described below. It is attached to mucin and can utilize prebiotics such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), has a carbon metabolism profile that adheres to the general patterns of lactic acid bacteria, and possesses antimicrobial properties against human pathogens. The probiotic product contains both the probiotic and a carrier, whereas the placebo contains only the carrier. The product packets containing the probiotic and the placebo appeared as a bright yellow powder. Both the probiotic and placebo products were stored in a storage temperature range of 30°C or lower, away from direct sunlight, according to the conditions recommended by the manufacturer. The intervention consisted of the oral administration of either the probiotic (9 log CFU / packet) or the placebo, one packet per day, for 12 weeks. The two products were composed. Regarding the composition of the two products, the probiotics (each bag) contained a total weight of 1.00g (0.63g of non-GMO corn starch; 0.27g of fructooligosaccharide; 0.10g of total Lactobacillus (9 log CFU of total Lactobacillus (log CFU / bag))), and the placebo (each bag) contained a total weight of 1.00g (0.70g of non-GMO corn starch; 0.30g of fructooligosaccharide).

[0139] Sample Collection

[0140] Vaginal swab samples were collected via self-swabs at weeks 0 and 12. To ensure consistency across all subjects, each swab was marked to indicate the depth of insertion into the vagina. Upon insertion, the swab was rotated 10 times inside the vagina. To determine the Nugent score, the swabs were applied to slides, and the swabs were collected in collection tubes containing RNAlater™ solution for microbiome analysis. All tubes were tightly closed and stored at -80°C until further analysis. For microbiome analysis, subjects' stool samples were collected at weeks 0 and 12 using stool collection tubes containing RNAlater™ solution and glass beads. All tubes were tightly closed and stored at -80°C until further analysis. For gene expression analysis, medical staff collected 3 ml of blood samples directly from the antecavitary vein at weeks 0 and 12. 2 It was collected using an EDTA tube.

[0141] Gene expression

[0142] Gene expression analysis was performed in the manner previously described (Hor et al., 2018). That is, K 2 Whole blood collected in an EDTA tube Prior to RNA extraction, the RNA was dissolved in Trisure reagent (Bioline, London, UK) according to the manufacturer's instructions. Isopropyl alcohol was added to precipitate the RNA, and the precipitated RNA was washed with 75% ethanol. The purity and concentration of the extracted RNA were determined by measuring absorbance at 260, 280, and 230 nm using a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, Waltham, MA, USA). First-strand cDNA was synthesized using the RevertAid RT Kit (Thermo Scientific) containing Random Hexamer primers according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed, and the reaction mixture was incubated at 25°C for 5 minutes, followed by amplification at 42°C for 60 minutes. The reaction was terminated by incubating the mixture at 70°C for 5 minutes. The cDNA was stored at -80°C until it was used directly as a template in qPCR or until use. The expression levels of the gene of interest were determined by qPCR using the Agilent AriaMx Realtime PCR system (Agilent Technologies, Santa Clara, CA, USA). A 20-microliter PCR reaction consisted of 10 μl of 2X SensiFAST SYBR mix (Bioline), 0.4 μl of 10 μM forward and reverse primers, and 100 ng of cDNA.

[0143] Primer sequences were prepared using the amplification conditions provided by the manufacturer and are shown in Table 1 below. Melting curve analysis was performed after each PCR amplification by measuring the dissociation of the PCR product between 50°C and 95°C using a reaction mixture without a cDNA template as a negative control. The threshold cycle (Ct) for each sample was recorded by measuring the number of cycles at which the release of SYBR green exceeded a critical level. Data were calculated using the comparative Ct (ΔCt) method for relative quantification and expressed as log2-fold changes, and the 18S rRNA gene was used as a housekeeping gene for data normalization.

[0144]

[0145] Table 1 above is a list of primer sequences used for quantitative real-time PCR. Here, 18S rRNA= 18S ribosomal RNA; IL-10= Interleukin-10; IL-1β= Interleukin-1 beta; TNF-α= Tumor necrosis factor-alpha; IFN-γ= Interferon gamma; CD56= Differentiation cluster 56; CD44= Differentiation cluster 44; NKp30= Natural killer cell P30; NKp46= Natural killer cell P46; NKp44= Natural killer cell P44; IL-4= Interleukin-4; CD8= Differentiation cluster 8; CD94= Differentiation cluster 94; CD117= Differentiation cluster 117; CD27= Differentiation cluster 27; CXCR5= CXC chemokine receptor type 5; FOXP3= Forkhead box P3; CD34= Differentiation cluster 34; CD44= Differentiation cluster 44; CD4 = Differentiation Cluster 4; IDO = Indoleamine-pyrrole 2,3-deoxygenase; CREB = cAMP-Reactive Factor Binding Protein; TH = Tyrosine Hydroxyhydroxylase; GAD-2 = Glutamate Decarboxylase 2; 5-HT6 = 5-Hydroxytryptamine Receptor-6; TPH-2 = Tryptophan Hydroxyhydroxylase-2; BDNF = Neurotrophic Factor. F = Forward Primer; R = Reverse Primer; a This is the NCBI access number.

[0146] Nugent score

[0147] Gram staining was performed on vaginal swab samples to determine the Nugent score. Specifically, the score was based on the number of various bacterial forms, such as Lactobacillus-like (large, uniform Gram-positive bacilli; scores ranging from 0 to 4), Gardnerella vaginalis-like (soda-shaped Gram-variable bacilli), Prevotella / Bacteroides-like (small Gram-negative bacilli) (scores ranging from 4 to 0), and Mobiluncus (bent Gram-variable bacilli; scores ranging from 2 to 0). A Nugent score of 7 or higher was interpreted as vaginitis according to inclusion criteria (Nugent et al., 1991).

[0148] Microbial analysis

[0149] DNA extraction and purification from vaginal swab and stool samples were performed as previously mentioned (Liu et al., 2020). The purified DNA was measured using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, NC, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified via thermocycler PCR (GeneAmp 9700, ABI, San Diego, California, USA). The PCR reaction was performed three times using a 20 μL mixture containing 4 μL of 5 x FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. The sequence was as follows: denaturation at 95°C for 3 minutes, 30 seconds at 95°C, 27 cycles, annealing at 55°C for 30 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 10 minutes. PCR products were extracted on a 2% agarose gel according to the manufacturer's protocol, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), and quantified using QuantiFluor .-ST (Promega, Madison, WI, USA). The purified amplicons were equimolarly pooled on the Illumina MiSeq platform (Illumina, San Diego, CA, USA) and paired-end sequencing (2 x 300). 16s rRNA gene sequences were processed using QIIME v.1.9.1 (see QIIME for high-throughput community sequencing data analysis) and USEARCH v.10.0 (see faster search and clustering order than BLAST).Raw FASTQ files were quality-filtered by Trimmomatic and merged by USEARCH based on criteria such as barcode and primer removal, filtering of low-quality reads, and finding non-duplicate reads. The number of merged raw reads was at least 50,000 per sample. Operational Tue Units (OTUs) were clustered using UPARSE with 97% similarity blocking. Taxonomy for each 16S rRNA gene sequence was analyzed against the Silva 132 16S rRNA database using the RDP Classifier algorithm ( / rdp.cme.msu.edu / ) with an 80% confidence threshold. Microbiota data were analyzed using the MicrobiomeAnalyst phyloseq-R package version 3.6.1 (https: / / www.microbiomeanalyst.ca / MicrobiomeAnalyst / home.xhtml).

[0150] Generally healthy women

[0151] Research subjects

[0152] Written informed consent was obtained from all women prior to the start of the study. Participants were recruited from the International Islamic University of Malaysia (IIUM) campus and community participation. Inclusion criteria included generally healthy women aged 18–80 who had a current Nugent score of less than 7.0, had no genitourinary infections, and were willing to participate throughout the study. Exclusion criteria included being pregnant; using vaginal suppositories within 4 weeks of study entry; using oral medications or vaginal treatments such as hormones and estrogens for vaginal conditions within 4 weeks of study entry; using long-term medication for a condition (> 6 months); using vaginal estrogen creams, rings, or tablets within 4 weeks of study entry; using vaginal moisturizers, lubricants, or homeopathic preparations within 4 weeks of study entry; using spermicides within 4 weeks of study entry; and having undergone pelvic or gynecological surgery within 6 months prior to study entry.

[0153] Research Protocol

[0154] This study was a double-blind, randomized, placebo-controlled design study. Randomization was performed considering inclusion and exclusion criteria. Qualified subjects were randomized in a 1:1 ratio to the two divisions of the study according to a computer-generated list and assigned to either the probiotics or placebo group with an individual code. Randomization was performed by a research statistician who had no contact with the participants. No member of the research team could use the assignment order until the study was completed. This study was conducted in accordance with the Declaration of Helsinki, the International Conference on Harmonisation of Clinical Practice (ICH-GCP), the Malaysian Guidelines for Clinical Practice, and the Council of International Organizations of Medical Sciences (CIOMS) International Ethics Guidelines. All procedures involving human subjects were approved by the IIUM Research Ethics Committee (Approval No. IIUM / IREC 2021-318) and registered on ClinicalTrials.gov (Identification No. NCT05302687).

[0155] The sample size was calculated for a parallel-group study design including one prevention division and one placebo division, based on power design analysis. For this study, a total of 112 subjects were required according to the calculations described below. A larger sample size is required when considering the sample sizes calculated for both the primary and secondary objectives. For the primary objective regarding vaginal microbiota and the secondary objective regarding gut microbiota, sample size calculations could not be obtained because most microbiota studies explain inter-group variation using relative taxonomic abundances instead of actual data. For the secondary objective, calculations were based on the need for an independent control group and a continuous response variable for subjects; the control-to-subject ratio was fixed at 1:1, the probability at 0.95, and the Type-I error probability at 0.05. For general women's health, a total of 100 subjects (rounded to the nearest even number) were required for this study. Each group consisted of a total of 50 subjects, including an additional 50% dropout (each group without dropouts, n=33). Since there were no publications regarding the use of probiotics and WHQ, the closest alternative is the use of the natural product French maritime pine bark extract (Pinus pinaster). As a result of administering this extract, the somatic-mental-physical interface score improved after 12 weeks in women with an average age of 47, with an within-group standard deviation of 0.50 observed, and an improvement of 0.45 was observed between the treatment and placebo groups (Yang et al., 2010). For the secondary objective of immunity, a total of 112 subjects (rounded to the nearest even number) were required for this study. It consisted of a total of 56 subjects in each group, including an additional 50% dropout (each group without dropouts, n=37). As a result of administering probiotics for 18 days to healthy free-living individuals with an average age of 63, salivary IgA concentrations were shown to increase, with an within-group standard deviation of 343.It was observed to be 5 μg / ml, and the concentration increased to 290 μg / ml (Lefevre et al., 2015).

