Uses of breast milk-derived rimosilactobacillus fermentum MSJK0025 in improving mammary gland health

Abstract

This application relates to the use of breast milk-derived rimosilactobacillus fermentum MSJK0025 in the field of microbial technology for improving breast health (improvement of mastitis, prevention and / or treatment of breast cancer). Specifically, it relates to pharmaceutical compositions for improving breast health containing breast milk-derived rimosilactobacillus fermentum MSJK0025, and probiotic formulations for improving breast health containing breast milk-derived rimosilactobacillus fermentum MSJK0025. This application provides novel uses for breast milk-derived rimosilactobacillus fermentum MSJK0025 in improving breast health and demonstrates that rimosilactobacillus fermentum MSJK0025 can effectively improve breast health.

Description

[Technical Field]

[0001] This application relates to the field of microbial technology, and more particularly to the use of breast milk-derived rimosilactobacillus fermentum MSJK0025 in improving mammary gland health. [Background technology]

[0002] Mastitis is an inflammatory disease of the mammary gland that is common in female mammals, including humans, and is classified into mastitis during lactation and mastitis during non-lactation. Due to the physiological structure of the breast, the mammary gland is an ecosystem that connects the external and internal environments, and is home to a large number of bacteria, normally forming a balanced microecosystem. There are two types of bacterial origins: (1) Exogenous origins are mainly microorganisms from the nipple skin and the infant's oral cavity that migrate to the mammary gland through the milk ducts. (2) Endogenous origins are bacteria from the digestive tract that migrate to the mammary gland (see Figure 1) through the "intestinal-mammary pathway". Overall, mastitis is a pathological process characterized by a disruption of the mammary gland microecosystem. Most cases of mastitis are caused by infection with Staphylococcus species, mainly Staphylococcus aureus and Staphylococcus epidermidis.

[0003] Currently, the primary treatment for mastitis is systemic antibiotic therapy. While this treatment is effective, it has various drawbacks, including the development of drug resistance, disruption of the bacterial flora balance, and the occurrence of side effects. Furthermore, in cases of mastitis during lactation, antibiotics can affect breastfeeding, raising concerns about adverse effects on the infant, thus posing a safety risk. Therefore, there is a need to explore alternatives to antibiotics for the improvement and treatment of mastitis.

[0004] In 2022, Rimosiractobacillus fermentum LC40 (CECT5716) was listed as a recommended treatment in the American Academy of Nursing Medicine (ABM) Clinical Guidelines #36: Mastitis Spectrum, 2022 Revised Edition. However, Rimosiractobacillus fermentum CECT5716 is the only probiotic strain in the world that clearly claims to have an effect on "improving mastitis." This strain is the flagship strain of Biosearch Life in Spain and was isolated from the breast milk of healthy women. The raw material powder prepared from this strain is currently produced in Spain, which is expensive, and the procurement of the powder is easily affected by the domestic environment, which has a significant impact on the continuity of production and cost of the final product.

[0005] Breast cancer is one of the most common malignant tumors affecting breast health in women, and it usually develops in the glandular epithelial tissue of the breast. Statistics show that it accounts for 7% to 10% of all malignant tumors in the body, and it is one of the most common malignant tumors that have a significant impact on the physical and mental health of women and can be life-threatening. Currently, the main treatments for breast cancer are surgery and Western medicine, but surgery carries certain risks and is not suitable for elderly or frail patients, while Western medicine is only symptomatic treatment and does not lead to a fundamental solution, its effectiveness is not clear, and it is expensive. Therefore, there is a need to develop new methods that are useful for improving and treating breast cancer.

[0006] The Rimosiractobacillus fermentum MSJK0025, the subject of this application, was isolated from the breast milk of healthy mothers and belongs to the same species as Rimosiractobacillus fermentum CECT5716, and the two are highly comparable. Studies have shown that Rimosiractobacillus fermentum MSJK0025 is superior in terms of gastric acid resistance, bile salt resistance, intestinal colonization ability, and growth rate. Since safety evaluation tests and large-scale commercial production have been completed, the prospects for industrialization are clear. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This application provides a use for breast milk-derived rimosilactobacillus fermentum MSJK0025 in improving mammary gland health (improvement of mastitis, prevention and / or treatment of breast cancer) in order to overcome the shortcomings of the prior art. Rimosilactobacillus fermentum MSJK0025 can effectively alleviate inflammation and pathological changes in the mammary gland, delay the premature growth of mammary gland tumors, and further effectively improve mammary gland health. [Means for solving the problem]

[0008] Therefore, in the first phase, this application provides a pharmaceutical composition for improving mammary gland health containing breast milk-derived limosilactobacillus fermentum MSJK0025.