[0156] Questionnaire

[0157] Two types of questionnaires in Malay were provided to eligible subjects who met all inclusion and exclusion criteria. (i) a demographic questionnaire (collected at reference week 0) translated and validated in a previous study (Chong et al., 2019a, 2019b) and (ii) a women's health questionnaire (WHQ; collected at week 0) 6, 12) were translated into Malay and validated by the Mapi Research Trust (OfficialWHQ Distributed by Mapi Research Trust | ePROVIDE, 2020).

[0158] The Women's Health Questionnaire (WHQ) is widely used in multinational clinical trials, epidemiological studies, and evaluations of non-medical treatments (M. Hunter, 2000). The WHQ has also been included as a measure of quality of life (QOL) in various trials involving preventive interventions for women. The WHQ is specific to the population in that it applies only to women. The WHQ consisted of a 36-item self-report validated inventory designed to measure depressive mood, including 9 domains (item numbers 3, 5, 7, 8, 10, 12, 25), somatic symptoms (item numbers 14, 15, 16, 18, 23, 30, 35), memory / focus (item numbers 20, 33, 36), vasomotor symptoms (item numbers 19, 27), anxiety / phobia (item numbers 2, 4, 6, 9), sexual behavior (item numbers 24, 31, 34), sleep problems (item numbers 1, 11, 29), physiological symptoms (item numbers 17, 22, 26, 28), and attractiveness (item numbers 21, 32). Here, the Likert scale was 1: not at all; 2: not or not often; 3: often or sometimes; 4: Distinguished as very so.

[0159] HPV-positive women

[0160] Research subjects

[0161] Written informed consent was obtained from all women prior to the start of the study. Subjects were recruited from Seberang Jaya Hospital (Penang, Malaysia), Raja Perempuan Zainab II Hospital (Kelantan, Malaysia), Universiti Malaysia Sarawak (Sarawak, Malaysia), and community participation. Inclusion criteria included sexually active women aged 26 years or older who tested positive for HPV for the L1 variant and were willing to participate throughout the study. Exclusion criteria included women who had received long-term drug treatment (6 months or more) for any disease, pregnant women, women who had undergone uterine and / or cervical removal, women who had not received the HPV vaccine, and women with cervical intraepithelial neoplasia.

[0162] Research Protocol

[0163] This study is a double-blind, randomized, placebo-controlled design study. Randomization was performed considering inclusion and exclusion criteria. Qualified subjects were randomized in a 1:1 ratio to the two divisions of the study according to a computer-generated list and assigned to either the probiotics or placebo group with an individual code. Randomization was performed by a research statistician who did not have contact with the participants. The assignment order was unavailable to any member of the research team until the study was completed. All procedures involving human subjects in this study were conducted in accordance with the Declaration of Helsinki, approved by the Board of Medical Research Ethics of the Ministry of Health of Malaysia (Approval No. NMRR-21-1819-61300), and registered on ClinicalTrials.gov (Identifier No. NCT05316064).

[0164] The sample size was calculated for a parallel group study design involving one participant in the prevention group and one in the placebo group, based on power design analysis. A total of 126 subjects are required for this study, based on the following calculations.

[0165] A Google Scholar search yielded few reports on the effects of probiotics on reducing HPV abundance and / or severity, with only three found. Ou et al. (2019) reported a reduction in mild abnormal cervical smears in the study probiotic group compared to the placebo group (p=0.017) in 121 women (probiotics n=62, placebo n=59), whereas Palma et al. (2018) reported a total HPV clearance rate of 11.6% in a short-term probiotic regimen compared to 31.2% in long-term users (p=0.044) in 117 women (probiotics n=60, probiotics n=57, 6 months). These results are similar to those of the present study, but this study did not include a placebo control group. Verhoven et al. (2013) reported that in a pilot study involving 51 women (probiotics n=24, placebo n=27), users of probiotics were twice as likely to eliminate cytological abnormalities compared to placebo (p=0.05). Considering that Ou et al. (2019) and Palma et al. (2018) both involved approximately 120 subjects, Verhoven et al. (2013)'s pilot study involved 102 subjects, and since there was no reference to the magnitude of the possible effect of probiotics based on primary outcomes, the average number of subjects decided to take was 121, 117, and 102, which yields 114 women (n=57 per group). An additional 10% dropout rate ultimately yields a total of 126 subjects (n=63 per group).

[0166] Questionnaire

[0167] Eligible subjects who met all inclusion and exclusion criteria were provided with two types of Malaysian questionnaires: (i) a demographic questionnaire translated and validated in previous studies (Chong et al., 2019a, 2019b) (collected at reference week 0) and (ii) a vaginal health and sexual function questionnaire (collected at weeks 0, 6, and 12). The vaginal health and sexual function questionnaire consists of three (3) types of questionnaires: i) the Vaginal Assessment Scale (VAS) and the Vulvar Assessment Scale (VuAS) are simple questionnaires with four items for each vaginal and vulvar symptom, used in the general healthy population and validated in the female cancer population (Eaton et al., 2017); ii) the general vaginal health questionnaire is based on the validated Vulvar Symptom Questionnaire (VSQ), consisting of 21 items, and covers aspects of vulvar and socioemotional symptoms (Erekson et al., 2013). The VSQ questionnaire was approved for use in a previous study by Bahasa Malaysia (USM / JEPeM / 18090421), and iii) female sexual function was evaluated using the 19-item Female Sexual Function Index (FSFI). The FSFI is a self-reporting tool consisting of 19 items covering six domains of female sexual function, such as desire, arousal, lubrication, orgasm, satisfaction, and pain. This scale has been validated in many languages ​​and is widely used in clinical and research settings (Rosen et al., 2000). A Bahasa Malaysian translation and validated version is available (Sidi et al., 2007) and is available with permission.

[0168] HPV positive, HPV genotype, and L1 detection

[0169] DNA extraction and purification from vaginal swab samples were performed according to the protocol of the High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany). Purified DNA was measured using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, NC, USA). An internal control for the human β-globin gene was performed. Nested PCR was performed using MY09 / MY11 as the external primers and GP5+ / GP6+ as the internal primers (Chong et al., 2010). The primers used are as follows.

[0170] (Forward) MY09: 5′- CGTCCMARRGGAWACTGATC -3′

[0171] (Reverse) MY11: 5′- GCMCAGGGWCATAAYAATGG -3′

[0172] (Forward) GP5+: 5′- TTTGTTACTGTGGTAGATACTAC -3′

[0173] (Reverse) GP6+: 5'- GAAAAATAAACTGTAAATCATATTC-3'

[0174] Samples with a band were confirmed to be HPV-positive. Sequences obtained through Sanger sequencing were compared with sequences from Genbank to identify HPV genotypes.

[0175] Quantitative real-time PCR was used to quantify the HPV L1 gene using the KAPA SYBR FAST qPCR Kit Master Mix (2x) Universal (KAPA reference dye BIOSEMS, MA, USA) and amplified using the Agilent AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA). A 20μL mixture containing 10μL of 2x KAPA SYBR Master Mix, 0.4μL of L1 forward and reverse primers (10μM), and 100ng of template DNA was used in a triple incubation. During the maintenance phase, the samples were incubated at 94°C for 5 minutes. Then, 30 cycles of amplification were performed on the samples for 30 seconds at 94°C and 59 seconds at 55°C, followed by an extension at 72°C for 59 seconds. The samples were further incubated at 95°C for 30 seconds, and then terminated at 95°C for 30 seconds during the melting curve phase. A reaction mixture without a cDNA template was used as a negative control. The critical cycle (Ct) of each sample was recorded by measuring the cycle in which the release of SYBR green exceeded a critical level. The universal primers for HPV gene L1 are as follows.

[0176] (Forward) MY09: 5′- CGTCCMARRGGAWACTGATC -3′

[0177] (Reverse) MY11: 5′- GCMCAGGGWCATAAYAATGG -3′

[0178] Statistical analysis

[0179] Data were analyzed using SPSS version 24.0 (SPSS Inc., Chicago, IL). The primary hypothesis of this study included differential effects between the two groups of probiotics and placebo. Considering the skewed distribution and non-parametric nature of the data, differences in scaled data were compared using the Mann-Whitney U test, and nominal data were compared using the chi-square test. Spearman's rank correlation was used in the non-parametric correlation analysis using rho(r) as the correlation coefficient. All tests were two-sided with a P<0.05 value, which is considered statistically significant, and data are expressed as mean ± standard error unless otherwise specified.

[0180] Results and Conclusions

[0181] Example 1: Effects of Lactobacillus plantarum Probio-87 on the promotion of mental, psychological, and emotional health in adult women

[0182] Clinical study results

[0183] The clinical study in humans was a double-blind, randomized, placebo-controlled design. Randomization was performed considering inclusion and exclusion criteria. Qualified subjects were randomly assigned to the two divisions of the study in a 1:1 ratio according to a computer-generated list and assigned to either the probiotics or placebo group using individual codes. Randomization was performed by research statisticians who did not have contact with the participants. The sample size was calculated for a parallel-group study design including one prevention division and one placebo division, based on power design analysis. A total of 112 subjects participated in this study, including 56 subjects in each group (probiotics and placebo) and an additional 50% dropout rate. This calculation was based on the need for independent controls and continuous response variables for the subjects. The ratio of controls to subjects was fixed at 1:1 with a probability of 0.9, and was performed based on the need for independent controls and continuous response variables for the subjects using the Type I error probability associated with this null trial (p<0.05).

[0184] Of the 121 subjects whose eligibility was reviewed, only 112 met the inclusion and exclusion criteria. Of the 112 recruited subjects, none dropped out or were unreachable during the 12 weeks. A total of 108 subjects provided complete answers to the WHQ, blood samples, and stool samples, while only 104 subjects provided vaginal swab samples (samples could not be collected due to menstruation). No adverse events were reported throughout the study, and no patients dropped out due to complications. Significant differences were observed in all demographic characteristics between the probiotic and placebo subjects (Table 2). Table 2 below shows the demographic characteristics of women (n=112) randomized to receive either placebo (n=54) or the probiotic Lactobacillus plantarum Probio-87 (n=58) in a double-blind study.

[0185]

[0186] Differences between groups were observed in the responses obtained through the WHQ (Table 3). Table 3 below shows the changes in Women's Health Questionnaire (WHQ) response scores of women (n=108) randomly assigned to double-blind administration of Lactobacillus plantarum Probio-87 (n=55).

[0187]

[0188] As can be seen in Table 3 above, in the depressed mood domain, probiotic administration showed improvement compared to placebo in item 8 after 6 weeks (P=0.047; feeling that life is not worth living). A minor placebo effect was observed in item 3 (P=0.064; miserable and sad) during the first 6 weeks, but this effect did not persist until the end of the study. After 6 weeks, slight improvement was also observed in item 10 of the probiotic group (P=0.071; good appetite), and this effect remained significant until the end of the study (P=0.012).

[0189] In the somatic symptoms domain, probiotic administration showed a slight improvement after 6 weeks compared to placebo on item number 14 (P=0.072; presence of headache). A placebo effect was observed within the first 6 weeks for item 23 (P=0.044; nausea) and item 35 (P=0.048; urinating more frequently than usual), but this effect did not persist until the end of the study. Instead, from week 6 until the end of the study, probiotic administration showed a significant improvement in item number 23 compared to placebo (P=0.018). In the anxiety or fear domain, probiotic administration showed improvement after 12 weeks on item 2 (P=0.066; feeling very scared or embarrassed for no reason) and item 9 (P=0.055; feeling good or hurt). A placebo effect was observed within the first 6 weeks for item number 9 (P=0.031), but this effect did not persist until the end of the study.

[0190] In the sexual activity domain, probiotic administration showed a slight improvement in item number 34 compared to the placebo group after 6 weeks (P=0.073; discomfort during intercourse due to vaginal dryness). In the sleep problems domain, probiotic administration was shown to improve item number 11 (P=0.009; anxiety and inability to sit still) after 6 weeks compared to placebo. In the menstrual symptoms domain, probiotic administration showed a slight improvement compared to placebo in item number 17 (P=0.055; chest pain or discomfort) after 6 weeks. A placebo effect was observed within the first 6 weeks in item number 22 (P=0.059; abdominal cramps or discomfort) and item number 28 (P=0.068; bloating), but this effect did not persist until the end of the study. Instead, from week 6 until the end of the study, probiotic administration showed a slight improvement compared to placebo in item number 22 (P=0.073).

[0191] The WHQ is the most frequently used tool for assessing the quality of life (QOL) of women, a specific population group. It can be used independently, as part of a QOL assessment, or for intervention and prevention strategies for women's health. This 36-item questionnaire covers aspects such as vasomotor symptoms, psychosocial factors, general health and / or aging, sleep, sexual issues, and cognitive impairment, and evaluates nine domains of physical and emotional health rated on a 4-point scale. Although initially developed for middle-aged women experiencing menopause or post-menopause (ages 45–65), the WHQ is currently applied to assess QOL in samples of healthy women, including young women (ages 23 and older), as well as through various medical and non-medical interventions and specific symptoms.

[0192] Changes in the expression of various genes were observed in both groups over 12 weeks, and the effect of the probiotic group was more dominant compared to the placebo group (Figure 1). Compared to the placebo group, the probiotic group showed higher expression of genes related to mental / physiological quality of life, such as BDNF (P<0.001), TPH-2 (P=0.001), 5-HT6 (P<0.001), GAD-2 (P=0.033), ghrelin (P=0.004), and leptin (P=0.003).

[0193] Neurotrophic factors such as BDNF often activate CREB by inducing intracellular signaling pathways that lead to increased CREB phosphorylation and transcriptional activity in pathways controlling neuronal survival, development, and functional aspects in both the central and peripheral nervous systems. However, according to the current data of this study, CREB genes showed no differences between groups, suggesting that BDNF may play a role beyond CREB activation for neuronal benefit. While BDNF is primarily associated with neuroplasticity and cognitive function, there is growing evidence that it can also influence inflammation and immune responses. BDNF regulates neuroinflammation, controls immune cells including microglia—resident immune cells of the brain—exerts anti-inflammatory effects in peripheral tissues, and influences the migration of immune cells during immune responses.

[0194] More importantly, BDNF has been reported to interact with neurotransmitters such as serotonin in immune regulatory processes. This is supported by the upregulation of serotonin-related genes within the probiotic group, namely TPH-2, which converts tryptophan to serotonin in the brain, and 5-HT6, which aids in serotonin release from neurons. In addition to serotonin, the administration of probiotics catalyzes the upregulation of the GAD-2 gene, which catalyzes the synthesis of gamma-aminobutyric acid (GABA) from the amino acid glutamate. Serotonin is a crucial neurotransmitter that regulates learning, memory, and happiness, as well as body temperature, sleep, sexual behavior, and hunger. Serotonin deficiency is associated with depression and anxiety. Meanwhile, the neurotransmitter GABA is associated with sedative effects that reduce anxiety, stress, and fear. All of this strongly supports the results of WHQ data showing that the administration of probiotics improved areas related to depressed mood, anxiety or fear, sexual behavior, and sleep problems.

[0195] Serotonin regulates the activity of ghrelin, a hormone produced in the stomach that elevates mood while simultaneously increasing hunger and appetite. This is also supported by WHQ data showing that the probiotics group experienced increased appetite compared to the placebo group over 12 weeks. On the other hand, leptin is a hormone produced by fat cells that plays a role in regulating energy balance through motivational behaviors aimed at increasing satiety and maintaining energy reserves. Although leptin and ghrelin have opposing effects in the physiology of hunger, leptin does not directly affect ghrelin levels. Given that the probiotics group showed upregulation of two genes related to ghrelin and leptin, it is hypothesized that while an increase in serotonin helps increase ghrelin and enhance appetite, all of this is balanced by increased leptin to suppress excess that could lead to unwanted weight gain.

[0196] conclusion

[0197] This study demonstrated that the administration of the probiotic KCTC 15755BP improved mental, psychological, and emotional health in adult women in areas related to depression, physical condition, anxiety, sexuality, sleep, and menstruation. The effects of the probiotic generally appeared after 6 weeks of administration, succeeding the placebo effect which was primarily reduced in the initial stages of administration. This is also supported by gene expression data showing that KCTC 15755BP enhanced mental health and physiology in particular through the action of the neurotransmitters serotonin and GABA along the gut-brain axis and hormones associated with regulating hunger and satiety. These results indicate that KCTC 15755BP meets the requirements for probiotic strains according to FAO / WHO recommendations and can be applied as a natural strategy to improve psychological functioning and mental health in adults.

[0198] Example 2: Effects of Lactobacillus plantarum Probio-87 on improving vaginal health in adult women through enhancement of immune and inflammatory parameters

[0199] Clinical study results

[0200] Figure 1 is a graph showing the changes in relative gene expression levels in the blood for pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ), anti-inflammatory cytokines (IL-10, IL-4), T cells (CD8, CD117, CD44, FOXP3, CD4), B cells (CD27, CXCR5), NK cells (CD56, CD94, CD34, Nkp30, Nkp46, Nkp44) and mental health / physiological parameters (CREB, BDNF, TPH-2, 5-HT6, IDO, TH, GAD-2, ghrelin, leptin) when a placebo (n=53) or Lactobacillus plantarum Probio-87 strain was administered for 12 weeks. The data in Figure 1 above are expressed as mean ± SEM, and the P-value indicates the difference between groups based on changes over 12 weeks using the ΔCT value through the Mann-Whitney test.

[0201] As shown in Figure 1, changes in various gene expressions were observed in both groups over 12 weeks, with more widespread effects observed in the probiotics group compared to the placebo group. The probiotics group showed higher gene upregulation compared to the placebo group for anti-inflammation-related genes (IL-10; P<0.001) and T cells (CD8 P=0.042, CD44 P<0.001). The most widespread effects were observed in genes related to the NK cell pathway, where the probiotics group showed higher gene upregulation compared to the placebo group (CD56 P=0.027, minimal CD94 P=0.085, minimal Nkp30 P=0.081, Nkp46 P=0.001, minimal Nkp44 P=0.074).

[0202] Most of the upregulated genes are associated with Natural Killer (NK) cells, a type of cytotoxic lymphocyte that plays a crucial role in the innate immune system. Nkp30, Nkp46, and Nkp44 are part of the Natural Cytotoxic Receptor (NCR) family and are primarily expressed on the surface of NK cells and some innate lymphocytes. These NCRs bind to ligands on target cells to induce signal activation in NK cells, thereby enhancing the cytotoxic function of NK cells by inducing apoptosis in target cells, including antigen-infected cells. Nkp30, Nkp46, and Nkp44 act as important activating receptors on the surface of NK cells, enhancing the ability of NK cells to detect and eliminate target cells during immune surveillance.

[0203] The CD56 and CD94 genes are expressed in various cell types, including NK cells, and are involved in cytokine production and immune regulation, possessing cytotoxicity that kills target cells. Their expression on the surface of NK cells can be regulated by various factors, including cytokines and activation signals.

[0204] IL-10 is an anti-inflammatory cytokine produced by various immune cells, including NK cells, in response to stimulation. IL-10 plays an important role in regulating immune responses by suppressing excessive inflammation, inhibiting the production of inflammatory cytokines, and promoting the function of regulatory T cells.