[0009] In some embodiments, the above-mentioned improvement of breast health includes the improvement of mastitis and the prevention and / or treatment of breast cancer.

[0010] The Rimosilactobacillus fermentum MSJK0025 used in the pharmaceutical composition described in this application is a strain isolated and selected in patent application number 202311070808.2, which is deposited with the General Microbiology Center of the China Microbial Species Preservation and Storage Administration, deposit number CGMCC No. 26426. The inventors of this application have found, through studying the strain, that it effectively alleviates inflammation and pathological changes in the mammary gland, also alleviates the premature growth of mammary tumors, and is applicable to pharmaceutical compositions for improving mammary health (improvement of mastitis, prevention and / or treatment of breast cancer).

[0011] In some embodiments, the mastitis described above is selected from at least one of non-lactational mastitis and lactational mastitis, and the breast cancer described above is early-stage breast cancer.

[0012] In the present application, Limosilactobacillus fermentum MSJK0025 can be used to improve both non-lactational mastitis and lactational mastitis, and in particular, it can be used to improve lactational (lactation period) mastitis.

[0013] In the present application, the above-mentioned early breast cancer (early breast cancer or early-stage breast cancer) refers to a patient whose tumor is less than 3 cm at the time of surgery, without axillary lymph node metastasis or distant metastasis, or with no metastasis or only micrometastasis in the ipsilateral axillary lymph nodes and without distant metastasis, including non-invasive cancer.

[0014] Limosilactobacillus fermentum MSJK0025 in the present application can delay the early growth of breast tumors (the tumor volume of female mice suffering from breast cancer in the experimental group administered with Limosilactobacillus fermentum MSJK0025 significantly decreased at the 13th week, and the tumor weight significantly decreased at both the 13th and 17th weeks), but there is no improvement effect on the malignant progression of breast tumors. Therefore, the treatment of breast cancer with the pharmaceutical composition described in the present application is preferably for early breast cancer.

[0015] In some embodiments, the above-mentioned pharmaceutical composition contains viable Limosilactobacillus fermentum MSJK0025.

[0016] According to the research of the inventors of the present application, it was discovered that the effect of improving breast health, particularly improving mastitis, by Limosilactobacillus fermentum MSJK0025 is mainly exerted in viable bacteria rather than in fermentation supernatants or the like.

[0017] In addition to Limosilactobacillus fermentum MSJK0025, the pharmaceutical composition in this application may further contain a pharmaceutically acceptable vector. In this application, the above-mentioned pharmaceutically acceptable vector includes, but is not limited to, any pharmaceutically acceptable excipient, surfactant, desiccant, diluent, carrier, adjuvant or additive. Suitable pharmaceutically acceptable vectors can be, for example, magnesium carbonate, lactose, pectin, dextrin, starch, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, cocoa butter, etc. In this application, the pharmaceutically acceptable vector can be solid, semi-solid or liquid.

[0018] In some embodiments, the dosing subjects of the above-mentioned pharmaceutical composition are female mammals including humans.

[0019] Limosilactobacillus fermentum MSJK0025 in the pharmaceutical composition of this application can effectively improve the symptoms of mastitis and delay the early growth of breast tumors. As a result of tissue observation, it was found that the inflammation and pathological changes of female mice suffering from mastitis in the experimental group administered with Limosilactobacillus fermentum MSJK0025 were effectively alleviated. As a result of HE staining, it was shown that female mice suffering from mastitis in the experimental group administered with Limosilactobacillus fermentum MSJK0025 had only a few inflammatory cells in the alveoli of the mammary glands, and the degree of inflammation was significantly reduced. As a result of the measurement of serum samples, it was shown that the TNF-α secretion level of female mice suffering from mastitis in the experimental group administered with Limosilactobacillus fermentum MSJK0025 was significantly decreased. In addition, the tumor volume of female mice suffering from breast cancer in the experimental group administered with Limosilactobacillus fermentum MSJK0025 significantly decreased at the 13th week, and the tumor weight significantly decreased at both the 13th and 17th weeks. Therefore, the pharmaceutical composition of this application can effectively improve breast health (improvement of mastitis, prevention and / or treatment of breast cancer), and can be effectively applied to female mammals including humans, especially female mammals with breast health problems (mastitis, early breast cancer, etc.).