[0205] CD44 and CD8 are expressed on the surface of T cells and regulate various aspects of the response, including recruiting T cells to the site of inflammation, forming immune synapses with target cells, and T cell activation and proliferation. They are also associated with memory T cells, which are crucial for initiating a rapid and effective immune response, particularly after an initial immune response, upon encountering previously encountered antigens again. However, considering that all other genes related to T and B cells analyzed showed no changes over time, the roles of CD44 and CD8 in the subjects are hypothesized to extend beyond the role of T cells. Furthermore, the surface glycoproteins CD44 and CD8 are also expressed on NK cells and facilitate interactions between NK cells and other cells, extracellular matrix components, and tissues, enabling NK cells to attach to endothelial cells and migrate into tissues to locate target cells. Therefore, they are upregulated upon NK cell activation and are also associated with memory-like NK cells, which exhibit an enhanced response upon encountering specific antigens again.

[0206] In response to antigen invasion, some of these genes interact with each other to activate various immune regulatory responses. Nkp46 interacts with IL-10 to limit excessive NK cell activity, thereby preventing immune-related tissue damage. Meanwhile, as part of its immune regulatory function, CD56 regulates IL-10 production by NK cells to modulate immune responses and maintain immune homeostasis. The balance between NK cell cytotoxic activity and cytokine production is critical to the ability to target infected or mutated cells while avoiding excessive tissue damage and the overall impact on the immune response.

[0207] Given that both groups experienced upregulation of immune-regulating genes, namely IL-10, CD44, and CD8, it can be assumed that antigenic intrusion occurred during the study period. Since most subjects remained healthy and did not report any events during clinical visits during the study period, they may have been asymptomatic or / or mild. However, because women in the probiotics group exhibited greater upregulation of NK-related immune genes—namely CD56, CD94, Nkp30, Nkp46, and Nkp44—than the placebo group, it is hypothesized that a greater defense against these antigenic intrusions was initiated compared to the placebo group. Although CD44 and CD8 are often associated with T cell activation, the data in this study did not show changes in other T cell (or B cell) related genes; therefore, it is hypothesized that this is more related to NK activation than to their upregulation. NK cells are the first line of immune defense before deeper defenses, such as T cells or B cells, are further activated. Therefore, according to the data of this study, the administration of probiotics was found to strengthen the first line of defense against antigen invasion more than the placebo group. In addition, considering that only IL-10 showed an effect, while other inflammation-inducing and anti-inflammatory genes in both groups remained unchanged over time, it is believed that the administration of probiotics acts as a buffer against excessive inflammatory effects from immune-mediated tissue damage.

[0208] conclusion

[0209] This study showed that the administration of probiotics improves vaginal health by exerting anti-inflammatory and immunomodulatory protection, particularly through the activity of NK cells. Consequently, this implies that KCTC 15755BP meets the probiotic strain requirements according to FAO / WHO recommendations and can be applied as a natural strategy for improving immunity in adults.

[0210] Example 3: Effects of Lactobacillus plantarum Probio-87 on the regulation of the vaginal environment regarding the unbalanced vaginal bacterial population in adult women

[0211] Clinical study results

[0212] Alpha diversity

[0213] Alpha diversity measures within-sample differences using various indices. The Chao1 index is a tool that estimates operational taxonomic unit (OUT) "richness" by treating frequent and rare OTUs equally. A higher Chao1 value indicates higher richness. The ACE index measures richness similar to Chao1, but it also considers non-rich OTUs to estimate the potential number of OTUs not found within the sample. A higher ACE index value indicates greater diversity influenced by richness and evenness.

[0214] Figure 2 is a graph showing alpha diversity in vaginal microbiota at baseline (week 0) and after week 12 upon administration of the Lactobacillus plantarum Probio-87 strain or a placebo. Figure 2 is based on the difference in diversity between groups measured by the Chao1 index for phylum (A) and genus (C), and the ACE index for phylum (B) and genus (D). No significant differences were observed between groups at baseline (P>0.10). The line inside the box represents the median, and the range line represents the lowest and highest values ​​within the interquartile range. Outliers and individual sample values ​​are indicated by dots. Statistical significance was analyzed using the Mann-Whitney U test. Here, n = 104 (KCTC 15755BP n = 53, placebo n = 51).

[0215] As shown in Figures 2A and 2C, at baseline, both groups exhibited similar Chao1 (P=0.899) and ACE (0.706) indices at the phylum level. However, at week 12, the placebo group showed higher Chao1 and ACE indices at the phylum level compared to the probiotics group (P=0.011 and P=0.021, respectively). Similarly, as shown in Figures 2B and 2D, at baseline, both groups exhibited similar Chao1 (P=0.912) and ACE (0.543) indices at the genus level. However, at week 12, the placebo group showed higher Chao1 and ACE indices at the genus level compared to the probiotics group (P=0.008 and P=0.003, respectively).

[0216] Observation indices measure abundance by representing the total number of OTUs observed within a sample without considering richness, providing the simplest measure of alpha diversity and offering a basis for the existing differences. Meanwhile, Shannon and Fisher indices measure both abundance and uniformity by considering the frequency of each OTU. Higher Shannon or Fisher index values ​​indicate higher diversity influenced by the number of OTUs and relative abundance.

[0217] Figure 3 is another graph showing alpha diversity in the vaginal microbiome at baseline (week 0) and after week 12 upon administration of the Lactobacillus plantarum Probio-87 strain or a placebo. Figure 3 is based on the difference diversity between groups measured by observed indices for (A) Phylum, (B) Family, and (C) Genus; and the Shannon index for (D) Genus and Fisher index for (E) Genus. At baseline, there were no significant differences between groups (P > 0.10). The line inside the box represents the median, and the range line represents the lowest and highest values ​​within the interquartile range. Outliers and individual sample values ​​are marked as dots. Statistical significance was analyzed using the Mann-Whitney U test. Here, n = 104 (KCTC 15755BP n = 53, placebo n = 51).

[0218] As shown in Figures 3A, B, and C, at baseline, both groups exhibited similar observed indices at the phylum (P=0.862), family (P=0.677), and genus (P=0.644) levels. However, at week 12, the placebo group showed higher observed indices compared to the probiotics group at the phylum, family, and genus levels (P=0.030, P=0.084, and P=0.068, respectively). Similarly, as shown in Figures 3D and E, at baseline, both groups exhibited similar Shannon (P=0.526) and Fisher (0.416) indices at the genus level. However, at week 12, the placebo group showed slightly higher Shannon and Fisher indices compared to the probiotics group at the genus level (P=0.075 and P=0.075, respectively).

[0219] Current data indicate that the administration of KCTC 15755BP prevented an increase in within-group ecological diversity across various taxa and relative abundances, which was clearly increased in the placebo group after 12 weeks. This also indicates the presence of specific dominant OTUs, which will be described in more detail in the subsequent analysis below. While a diverse and balanced vaginal microbiome is generally associated with improved vaginal health, increased diversity can lead to microbial imbalances associated with disease and infection. The composition of the vaginal microbiome plays a crucial role in maintaining acidic pH and preventing the overgrowth of harmful microorganisms. Women with bacterial vaginosis (BV) have a more diverse and heterogeneous community than women without BV, possessing higher levels of species richness and bacterial diversity.

[0220] Beta diversity

[0221] Beta diversity measures differences between samples. The Bray-Curtis index considers both the co-occurrence and differential abundance of OTUs. Beta diversity is often measured by analysis of principal coordinates (PCoA) using PERMANOVA or ANOSIM. PERMANOVA tests for differences between groups in terms of multivariate variance, whereas ANOSIM tests for differences in the rank of means between groups.

[0222] Figure 4 is a graph of principal coordinate analysis (PCoA) measured by PERMANOVA for (A) Phylum (PCoA P=0.991), (B) Class (PCoA P=0.982), (C) Order (PCoA P=0.808), (D) Family (PCoA P=0.587), and (E) Genus (PCoA P=0.551), and Figure 5 is a graph of principal coordinate analysis (PCoA) measured by ANOSIM for (A) Phylum (PCoA P=0.999), (B) Class (PCoA P=0.986), (C) Order (PCoA P=0.960), (D) Family (PCoA P=0.796), and (E) Genus (PCoA P=0.759).

[0223] In Figures 4 and 5, beta diversity measured by the Bray-Curtis non-discordance index is shown at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered. Here, n = 104 (KCTC 15755BP n = 53 and placebo n = 51). As shown in Figures 4 and 5, both PERMANOVA and ANOSIM obtained similar results, and the placebo group showed a difference from the probiotic group at week 12, despite a minimal difference at baseline. This was observed at all taxonomic levels studied, namely in PERMANOVA at the phylum (P=0.058), class (P=0.036), order (P=0.075), family (P=0.025), and genus (P=0.027) levels (Fig. 4 AE), and in ANOSIM at the phylum (P=0.079), class (P=0.055), order (P=0.080), family (P=0.036), and genus (P=0.023) levels (Fig. 5 AE).

[0224] Change in composition

[0225] Alpha and beta diversity assessments indicate group differences in the vaginal microbiome of healthy women. According to the data from this study, changes occurred in the placebo group over time, generally resulting in increased abundance across various taxonomic levels and the inclusion of specific bacterial groups typically associated with vaginal infections. Many women are unaware of the onset of vaginal infections, an increase in vaginal pathogens, and / or a decrease in the growth of beneficial vaginal bacteria, particularly when symptoms are absent. Based on this, microbiome profiling was used to further detect specific compositional changes in the vaginal bacterial population.

[0226] The placebo group showed an increase in vaginal Bacteroidaceae abundance, whereas the probiotic group showed a decrease over 12 weeks (P=0.010). Similar trends were consistently observed at lower taxonomic levels for the genus Bacteroides (P=0.010) and for other individual genera within other families, such as Paraprevotella (P=0.008) and Parabacteroides (P=0.027). Higher levels of Bacteroides are present in the vaginal microbiome of women infected with HPV, which is also associated with urinary incontinence in women with urge incontinence. Meanwhile, Parabacteria are found in human feces and generally inhabit the gastrointestinal tract of numerous species. They are also one of the dominant endometrial microbial communities. Their roles for the host are contradictory, ranging from tissue lesions in Crohn's disease patients to protective effects against IBD.