[0020] In this application, mammals may include, for example, humans, mice, pigs, cows, sheep, dogs, cats, rabbits, and the like.

[0021] In the second aspect, this application provides a probiotic formulation for improving breast health containing breast milk-derived limosilactobacillus fermentum MSJK0025.

[0022] In some embodiments, the above-mentioned improvement of breast health includes the improvement of mastitis and the prevention and / or treatment of breast cancer.

[0023] The probiotic formulation described in this application includes Rimosiractobacillus fermentum MSJK0025, and the inventors of this application have shown through research that the strain effectively alleviates inflammation and pathological changes in the mammary gland, also alleviates the premature growth of mammary gland tumors, and is superior in terms of gastric acid resistance, bile salt resistance, intestinal colonization ability, and growth rate, and has completed safety evaluation tests and large-scale commercial production, and can be applied to probiotic formulations for improving mammary gland health (improvement of mastitis, prevention and / or treatment of breast cancer).

[0024] In some embodiments, the mastitis described above is selected from at least one of non-lactational mastitis and lactational mastitis, and the breast cancer described above is early-stage breast cancer.

[0025] In some embodiments, the above-described probiotic formulation contains live Rimosiractobacillus fermentum MSJK0025 bacteria.

[0026] The probiotic formulations in this application may include, but are not limited to, rimosilactobacillus fermentum MSJK0025, other probiotics, etc.

[0027] In some embodiments, the target population for the above-mentioned probiotic formulations is female mammals, including humans. [Effects of the Invention]

[0028] The advantageous technical effect of this application is that it provides a novel use for breast milk-derived rimosiractobacillus fermentum MSJK0025 in improving mammary gland health (improvement of mastitis, prevention and / or treatment of breast cancer). Inflammation and pathological changes were effectively alleviated in female mice with mastitis in the experimental group administered rimosiractobacillus fermentum MSJK0025. HE staining results showed that female mice with mastitis in the experimental group administered rimosiractobacillus fermentum MSJK0025 had only a small amount of inflammatory cells in the mammary acini, indicating a clear reduction in the degree of inflammation. Serum sample measurements showed a significant decrease in TNF-α secretion levels in female mice with mastitis in the experimental group administered rimosiractobacillus fermentum MSJK0025. Furthermore, in the experimental group of female mice with breast cancer administered rimosiractobacillus fermentum MSJK0025, tumor volume significantly decreased at week 13, and tumor weight significantly decreased at both week 13 and week 17. Therefore, rimosiractobacillus fermentum MSJK0025 can be effectively applied to improve mammary gland health (improvement of mastitis, prevention and / or treatment of breast cancer). [Brief explanation of the drawing]

[0029] [Figure 1] This is a diagram showing the bacterial migration pathway from the intestinal tract to the mammary gland. [Figure 2] These are photographic images of the mammary gland tissue of female mice in each group in Example 1, "Construction of a Staphylococcus aureus-infected mastitis model in mice." Here, A is the blank control group, B is the Staphylococcus aureus 103 CFU / mL group, C is the Staphylococcus aureus 104 CFU / mL group, and D is the Staphylococcus aureus 105 CFU / mL group. The arrows indicate the mammary gland. [Figure 3]These are HE-stained (200x magnification) images of mammary tissue from female mice in each group in Example 1, "Construction of a Staphylococcus aureus-infected mastitis model in mice." Here, A is the blank control group, B is the Staphylococcus aureus 103 CFU / mL group, C is the Staphylococcus aureus 104 CFU / mL group, and D is the Staphylococcus aureus 105 CFU / mL group. Arrows indicate inflammatory cells. [Figure 4] These are photographic images of the mammary gland tissue of mice in each group in Example 2, "Evaluation of the effect of rimosiractobacillus fermentum MSJK0025 on improving mastitis." Here, A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is the rimosiractobacillus fermentum MSJK0025 live bacteria 107 CFU / mL group, E is the rimosiractobacillus fermentum MSJK0025 live bacteria 108 CFU / mL group, and F is the rimosiractobacillus fermentum MSJK0025 live bacteria 109 CFU / mL group. The arrows indicate the mammary gland. [Figure 5] These are HE-stained (200x magnification) images of mammary gland tissue from mice in each group in Example 2, "Evaluation of the effect of rimosilactobacillus fermentum MSJK0025 on improving mastitis." Here, A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is the rimosilactobacillus fermentum MSJK0025 live bacteria 107 CFU / mL group, E is the rimosilactobacillus fermentum MSJK0025 live bacteria 108 CFU / mL group, and F is the rimosilactobacillus fermentum MSJK0025 live bacteria 109 CFU / mL group. Arrows indicate inflammatory cells. [Figure 6]These are photographic images of the mammary gland tissue of mice in each group in Example 3, "Evaluation of the active ingredient in the improvement of mastitis by Rimosiractobacillus fermentum MSJK0025," where A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is experimental group 1 (live Rimosiractobacillus fermentum MSJK0025), E is experimental group 2 (live Rimosiractobacillus fermentum CECT5716), F is experimental group 3 (fermented supernatant of Rimosiractobacillus fermentum MSJK0025), and G is experimental group 4 (fermented supernatant of Rimosiractobacillus fermentum CECT5716). [Figure 7] These are HE-stained (200x magnification) images of mammary gland tissue from mice in each group in Example 3, "Evaluation of the active ingredient in the improvement of mastitis by Rimosiractobacillus fermentum MSJK0025," where A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is experimental group 1 (live Rimosiractobacillus fermentum MSJK0025), E is experimental group 2 (live Rimosiractobacillus fermentum CECT5716), F is experimental group 3 (fermented supernatant of Rimosiractobacillus fermentum MSJK0025), and G is experimental group 4 (fermented supernatant of Rimosiractobacillus fermentum CECT5716). [Figure 8] These are photographic images of the mammary gland tissue of mice in each group in Example 4, "Evaluation of the effect of rimosilactobacillus fermentum MSJK0025 on preventing mastitis," where A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is experimental group 1 (live rimosilactobacillus fermentum MSJK0025), and E is experimental group 2 (live rimosilactobacillus fermentum CECT5716). [Figure 9]These are HE-stained (200x magnification) images of mammary gland tissue from mice in each group in Example 4, "Evaluation of the effect of Rimosiractobacillus fermentum MSJK0025 on preventing mastitis." Here, A is the blank control group, B is the model control group, C is the positive drug (levofloxacin) group, D is experimental group 1 (live Rimosiractobacillus fermentum MSJK0025), and E is experimental group 2 (live Rimosiractobacillus fermentum CECT5716). [Figure 10] This is a comparison chart of mouse tumor volumes between the model control group and the rimosilactobacillus fermentum group in Example 5. [Figure 11] This is a comparison chart of mouse tumor weights between the model control group and the rimosiractobacillus fermentum group in Example 5. [Figure 12] These are images of the whole-body tumor appearance of mice in the model control group and the rimosiractobacillus fermentum group in Example 5, where A represents the model control group and B represents the rimosiractobacillus fermentum group.

[0030] Biological deposit information Limosilactobacillus fermentum MSJK0025 has deposit number CGMCC No. 26426, is deposited at the General Microbiology Center of the China Microbial Species Preservation and Storage Administration (CGMCC, address: 1-3 Beichen West Road, Chaoyang District, Beijing), and was deposited on January 9, 2023. [Modes for carrying out the invention]

[0031] Hereinafter, this application will be described in more detail with reference to examples to facilitate understanding.

[0032] The experimental animals in Examples 1-4 below were provided by Sibeifu (Suzhou) Biotechnology Co., Ltd. (China), with certificate number: SCXK(Su)2022-0006. The experimental animals in Example 5 below are MMTV-PyMT genetically modified mice (female, 6 weeks old), provided by Jiangsu Hua Chuang Xin Nuo Pharmaceutical Technology Co., Ltd., with certificate number: SCXK(Su)2020-0009.

[0033] Example 1: Construction of a mouse model of mastitis caused by Staphylococcus aureus infection.

[0034] 1. Staphylococcus aureus strain (ATCC-29213, General Microbiology Center, China Microbial Species Preservation and Storage Administration) was inoculated onto a slant of nutrient agar and cultured at 37°C.

[0035] 2. After 24 hours of incubation, the cultures were inoculated into 100 mL of nutrient broth liquid medium and continued to be incubated at 37°C.