[0227] Firmicutes included the largest genera in which differences were observed between treatment groups, with the placebo group showing increased abundance while the probiotic group showed decreased abundance over 12 weeks. These included Marvin Bryantia (P=0.008), Dorea (P=0.006), and Lachnoclostridium (P=0.007) of the Lachnospiridae family, Eubacterium syraum group (P=0.008) and Pastidiosifera genus (P=0.045) of the Ruminococcidae family, Clostridium bacteria KA00274 (P=0.049) of the Hungarianclostridium family, and Pachlamia genus (P=0.006) and Pachlamia hominis CCUG36813 (P=0.006) of the Aerococcidae family. In addition, other genera such as Negatyvivacillus (P=0.056), Subdoligranulum (P=0.082), Blautia (P=0.093), and Stomatobakulum (P=0.096) showed similar trends. Dorea is positively correlated with BV, while Marvinbriantia is detected in the urine of women with acute urinary incontinence. Eubacterium siraeum is positively correlated with vaginal group-B Streptococcus, whereas Negatyvivacillus is generally known as a butyrate producer in the intestines but causes microbial infection and inflammation in the vagina. Pastidiosifilla is generally positively correlated with high-risk BV subtypes and incidence rates, while Lachnoclostridium is positively correlated with cervical cancer in women. Blautia is one of the dominant endometrial microbiota, whereas Stomatobakulum is part of the oral microbiota of healthy individuals. Meanwhile, the genus Subdoliculum was confirmed to be common.

[0228] Several genera under the phylum Actinobacteria showed increased abundance in the placebo group after 12 weeks, while they showed decreased abundance in the probiotics group. These included Actinomyces (P=0.014), Brevacterium (P=0.047), Micrococus (P=0.045), and Coriobacteriaaceae (P=0.014). The species Corynebacterium simulans (P=0.047) showed a similar trend, and the genus Trueppelella showed a marginal effect (P=0.075). Actinomyces is known to cause pelvic inflammatory disease in women with intrauterine devices and is associated with urinary incontinence in women with acute urinary incontinence. Several species of Actinomyces are known as oral and dental pathogens. Meanwhile, women infected with Clamydia trachomatis have been reported to possess higher levels of vaginal Cholinesella. While Brevibacterium species are associated with BV due to their biofilm-producing capabilities (Alves et al., 2014), an increased prevalence of Micrococcus within the endometrial microbiome is associated with inflammation in women with endometrial cancer. Higher levels of Troperella have been reported in women with BV compared to healthy controls. Corynebacterium simulans is primarily found on the skin and has been reported to be highly resistant to numerous antibiotics, such as β-lactams, cehemes, carbapenems, and quinolones. An increase in opportunistic infections caused by Corynebacterium species has been reported in the elderly and immunocompromised patients.

[0229] Desulfovibryonian class (P=0.032) and lower taxonomic levels were found to have increased abundance in the placebo group, whereas their abundance in the probiotics group decreased over 12 weeks. These included Desulfovibryones (P=0.032), Desulfovibryonian family (P=0.032), and Bilopila genus (P=0.034). Bilopila species are less frequently encountered in clinical practice but have been reported to cause infections and inhabit the human coropharynx, gastrointestinal tract, and genitourinary tract.

[0230] The genus *Cupriavidus* under the phylum Proteobacteria increased abundantly in the placebo group for 12 weeks but decreased abundantly in the probiotics group (P=0.003). A similar trend was observed in the genus *Aeromonas* (P=0.047), the family Aeromonidae (P=0.047), and the genus *Encipher* (P=0.043). The order Xanthomonadales under the same phylum showed a similar trend (P=0.023), followed by the taxonomic level of the family Xanthomonadales (P=0.23) and the marginal effect of the genus *Stenotrophomonas* (P=0.082). While *Aeromonas* species were isolated from urine samples of women with UTI, *Cupriavidus* was found more frequently in women with BV than in the normal group. Ensifer is present in the environment and on human skin, but has recently been reported to have high predictive value for embryonic arrest, whereas Stenotrophomonas is considered a human symbiont and is a microorganism frequently extracted from urine samples.

[0231] conclusion

[0232] The Lactobacillus plantarum Probio-87 strain according to the present invention was shown to prevent changes in microbial community composition over time that were evident in the placebo group. The results indicate that the Lactobacillus plantarum Probio-87 strain meets the requirements for probiotic strains according to FAO / WHO recommendations and can be applied as a natural strategy to regulate the vaginal environment for unbalanced vaginal bacterial populations in adult women.

[0233] Example 4: Effects of Lactobacillus plantarum Probio-87 on Gut Regulation of Unbalanced Gut Microbiota in Adult Women

[0234] Clinical study results

[0235] Alpha diversity

[0236] Figure 6 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups measured by the Chao1 index for (A) Phylum (W0 P=0.263), (B) Class (W0 P=0.342), (C) Order (W0 P=0.216), (D) Family (W0 P=0.341), and (E) Genus (W0 P=0.827). Figure 7 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by observation indices for (A) phylum (W0 P=0.429), (B) class (W0 P=0.212), (C) order (W0 P=0.376), (D) family (W0 P=0.721), and (E) genus (W0 P=0.941). Figure 8 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by the ACE index for (A) phylum (W0 P=0.586), (B) class (W0 P=0.435), (C) order (W0 P=0.386), (D) family (W0 P=0.428), and (E) genus (W0 P=0.801). In addition, Figure 9 is a graph showing alpha diversity for stool microbiota at baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered, showing the difference in diversity between groups as measured by Fisher's index for (A) phylum (W0 P=0.507), (B) class (W0 P=0.221), (C) order (W0 P=0.370), (D) family (W0 P=0.685), and (E) genus (W0 P=1.000).

[0237] In Figures 6 through 9, the line inside the box represents the median, the range line represents the lowest and highest values ​​within the interquartile range, and outliers and individual sample values ​​are marked as points. The statistical significance of the above results was analyzed using the Mann-Whitney U test, with n = 104 (KCTC 15755BP n = 53, placebo n = 51).

[0238] As illustrated in Figures 6 through 9, over 12 weeks, the probiotics group demonstrated significantly higher abundance and evenness compared to the placebo group through the Chao1 (Figure 6), Observed (Figure 7), ACE (Figure 8), and Fisher (Figure 9) indices, and it was found that this was present across various taxonomic levels, including phylum, class, order, family, and genus. Meanwhile, there were no significant differences between the groups at week 0 across all taxonomic levels studied (P>0.05).

[0239] The data above showed that the administration of the Lactobacillus plantarum Probio-87 strain prevented the loss of ecological diversity within the group across various taxa and relative abundances, which was not evident in the placebo group. A healthy gut microbiome is often associated with diversity. Meanwhile, bacterial imbalances and other gut-related disorders have been shown to alter the gut microenvironment and reduce diversity.

[0240] Beta diversity

[0241] Figure 10 is a graph of the principal coordinate analysis (PCoA) measured by PERMANOVA for (A) phylum, (B) class, (C) order, (D) family, and (E) genus, and Figure 11 is the N-metric multidimensional scaling (NMDS) measured by PERMANOVA for (A) phylum, (B) class, (C) order, (D) family, and (E) genus. Figures 10 and 11 show beta diversity measured using the Bray-Curtis non-disjointity index, distinguished by baseline (week 0) and after week 12 when Lactobacillus plantarum Probio-87 strain or placebo was administered.

[0242] As shown in Figure 10, no significant difference was observed between the groups at baseline (week 0). At week 12, significant differences were observed between the probiotic group and the placebo group for class (P=0.033) and genus (P=0.032), while marginal differences were observed for phylum (P=0.072), order (0.091), and family (P=0.088). Here, n = 108 (KCTC 15755BP n = 55, placebo n = 53).

[0243] As shown in Figure 11, no significant difference was observed between the groups at baseline (week 0). At week 12, significant differences were observed between the probiotic group and the placebo group for Phylum (P=0.045), Class (P=0.028), and Genus (P=0.033), and marginal differences were observed at the Order (P=0.087) and Family (P=0.085) levels. Here, n = 108 (KCTC 15755BP n = 55, placebo n = 53).

[0244] Principal Coordinate Analysis (PCoA) and Non-Metric Multidimensional Scaling (NMDS) for various taxonomic levels of phylum, class, order, family, and genus as measured by PERMANOVA showed significant changes from baseline between groups. However, at week 12, significant differences were observed between the probiotic and placebo groups for PCoA at the class (P=0.033) and genus (P=0.032) levels, while marginal differences were observed for phylum (P=0.072), order (0.091), and family (P=0.088) levels (Fig. 10). Similarly, despite significant differences between groups at week 0, differences in NMDS between the probiotic and placebo groups were observed at week 12 at the phylum (P=0.045), class (P=0.028), and genus (P=0.033) levels, and marginal differences were observed at the order (P=0.087) and family (P=0.085) levels (Fig. 11). All of this demonstrates that the Lactobacillus plantarum Probio-87 strain contributed to changes in the composition of the gut microbial community after 12 weeks compared to placebo.

[0245] Change in composition

[0246] Alpha and beta diversity assessments both indicate intergroup differences in bacterial diversity in stool samples from healthy women across different taxa. Therefore, additional analyses were performed for each individual taxon to confirm changes in composition and profile.

[0247] At the phylum level, only phylum Firmicutes showed differences between groups, where the probiotic group showed increased abundance over 12 weeks (P=0.071). Similar trends were observed at lower taxonomic levels in the classes Clostridia (P=0.059) and Bacillae (P=0.096), Lachnospirales (P=0.013), Monoglobaceae (P=0.012), Lachnospiraceae (P=0.013), and Erysipelotrichales (P=0.007). Here, Dorea (P=0.001), Eubacterium halyi group (P=0.031), Eubacterium nodatum group (Mimi, P=0.093), Agatobacter (P=0.003), Lachnospira and ND3007 group (P=0.003), Luminococcus (P=0.016), Luminococcus gobraui group (P=0.002), Luminococcus toc group (Mimi, P=0.081), Oscillybacter (P=0.029), Monoglobus (P=0.012), Lactococcus (Mimi, P=0.083), and Delma (Mimi, P=0.083) showed the same trend. While Dorea is a common genus in the human gut microbiome, Dorea formicigenerans is often isolated from human feces and typically produces butyrate (Downes et al., 2002). Eubacterium hallii is a butyrate producer and is naturally found in the feces of healthy humans (Pant & Das, 2022). Although E. hallii has been shown to improve insulin resistance and energy metabolism through the production of satiety hormones in obesity and diabetes models (Aruda et al., 2022), this species may also synthesize cobalamin, which is important for the formation of red blood cells and DNA for the function and development of brain and nerve cells (Engels et al., 2016). Meanwhile, the Eubacterium nodatum group has a negative association with liver cancer (Xie et al., 2023).Eubacterium rectale is a butyrate producer, and the reduced abundance observed in patients with immune disorders has had a negative impact on penetration into many organs, including the intestines (Payen & Rousseau, 2019). Although there is not much information on the Dielma, Lachnospiraceae ND3007 group and Lachnoclostridium species, they are isolated from human feces and are typically butyrate producers (Downes et al., 2002; Nishiwaki et al., 2020). Agatobacter also produces butyrate (Russell et al., 2011), and reduced abundance is associated with sleep disorders in children (Hua et al., 2020). Luminococcus is a butyrate producer and an important gut microbial symbiont by breaking down and converting complex polysaccharides into various nutrients for the host (La Lau & Suen, 2018). Ruminococcus bicirculans can produce acetate, an intestinal protective SCFA against pathogenic toxins, using various carbohydrates such as soluble starch, hemicellulose, barley beta-glucan, (1,4)-beta-D-mannan, and xyloglucan (Park, 2018). Oscillibacter produces valeric acid, which is important in the intestines for regulating Clostridium difficile infection (McDonald et al., 2018). Monoglobules specialized for pectin degradation have been shown to be negatively associated with neutrophil inflammation (Zha et al., 2022), and a decrease in their abundance is associated with intensified systemic inflammation (Dang et al., 2022), which is observed in patients with liver disease caused by the hepatitis B virus (Li et al., 2022). Lactococcus species generally have low toxicity and are rarely considered pathogenic, and several species are used as probiotics (Firmani et al., 2022).Lactobacillus ruminis is a symbiotic motile lactic acid bacterium that lives in the intestines of humans and animals (Wang et al., 2020). Overall, the significant increase in the abundance of these genera and species in the probiotics group over time compared to the placebo group demonstrates the important regulation of the gut microbiome associated with beneficial metabolites such as SCFAs.

[0248] Lower taxonomic levels of the Actinobacteria phylum showed a greater decrease in abundance in the placebo group compared to the probiotic group. These included the class *Choriobacteria* (P=0.018), *Choriobacteria* (P=0.018), *Choriobacteria* (P=0.024), *Chorisophylla* (P=0.026), *Rhaultibacter* (minimal, P=0.091), and the species *Chorisophylla aerofaciens* (P=0.028). Meanwhile, the species *Desulfovibrio piger* under the *Desulfobacteria* phylum increased in abundance over time in the probiotic group but decreased in the placebo group (P=0.009). Similar trends were observed at lower taxonomic levels within the phylum Bacteroidota, namely the genus Odoribacter (P=0.029) and its species Odoribacter splanticus (P=0.008), the family Vanciellaceae (P=0.014), and the genus Coprobacter (minimal, P=0.073). Colincella aerofaciens is a common gut species known in healthy individuals (Ahrodia et al., 2022) and has been shown to be abundant in patients responding positively to cancer immunotherapy (Russo et al., 2022). Desulfovibrio figurus is the most common sulfate-reducing bacterium among healthy adults. They utilize hydrogen as an electron donor to reduce sulfates or other oxidized sulfur compounds commonly found in diets containing preservatives, antioxidants, and food additives (Rey et al., 2013). Odoribacter is a common SCFA-producing member of the human gut microbiome. A decrease in the abundance of Odoriibacter is associated with various microbiome-related diseases such as non-alcoholic fatty liver disease, cystic fibrosis, and inflammatory bowel disease (Hippala et al., 2020).

[0249] conclusion

[0250] The Lactobacillus plantarum Probio-87 strain has been shown to be effective in regulating the gut regarding unbalanced gut bacterial populations, that is, in improving gut bacterial imbalance. The results indicate that the Lactobacillus plantarum Probio-87 strain meets the probiotic strain requirements according to FAO / WHO recommendations and can be applied as a natural strategy to improve gut health in adults.

[0251] Example 5: Growth inhibition of Lactobacillus plantarum Probio-87 and growth inhibitory effect of pathogenic Candida fungi through co-aggregation

[0252] In vitro study results

[0253] Vaginal infections are a widespread gynecological problem globally, with bacteria and yeast being the primary causes. The most common pathogenic yeasts isolated from vaginal candidiasis are Candida albicans, Candida glabrata, Candida parasilopsis, Candida krusei, and Candida tropicalis. These infections are significant in obstetrics due to the potential transmission of vaginal pathogens from mother to newborn during vaginal delivery. This transmission plays a role in shaping the infant's microbiome, which is important for gastrointestinal health but can also pose a risk of disease. Pregnancy increases the risk of vaginal Candida colonization due to increased estrogen and vaginal glycogen levels. Azole antifungals are the primary treatment, but concerns are being raised by the emergence of drug-resistant yeast strains. Drug resistance and changes in vaginal yeast have been observed, particularly in cases of frequent use of over-the-counter medications. Probiotics, known to be beneficial for gut health, are being increasingly studied for their role in managing the vaginal microbiome and inhibiting pathogens.

[0254] Figure 12 is a graph showing the antimicrobial activity of postbiotics, including the Lactobacillus plantarum Probio-87 strain and other probiotic strains, against (A) Candida albicans, (B) Candida glabrata, (C) Candida tropicalis, (D) Candida krusei, and (E) Candida parasilopsis. In Figure 12, the untreated control group was designed to grow pathogenic yeast in MRS without the treatment compound. Pathogenic yeast was treated with the antibiotic clotrimazole (50 μM and 200 μM) as a positive control. All data are expressed as means, and error bars represent the standard error of the mean (n=6). Here, abc This is the statistical difference between groups (P<0.05) determined using repeated measures ANOVA.

[0255] As shown in Fig. 12, the postbiotic of the Lactobacillus plantarum Probio-87 strain and all studied commercial probiotic strains exhibited growth inhibition similar to that of the antibiotic clotrimazole against Candida albicans and Candida parasilopsis at concentrations of 50 μM and 200 μM compared to the untreated control (P<0.05). However, the Lactobacillus plantarum Probio-87 strain exhibited the most potent growth inhibitory effect against Candida glabrata and Candida krusei compared to other commercial probiotics and antibiotics at different concentrations (P<0.05). The Lactobacillus plantarum Probio-87 strain had little effect on the growth of Candida tropicalis and showed an inhibitory effect similar to that of the untreated control and antibiotics.

[0256] FIG. 13 shows (A) living probiotic cells (10 6 Candida albicans cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0257] FIG. 14 shows (A) living probiotic cells (10 6 Candida glabrata cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0258] FIG. 15 shows (A) living probiotic cells (10 6 Candida tropicalis cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0259] FIG. 16 shows (A) living probiotic cells (10 6 Candida krusei cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0260] FIG. 17 shows (A) living probiotic cells (10 6 Candida parasilopsis cultured for 8 hours at 37°C when CFU / mL) or a control group was present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0261] (A) of FIGS. 13 to 17 above is abc Statistical differences between groups determined using one-way ANOVA (P<0.05). They were cultured with live probiotic cells and used YEPG medium (untreated control) or clotrimazole (100 μM or 400 μM) as positive controls. Additionally, (B) to (I) in FIGS. 13 to 17 represent (B) Lactobacillus plantarum Probio-87; (C) Lactiplantibacillus plantarum GC; (D) Lacticaseibacillus rhamnosus FD; (E) Lacticaseibacillus rhamnosus VP; and (F) Lactobacillus crispatus VP; (G) Untreated control group; (H) Clotrimazole (100 μM); (I) Image of clotrimazole (400 μM).

[0262] As illustrated in FIGS. 13 to 17, the live strain of Lactobacillus plantarum Probio-87 according to the present invention exhibited the highest co-aggregation ability compared to all commercial probiotic strains studied against Candida albicans (C. albicans) (Fig. 13), Candida glabrata (C. glabrata) (Fig. 14), Candida tropicalis (C. tropicalis) (Fig. 15), Candida krusei (C. krusei) (Fig. 16), and Candida parasilopsis (C. parasilopsis) (Fig. 17) (P<0.05).

[0263] FIG. 18 shows (A) Candida albicans cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v) (10 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0264] FIG. 19 shows (A) Candida glabrata (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0265] FIG. 20 shows (A) Candida tropicalis (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0266] FIG. 21 shows (A) Candida krusei (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0267] FIG. 22 shows (A) Candida parasilopsis (10) cultured at 37°C for 8 hours when postbiotics or a control were present in a 1:1 ratio (v:v). 6 This is a graph showing the results of the aggregation percentage (%) of CFU / mL, with results expressed as mean ± SEM (n=6). (B) to (I) are Gram staining images observed with an optical microscope at ×100 magnification.

[0268] (A) of FIGS. 18 to 22 above is abc Statistical difference between groups determined using one-way ANOVA analysis (P<0.05). Live probiotic cells were cultured and used as YEPG medium (untreated control) or clotrimazole (100 μM or 400 μM) as a positive control. Additionally, (B) to (I) in FIGS. 18 to 22 respectively represent (B) Lactobacillus plantarum Probio-87; (C) Lactiplantibacillus plantarum GC; (D) Lacticaseibacillus rhamnosus FD; (E) Lacticaseibacillus rhamnosus VP; (F) Lactobacillus crispatus VP; (G) Untreated control group; (H) Clotrimazole (100 μM); (I) Image of clotrimazole (400 μM).

[0269] As shown in FIGS. 18 to 22, the postbiotic of Lactobacillus plantarum Probio-87 according to the present invention exhibited the highest co-aggregation ability compared to all commercial probiotic strains studied against Candida albicans (C. albicans) (Fig. 18), Candida glabrata (C. glabrata) (Fig. 19), Candida tropicalis (C. tropicalis) (Fig. 20), Candida krusei (C. krusei) (Fig. 21), and Candida parasilopsis (C. parasilopsis) (Fig. 22) (P<0.05).