[0036] 3. After culturing for another 16 hours, 10 mL of the Staphylococcus aureus suspension was aspirated, centrifuged at 3000 rpm / min for 5 minutes, the supernatant was discarded, the suspension was resuspended in an equal volume of physiological saline, centrifuged again, the supernatant was discarded, and 10 mL of sterile physiological saline was added and mixed uniformly to obtain the initial Staphylococcus aureus suspension.

[0037] 4. The concentration of Staphylococcus aureus in the suspension was determined by counting the number of viable cells. The required concentration was then adjusted with sterile physiological saline, and the suspension was stored until use.

[0038] 5. Two hours before inoculation, pup mice that had been breastfed for 10 days were isolated from the female mice. The female mice were anesthetized, tied up on their backs, and the fourth mammary gland was fully exposed. The mammary gland was wiped and disinfected with 75% alcohol, and the tip of the nipple was cut off by approximately 1 mm using micro-scissors.

[0039] 6. Insert a blunt micro syringe (less than 30G) slowly about 2 mm from the lactiferous duct part and inject 50 μL of Staphylococcus aureus suspension. The concentration of the Staphylococcus aureus suspension is divided into three dosage explorations (10 3 CFU / mL, 10 4 CFU / mL, 10 5C FU / mL), and the details are shown in Table 1.

[0040] 7. Three hours after inoculation, the neonatal mice were returned to the female mice. Twenty-four hours after infection, the mammary gland tissues of the female mice were dissected and observed, and the results are shown in Figures 2 and 3.

[0041] Table 1: Grouping of exploration concentrations for Staphylococcus aureus infectious mastitis model

Table 1

[0042] From Figure 2, it was revealed that the mammary gland tissues of the mice infected with the 10 4 CFU / mL group and the 10 5 CFU / mL group of Staphylococcus aureus were slightly darkened and accompanied by slight bleeding compared with the blank control group. From Figure 3, it was revealed that compared with the blank control group, the mice infected with the 10 3 CFU / mL, 10 4 CFU / mL group and the 10 5 CFU / mL group of Staphylococcus aureus had obvious inflammatory cells in the alveolar lumen, and it was clear that the number increased with the increase in the concentration of the infected bacteria. As a result of the model exploration, it was determined that 10 5 CFU / mL would be the concentration of the infected bacteria for mastitis caused by Staphylococcus aureus.

[0043] Example 2: Evaluation of the effect of Lactobacillus fermentum MSJK0025 on improving mastitis

[0044] 1. Limosilactobacillus fermentum MSJK0025 (CGMCC No. 26426, General Microbiology Center, China Microbial Species Preservation and Management Committee) was inoculated onto a slant of MRS agar plate and incubated at 37°C for 24 hours. Then, it was inoculated onto 100 mL of MRS broth and incubated at 37°C for another 16 hours. 10 mL of the Staphylococcus aureus suspension was then aspirated, centrifuged at 3000 rpm / min for 5 minutes, the supernatant was discarded, and the suspension was resuspended in an equal volume of physiological saline. It was centrifuged again, the supernatant was discarded, and 10 mL of sterile physiological saline was added and mixed uniformly to obtain the initial Staphylococcus aureus suspension. The viable cell count of the initial Staphylococcus aureus suspension was determined by counting the viable cells. The required concentration was then adjusted with sterile physiological saline and stored until use.

[0045] 2. Based on the experimental results of Example 1, the bacterial concentration of the mastitis model was determined, and then 48 lactating mice were randomly divided into six groups: one blank control group, one model control group, one positive drug group, and three experimental groups (three doses), with eight mice in each group.

[0046] 3. Three days prior to the construction of the mastitis model, the positive drug group received levofloxacin 55 mg / kg, and the three experimental groups received 0.5 mL of live rimosilactobacillus fermentum MSJK0025, each group receiving 10 7 CFU / mL, 10 8 CFU / mL, 10 9 The drug was administered orally at a dose of CFU / mL, while the model control group and the blank control group received 0.5 mL of physiological saline once daily.

[0047] 4. All lactating mice, excluding the blank control group, were used to construct a Staphylococcus aureus-infected mastitis model according to the procedure in Example 1. 5 CFU / mL was defined as the bacterial concentration for Staphylococcus aureus causing mastitis.

[0048] 5. After the model was constructed, the positive drug group continued daily oral intervention with levofloxacin, while the experimental group continued daily oral intervention with live rimosilactobacillus fermentum MSJK0025 bacteria. The model control group and the blank control group were each administered the same amount of saline solution once a day for a total of 10 interventions (including before model construction).

[0049] 6. Twenty-four hours after the final intervention, blood samples were taken from each group of mice, they were sacrificed, their skin was disinfected with alcohol, their abdominal skin was incised to expose the mammary gland tissue on both sides, and the appearance of the mammary gland tissue (color, texture, presence or absence of bleeding) was observed, photographed, and recorded. The results are shown in Figure 4. The mammary gland tissue was then excised for subsequent pathological HE staining and measurement. The results are shown in Figure 5.

[0050] 7. Twenty-four hours after the final intervention, blood was collected from the mouse orbits, and TNF-α levels in the mouse serum were measured using an ELISA kit. The results are shown in Table 2.

[0051] Table 2: TNF-α levels in mouse serum samples from each group [Table 2] ***, p<0.001; **, p<0.005

[0052] Figure 4 clearly shows that, compared to the blank control group, the mammary gland tissue of the model group mice was slightly darker and accompanied by mild hemorrhage. Compared to the model group, the positive drug group and the rimosilactobacillus fermentum MSJK0025 live bacteria 10 9 The mammary gland tissue of mice in the CFU / mL group showed no obvious inflammation or pathological changes, and simultaneously, compared to the model group, the amount of live Rimosilactobacillus fermentum MSJK0025 was 10. 7 CFU / mL group, 10 live Rimosilactobacillus fermentum MSJK0025 8 The mammary gland tissue of mice in the CFU / mL group showed some degree of reduction in external inflammation and pathological changes.

[0053] Figure 5 shows that HE staining results indicate a significant increase in inflammatory cells in the acinar lumen of the model control group mice compared to the blank control group, and compared to the model control group mice, there were no obvious inflammatory cells in the mammary acinar lumen of the positive drug group mice. 8 , 10 9C The study revealed that a small number of inflammatory cells were present in the mammary acini of mice in the FU / mL group, indicating a significant reduction in the degree of inflammation.

[0054] Table 2 shows that, compared to the blank control group, the TNF-α secretion level in the model control group's mouse serum was significantly increased, and the rimosilactobacillus fermentum MSJK0025 live bacteria 10 7 Excluding the CFU / mL group, compared to the model control group, the positive drug group and Rimosilactobacillus fermentum MSJK0025 live bacteria 10 8 , 10 9 It was revealed that the TNF-α secretion levels in the serum of mice in the CFU / mL group were significantly reduced.

[0055] Based on the above results, live Rimosiractobacillus fermentum MSJK0025 effectively improves the symptoms of mastitis in mice, and 10 live Rimosiractobacillus fermentum MSJK0025 9 The improvement in the CFU / mL group was the most effective.

[0056] Example 3: Evaluation of the active ingredient in the improvement of mastitis by Rimosilactobacillus fermentum MSJK0025

[0057] I. Sample Formulation

[0058] Preparation of Staphylococcus aureus suspension and fermentation supernatant: Rimosilactobacillus fermentum strain (MSJK0025 or CECT5716) was inoculated onto MRS slant medium and incubated at 37°C for 24 hours. The inoculated strain was then transferred to 100 mL of MRS broth medium and inoculated again. Fermentation incubation continued at 37°C for 16 hours. 10 mL of the Staphylococcus aureus suspension was aspirated, centrifuged at 3000 rpm / min for 5 minutes, and the fermentation supernatant was collected and stored until use. The cells were resuspended in an equal volume of sterile saline, centrifuged again, the supernatant was discarded, and an equal volume of sterile saline was added and mixed uniformly to obtain the initial Staphylococcus aureus suspension. The viable cell concentration was then reduced to 10% with sterile saline. 9 It was adjusted to CFU / mL and stored until use.

[0059] Preparation of the pharmaceutical solution: Levofloxacin was weighed, dissolved in sterile water, and mixed into a 2.2 mg / mL solution, which was then stored until use.