[0270] Beneficial native genitourinary lactic acid bacteria often exhibit strong adhesion to pathogens, promoting cell aggregation that eliminates pathogens during genitourinary infections; this stronger adhesion leads to the formation of much larger aggregates. Aggregated microbial cells have a reduced ability to adhere to the host's epithelial surface, resulting in greater removal from the host's system.

[0271] Increasing evidence suggests that microbiota are transferred from mother to newborn during natural delivery, serving as an important early seeding source that forms the basis of the early neonatal microbiota formation, and that the microbial lineage shared between mother and newborn continues to be detected for three months after birth. It is worth noting that while bacterial vaginosis is common in women, vaginal candidiasis occurs more frequently in pregnant women due to changes in pH and hormones that occur during pregnancy.

[0272] conclusion

[0273] The Lactobacillus plantarum Probio-87 strain demonstrated inhibitory effects against five major Candida species found in the vaginas of women with candidiasis, compared to unstudied controls and most commercial probiotic strains. These antimicrobial effects were demonstrated through two methods: direct growth inhibition and cell aggregation potential. Furthermore, cell aggregation ability was observed in both the live and postbiotic forms of Lactobacillus plantarum Probio-87, implying that the active live and postbiotic forms of Lactobacillus plantarum Probio-87 according to the present invention have a lethal effect on these pathogenic yeasts. These results demonstrate that the Lactobacillus plantarum Probio-87 strain meets the requirements for probiotic strains according to FAO / WHO recommendations and can be applied as a natural strategy to protect women from vaginal candidiasis, and subsequently as a natural strategy to protect women from the transmission of pathogenic microorganisms to newborns and / or infection of newborns.

[0274] Example 6: Growth inhibitory effect of vaginal pathogenic bacteria through the growth inhibition of Lactobacillus plantarum Probio-87

[0275] In vitro study results

[0276] We evaluated the effects of using postbiotics of Lactobacillus plantarum Probio-87 and CFS to inhibit the growth of primary pathogenic bacteria such as Gardnerella vagninalis, Escherichia coli, and Staphylococcus aureus, and to the growth of beneficial vaginal bacteria such as Lactobacillus iners and Lactobacillus Crispatus, which are abundant in the vaginas of women with bacterial vaginosis.

[0277] Figure 23 is a graph showing the antimicrobial activity of CFS derived from Lactobacillus plantarum Probio-87 against (A) Staphylococcus aureus, (B) Escherichia coli, (C) Gardnerella vaginalis, and (D) Lactobacillus iners, and (E) a graph showing the symbiotic activity of Lactobacillus plantarum Probio-87 and Lactobacillus crispatus. As a negative control, Figure 23 used pathogens grown without CFS of Lactobacillus plantarum Probio-87. Streptomycin (10 μg / ml), amoxicillin (1.5 μg / ml), ampicillin (0.03 μg / ml), and clindamycin (4 μg / ml) were used as positive controls against Staphylococcus aureus. Additionally, CFS of Lactobacillus crispatus was added to compare antimicrobial activity against Escherichia coli, Gardnerella vaginalis, and the lactic acid bacterium Lactobacillus iners. The data are expressed as means, and error bars represent the standard error of the mean (n=6). abc Between groups determined using repeated measures ANOVA It is a statistical difference (P<0.05).

[0278] As shown in Fig. 23, Lactobacillus plantarum Probio-87 exhibited antimicrobial activity against common intestinal pathogens such as Staphylococcus aureus (S. aureus) (Fig. 23 A), Escherichia coli (E. coli) (Fig. 23 B), and vaginal pathogenic Gardnerella vaginalis (G. Vaginalis) (Fig. 23 C), which was similar to or superior to the antibiotic control group, and showed antimicrobial activity against common intestinal pathogens compared to the untreated control group (P<0.05). Lactobacillus plantarum Probio-87 and Lactobacillus Crispatus also inhibited Lactobacillus iners (L. iners), a lactic acid bacterium known to be more abundant in the vaginas of women with bacterial vaginosis (BV), with an effect similar to that of the antibiotic control group (Fig. 23 D; P<0.05). Lactobacillus plantarum Probio-87 slightly inhibited the growth of Lactobacillus cripatus (L. cripatus) compared to the untreated control group, but not as much as the antibiotic control group (Fig. 23 E). BV is a very widespread condition in women caused by a bacterial imbalance in the vagina. While BV is primarily caused by pathogens such as Gardnerella vaginalis (G. vaginalis) and Mobiluncus, the risk of bacterial vaginosis increases when there is a loss of health-related lactic acid bacteria species, such as Lactobacillus cripatus (L. cripatus), and replacement with suboptimal lactic acid bacteria, such as Lactobacillus inus (L. iners), often due to the presence of Actinomyces.

[0279] conclusion

[0280] The Lactobacillus plantarum Probio-87 strain has been shown to exert antimicrobial effects against vaginal pathogenic bacteria, thereby regulating a healthier vaginal environment. The results indicate that the Lactobacillus plantarum Probio-87 strain meets the probiotic strain requirements according to FAO / WHO recommendations and can be applied as a natural strategy to regulate the vaginal environment against imbalanced vaginal pathogens in adult women.

[0281] Example 7: Effect of Lactobacillus plantarum Probio-87 on suppressing vaginal HPV populations in HPV-positive adult women

[0282] Clinical study results

[0283] HPV is a double-stranded circular DNA virus containing approximately 8,000 base pairs and possessing an open read frame that encodes functional early proteins such as E1, E2, E4, E5, E6, and E7, as well as late capsid proteins such as L1 and L2. Unique HPV genotypes are recognized based on the sequence of the L1 gene. During infection, the L1 protein accompanies the L2 protein and the viral genome into the host nucleus. The L1 and L2 proteins and the viral genome move as a subviral complex to the mitotic chromosome present in transport vesicles and are maintained in the nucleus of interphase cells. The full length of the L1 protein was detected in HPV-carrying vesicles throughout mitosis; while the viral genome detaches after being released from the transport vesicle and becoming accessible in the nucleus, the L2 protein remains for a longer period. After the completion of mitosis, both L1 and L2 proteins detach from the viral genome, but the loss of the L1 protein is associated with the viral genome exiting the transport vesicle. It is hypothesized that the L1 protein is required during transport to stabilize and maintain the subviral complex. Once the viral genome is safely released from the nucleus, the L1 protein is no longer needed. In contrast, the L2 protein appears to remain within the nucleus and remain associated with the viral genome for an extended period. The longer persistence of the L2 protein after the viral genome has been delivered suggests that it may play a crucial role in subsequent events, such as the establishment of infection. The L2 protein is located alongside the viral genome within the nucleus of the infected cell, unlike the L1 protein. During late infection, the complete loss of the L1 protein was observed in relation to the leakage of the viral genome from transport vesicles. Considering that the L1 protein plays a more critical role in accompanying the viral genome to the nucleus after infectious entry, it remains bound to the viral genome until mitosis is complete and detaches once the viral genome becomes accessible within the newly formed nucleus.L1 protein is more involved in trafficking events than L2 protein and better indicates the abundance of active HPV viruses within host cells. Based on this, the present invention utilized the quantification of the L1 gene, which indicates HPV abundance in the vagina of HPV-positive women.

[0284]

[0285] Table 4 above shows the Ct values ​​of the L1 gene collected from vaginal swabs, determined by RT-qPCR and Nugent scores, for HPV-positive women (n=82) who consumed either a placebo (n=43) or the probiotic Lactobacillus plantarum Probio-87 (n=39). As shown in Table 4, when HPV-positive women consumed probiotics for 12 weeks, the presence of vaginal HPV was lower compared to the placebo group at week 12 (higher Ct values; P=0.001). Changes over the 12 weeks between the groups also showed a significant difference (P=0.015). The probiotic group showed a higher increase in vaginal HPV presence (lower Ct values), while the placebo group showed a lower increase in presence over time (higher Ct values). After 12 weeks, the placebo group showed a higher Nugent score compared to the probiotics group (Table 6; P<0.001). Changes in each group over 12 weeks also showed significant differences between the groups, with the probiotics group showing a decrease while the placebo group showed an increase (P<0.001).

[0286] conclusion

[0287] The Lactobacillus plantarum Probio-87 strain suppressed the vaginal HPV population in HPV-positive adult women, as demonstrated in a randomized, double-blind, placebo-controlled study. The results indicate that the Lactobacillus plantarum Probio-87 strain meets the requirements for probiotic strains according to FAO / WHO recommendations and can be applied as a natural strategy to regulate the vaginal environment against the HPV population in adult women.

[0288] Example 8: Preventive effect of Lactobacillus plantarum Probio-87 on HPV-mediated cervical cancer through inhibition of HPV-mediated cervical cancer cell growth

[0289] In vitro study results

[0290] MTT analysis

[0291] Figure 24 is a graph showing cell viability confirmed by MTT assay after treating (A) the non-HPV cervical cancer cell line C33A, (B) the HPV-16-mediated cervical cancer cell line CaSki, and (C) the HPV-18-mediated cervical cancer cell line HeLa with Lactobacillus plantarum (L. plantarum) Probio-87 and other probiotic strains (untreated control group) excluding postbiotics. Results are expressed as mean ± SEM (standard error of mean) (n=3). abP<0.05 one-way ANOVA.

[0292] Postbiotics from the Lactobacillus plantarum Probio-87 strain showed an inhibitory effect on the non-HPV cervical cancer cell line C33A compared to the untreated control (Fig. 24 A). The same effect was observed for the postbiotics in all strain studies. The postbiotics from the Lactobacillus plantarum Probio-87 strain showed a better inhibitory effect on the HPV-16-mediated cervical cancer cell line CaSki (Fig. 24 B) and the HPV-18 medicinal cervical cancer cell line HeLa (Fig. 24 C) compared to the untreated control and most commercial strains.

[0293] Among all strains studied, C33A showed a similar inhibitory effect, whereas differences were observed in HPV-mediated cell lines, indicating that KCTC 15755BP exerts an inhibitory effect on cervical cancer cell proliferation along HPV-related pathways.