[0060] II. Evaluation Process

[0061] 1. Before the start of the experiment, the female mice to be used in the experiment (accompanied by their pups) were reared for 7 days. Then, healthy female mice 3 to 5 days postpartum were divided into 7 groups (1 blank control group, 1 model control group, 1 positive drug group, and 4 experimental groups), with 8 mice in each group. Experimental group 1 was given 0.5 mL of live rimosilactobacillus fermentum MSJK0025 (10 9 In experimental group 2, the test rimosilactobacillus fermentum CECT5716 live bacteria 0.5 mL (10 9 In the experimental group, a CFU / mL dose was administered orally daily. In experimental group 3, 0.5 mL of the fermented supernatant of Rimosiractobacillus fermentum MSJK0025 was administered orally daily. In experimental group 4, 0.5 mL of the fermented supernatant of Rimosiractobacillus fermentum CECT5716 was administered orally daily. In the positive drug group, levofloxacin 55 mg / kg was administered orally daily. In the model control group and blank control group, 0.5 mL of physiological saline was administered. Premedication was given once daily for three consecutive days.

[0062] 2. After three consecutive days of premedication, all female mice except the blank control group were inoculated with Staphylococcus aureus to construct a model (the model construction method was the same as in Example 2).

[0063] 3. After vaccination, each group continued the medication intervention once daily for a total of 10 times, in accordance with the requirements of Procedure 1 (including premedication).

[0064] 4. Twenty-four hours after the final intervention, mice from each group were sacrificed and dissected for histopathological observation (interstitial edema and inflammatory infiltration in the mammary epithelium and acini), photographs were taken, and records were made. The results are shown in Figure 6. Mammary tissue was excised for subsequent pathological HE staining and measurement, and the results are shown in Figure 7.

[0065] 5. Twenty-four hours after the final intervention, blood was collected from the mouse orbits, and TNF-α levels in the mouse serum were measured using an ELISA kit. The results are shown in Table 3.

[0066] Table 3: TNF-α levels in mouse serum samples from each group [Table 3] Note: Comparison with the model control group, **p<0.01

[0067] Figure 6 shows that, compared to the blank control group, the mammary gland tissue of the mice in the model control group, experimental group 2, experimental group 3, and experimental group 4 was somewhat redder, more swollen, and harder in texture. Compared to the model control group, the positive drug group and experimental group 1 (Rimosilactobacillus fermentum MSJK0025 live bacteria 10) showed improved coloration and texture. 9 It was revealed that the mammary gland tissue of mice treated with CFU / mL showed no obvious inflammation or pathological changes.

[0068] Figure 7 shows that HE staining results revealed that the blank control group mice had a complete structure of the acinar lumen of the mammary gland with no obvious inflammatory cell infiltration within the lumen. In contrast to the blank control group, the model control group mice had an indistinct histological structure of the mammary gland, a significant increase in inflammatory cells in the acinar lumen, and scattered hemorrhage. Compared to the model control group, the positive drug group and experimental group 1 (Limosiractobacillus fermentum MSJK0025 live bacteria 10 9 It was revealed that mice in experimental group 2 (CFU / mL) had well-defined mammary gland structures and a significant reduction in inflammatory cells within the acinar lumen, mice in experimental group 3 had poorly defined mammary gland structures and a large number of inflammatory cells in the acinar lumen, mice in experimental group 3 had a large number of inflammatory cells in the acinar lumen of the mammary gland, and mice in experimental group 4 showed shedding of epithelial cells in the acinar wall of the mammary gland tissue and a large number of inflammatory cells in the acinar lumen.

[0069] Table 3 clearly shows that, based on serum measurements by ELISA for each group, the TNF-α levels in the model control group mice were significantly elevated compared to the blank control group. Compared to the model control group, the positive drug group and experimental group 1 (Limosilactobacillus fermentum MSJK0025 live bacteria 10 9 The study revealed that TNF-α levels in mouse serum (CFU / mL) decreased significantly (p<0.01), and while there was a slight decrease in TNF-α levels in the serum of mouse group 3, the difference was not statistically significant, indicating that no decrease in TNF-α levels was observed in the serum of experimental groups 2 and 4.

[0070] The results above indicate that live Rimosiractobacillus fermentum MSJK0025 improved symptoms such as redness, swelling, and hardening of the mammary gland due to Staphylococcus aureus infection, significantly reduced serum inflammatory factor levels (p<0.01), and had an effect on improving mastitis during lactation caused by Staphylococcus aureus infection. This improvement effect was mainly expressed in the active bacteria, while the supernatant of Rimosiractobacillus fermentum MSJK0025 did not have an effect on improving mastitis. Furthermore, both live Rimosiractobacillus fermentum CECT5716 and the fermented supernatant failed to improve the development of mastitis caused by Staphylococcus aureus infection during lactation.