[0294] Phase contrast microscope

[0295] Figure 25 is a photograph showing the morphological analysis of non-HPV cervical cancer cell line C33A cells treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope; Figure 26 is a photograph showing the morphological analysis of HPV-16-mediated cervical cancer cell CaSki cells treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope; and Figure 27 is a photograph showing the morphological analysis of HPV-18-mediated cervical cancer cell HeLa-treated cells treated with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control) using a 50 μM phase-contrast microscope. It is a photo.

[0296] As illustrated in Figs. 25 to 27, phase-contrast microscopy analysis showed that the morphological changes in C33A (Fig. 25), CaSki (Fig. 26), and HeLa (Fig. 27) upon postbiotic treatment of probiotic strains followed trends similar to those observed via MTT analysis. A more widespread effect was observed upon postbiotic treatment of the KCTC 15755BP strain compared to the untreated control and other strains studied. This includes changes in cell shape, such as a decrease in cell number, shrinkage, and surface exfoliation.

[0297] angiogenesis analysis

[0298] Figure 28 is a graph showing the concentrations of angiogenin, angiopoietin-2 (ANG-2), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) proteins expressed in (A) HeLa, (B) CaSki, and (C) C33A cells with or without CFS treatment with Lactobacillus plantarum (L. plantarum) Probio-87.

[0299] As shown in Fig. 28, the CFS of Lactobacillus plantarum (L. plantarum) Probio-87 regulated potent angiogenesis promoters in HeLa (Fig. 28 A), CaSki (Fig. 28 B), and C33A (Fig. 28 C) cells, particularly compared to VEGF, ANG-2, and Angiogenin. EGF and HGF showed a significant decrease only in C33A cells.

[0300] Cancer pathway and dual luciferase test

[0301] Figure 29 is a graph showing the ratio of changes in transcription factor regulation when (A) HeLa, (B) CaSki, and (C) C33A cells were treated with the CFS of Lactobacillus plantarum (L. plantarum) Probio-87. Here, values ​​greater than 1 indicate upregulation of a specific transcription factor, while values ​​less than 1 indicate downregulation of the target.

[0302] As shown in Fig. 29, in contrast to VC-treated cells, CFS-treated cells showed upregulation of the p53, TGF-β, NFκ-B, Myc / Max, and MAPK / ERK genes in Hela cells (Fig. 29 A), and upregulation of Notch, p53, and Myc / Max in CaSki cells (Fig. 29 B). Meanwhile, no significant changes were observed in C33A cells (Fig. 29 C).

[0303] Figure 30 is a graph showing the changes in the relative gene expression levels of tumor suppressor factors p21 and ARF in HeLa, CaSki, and C33A cells upon CFS treatment with Lactobacillus plantarum (L. plantarum) Probio-87.

[0304] As shown in Fig. 30, p53 can activate the ERK, Notch, and NFκB pathways, which in turn mutually influence p53 and ultimately activate p21 in cancer cells to induce apoptosis. Additionally, c-Myc can induce p53 expression by generating ARF, a potent promoter of apoptosis. ERK and NFκB can induce apoptosis independently, without relying on the p53 pathway. Introducing the CFS of Lactobacillus plantarum Probio-87 into Hela and CaSki affected two common pathways. p53 and Myc / Max., which are important for cancer cell apoptosis primarily through the ARF and p21 pathways. To verify the hypothesis of activation of the ARF and p21 pathways, gene expression analysis for ARF and p21 genes was performed in three different cervical cancer cell lines. After CFS treatment with Lactobacillus plantarum Probio-87, p21 was upregulated in all three cancer cell lines, whereas ARF was upregulated only in Hela and CaSki. These results showed that the effect of Lactobacillus plantarum Probio-87 was mainly observed in HPV-treated cell lines such as HeLa and CaSki, and had less effect on HPV-non-mediated cancer cell lines such as C33A.

[0305] conclusion

[0306] It has been revealed that Lactobacillus plantarum Probio-87 inhibits cell proliferation pathways associated with HPV-mediated cells and suppresses the growth of cervical cancer cells through angiogenesis. The results indicate that Lactobacillus plantarum Probio-87 meets the requirements for probiotic strains according to FAO / WHO recommendations and can be applied as a natural strategy for the prevention of cervical cancer in women.

[0307] Example 9: Preventive effect of Lactobacillus plantarum Probio-87 on breast cancer cell growth

[0308] In vitro study results

[0309] MTT analysis

[0310] Figure 31 is a graph showing the cell viability of human breast cancer cell lines MDA-MB-231 (A) and T47D (B) upon treatment with Lactobacillus plantarum (L. plantarum) Probio-87, other probiotic strains, or postbiotics (untreated control). Results were obtained via one-way ANOVA with mean ± SEM (standard error of mean), n=3, and abP<0.05.

[0311] As shown in Fig. 31, the postbiotic strain Lactobacillus plantarum Probio-87 exhibited an inhibitory effect on MDA-MB-231 compared to the untreated control (Fig. 31 A), and this effect was superior to most commercial strains studied. Lactobacillus plantarum Probio-87 also exhibited an inhibitory effect on T47D compared to the untreated control, and was the only strain among all studied to exhibit this effect, whereas all other commercial strains studied were similar to the untreated control (Fig. 31 B).

[0312] MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer cell line due to the absence of human epidermal growth factor receptor-2 amplification as well as the expression of estrogen and progesterone receptors. Like other invasive cell lines, the invasiveness of MDA-MB-231 cells is mediated by the proteolysis of the extracellular matrix. This cell line exhibits an endothelium-like morphology and is distinguished by an invasive phenotype, often featuring star-shaped projections connecting multiple cell colonies. Triple-negative breast cancer is an aggressive form of breast cancer with limited treatment options. MDA-MB-231 is commonly used to modulate advanced breast cancer, including studies on bone, brain, and lung metastases. Meanwhile, T47D is a non-metastatic cell line dependent on hormones, primarily progesterone. Furthermore, T47D has been shown to be estrogen-sensitive via estrogen receptor alpha, exhibiting tumorigenic properties in the presence of estrogen.

[0313] Phase contrast microscope

[0314] Figure 32 is a photograph showing the morphological analysis of human breast cancer cells MDA-MB-231 treated with Lactobacillus plantarum Probio-87, other probiotic strains, or postbiotics (untreated control group) without postbiotics, performed using a phase-contrast microscope (100 μM), and Figure 33 is a photograph showing the morphological analysis of human breast cancer cells T47D treated with Lactobacillus plantarum Probio-87, other probiotic strains, or postbiotics (untreated control group) without postbiotics, performed using a phase-contrast microscope (100 μM).

[0315] As illustrated in Figs. 32 and 33, phase-contrast microscopy analysis via MTT assa showed that the morphological changes of MDA-MB-231 (Fig. 32) and T47D (Fig. 33) cell lines upon postbiotic treatment with probiotics exhibited similar trends. Postbiotic treatment with the Lactobacillus plantarum Probio-87 strain showed greater effects compared to the untreated control strain. These included morphological changes such as a decrease in cell number and shrinkage and detachment from the surface. In the case of MDA-MB-231 cells, which exhibit an invasive phenotype through stellate projections connecting cells, postbiotic treatment with Lactobacillus plantarum Probio-87 also resulted in reduced or severed connection formation.

[0316] conclusion

[0317] Lactobacillus plantarum Probio-87 has been shown to have the ability to inhibit metastatic (MDA-MB-231) and non-metastatic (T47D) human breast cancer cells. Additionally, Lactobacillus plantarum Probio-87 exhibited tumorigenic properties by inhibiting hormones, which caused estrogen-dependent human breast cancer cells (T47D) to form tumors in the presence of estrogen or estrogen-independent (MDA-MB-231 is estrogen-negative) cells. This is an important feature demonstrating that Lactobacillus plantarum Probio-87 has potential against a wide range of breast cancer cell types.

[0318] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, it will be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of protection of the present application should be interpreted as including all modifications or variations that can be derived from the meaning, scope, and equivalents of the claims set forth below, rather than from the detailed description above.

[0319] ctg000000:126255-127818(+)

[0320] TATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCAT TAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGA GTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT CACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCTGCCTAAGGTGGGACAGATGATTAGGGTTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT

Claims

1. Lactobacillus plantarum Probio-87 strain (KCTC15755BP).

2. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for regulating an unbalanced vaginal microbiome and preventing vaginal bacterial imbalance.

3. In paragraph 2, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for inhibiting the proliferation of disease-pathogenic Candida species selected from the group consisting of Candida albicans, Candida glabrata, Candida krusei, Candida parasilopsis, and Candida tropicalis.

4. In paragraph 2, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for inhibiting the growth of bacteria selected from the group consisting of Gardnerella vagninalis, Escherichia coli, and Staphylococcus aureus.

5. In paragraph 2, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for suppressing vaginal HPV (Human Papillonia virus) populations.

6. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for anti-inflammatory and immune-modulating protection of the vagina.

7. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for regulating unbalanced intestinal microorganisms and preventing intestinal bacterial imbalance.

8. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for use in the prevention of HPV (Human Papillonia virus)-mediated cervical cancer.

9. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for use in the prevention of breast cancer.

10. In claim 1, the strain is a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) for use in improving mental and emotional health.

11. It is a freeze-dried living cell, and 10 of the total composition 4 to 10 12 A composition comprising a Lactobacillus plantarum Probio-87 strain (KCTC15755BP) characterized by containing an amount of cfu / g.

12. In claim 11, the above composition is 10 4 to 10 12 CFU / ml A composition comprising the Lactobacillus plantarum Probio-87 strain (KCTC15755BP), characterized by having a liquid form postbiotic containing an amount of [amount] or an equivalent dry form.

13. A composition comprising the Lactobacillus plantarum Probio-87 strain (KCTC15755BP) according to claim 11, characterized in that the composition is selected from the group consisting of food supplements, medicines, infant formulas, edible products, food, and agricultural products.

14. A composition comprising the Lactobacillus plantarum Probio-87 strain (KCTC15755BP) according to claim 12, characterized in that the composition is selected from the group consisting of tablets, capsules, pills, and liquid formulations.

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