[0071] Example 4: Evaluation of the efficacy of rimosilactobacillus fermentum MSJK0025 in preventing mastitis.

[0072] I. Sample Formulation

[0073] Same example 3.

[0074] II. Evaluation Process

[0075] 1. Before the start of the experiment, the female mice to be used in the experiment (accompanied by their pups) were reared for 7 days. Then, healthy female mice 3 to 5 days postpartum were divided into 5 groups (1 blank control group, 1 model control group, 1 positive drug group, and 2 experimental groups), with 8 mice in each group. Experimental group 1 was given 0.5 mL of the test rimosilactobacillus fermentum MSJK0025 live bacteria (10 9 In experimental group 2, the test rimosilactobacillus fermentum CECT5716 live bacteria 0.5 mL (10 9 The positive drug group received daily oral administration of a CFU / mL dose, while the model control group and blank control group received daily oral administration of levofloxacin 55 mg / kg. Premedication was administered once daily for 10 consecutive days.

[0076] 2. After the 10-day intervention period, all female mice except for the blank control group were inoculated with Staphylococcus aureus to construct a model (the model construction method was the same as in Example 2).

[0077] 3. 24 hours after inoculation, mice from each group were sacrificed and dissected for histopathological observation (interstitial edema, inflammatory infiltration, etc., in the mammary epithelium and acini), and photographs and records were taken. The results are shown in Figure 8. Mammary tissue was excised for subsequent pathological HE staining and measurement, and the results are shown in Figure 9.

[0078] 4. 24 hours after inoculation, blood was collected from the mouse orbits, and TNF-α levels in the mouse serum were measured using an ELISA kit. The results are shown in Table 4.

[0079] Table 4: TNF-α levels in mouse serum samples from each group [Table 4] Note: Comparison with the model control group, *p<0.05

[0080] Figure 8 shows that, compared to the blank control group, mice in the positive drug group exhibited dark red mammary gland tissue, while mice in the other groups showed some degree of redness and swelling in the mammary gland tissue compared to the blank control group. No significant differences were observed between the groups.

[0081] Figure 9 shows that HE staining results revealed that the blank control group mice had a complete structure of mammary acinar lumen and no obvious inflammatory cell infiltration within the lumen. Compared to the blank control group, the model control group, experimental group 1, and experimental group 2 mice had poorly defined mammary histological structures, a significant increase in inflammatory cells in the mammary lumen, and scattered hemorrhage. No significant differences were observed among the three groups. Compared to the model control group, the mice in the positive drug group showed a significant reduction in inflammatory cells within the mammary acinar lumen.

[0082] Table 4 shows that, based on serum measurements of each group using ELISA, the TNF-α levels in the serum of the model control group mice were significantly elevated compared to the blank control group, and the TNF-α levels in the serum of the positive drug group mice were significantly lower compared to the model control group (p<0.05). In addition, the TNF-α levels in the serum of experimental groups 1 and 2 were slightly lower, but the difference was not statistically significant, and there was no significant difference between the two experimental groups.

[0083] The results above indicate that, under the conditions of this study, live Rimosiractobacillus fermentum MSJK0025 has an effect in improving mastitis during lactation caused by Staphylococcus aureus infection, but it cannot prevent the onset of mastitis during lactation caused by Staphylococcus aureus infection. This effect is mainly observed in the active bacteria, not in the fermentation supernatant. Under the conditions of this study, neither live Rimosiractobacillus fermentum CECT5716 nor the fermentation supernatant showed any effect in improving or preventing mastitis during lactation caused by Staphylococcus aureus infection.

[0084] Example 5: Evaluation of the preventive and therapeutic effects of rimosilactobacillus fermentum MSJK0025 on breast cancer.

[0085] 1. Animal grouping and medication

[0086] After acclimatizing MMTV-PyMT genetically modified mice (female, 6 weeks old), the mice were randomly divided into two groups: a model control group and a rimosilactobacillus fermentum group, with 32 mice in each group, for a total of 64 mice.

[0087] From 6 weeks of age, the mice were administered the sample to both a model control group and a rimosiractobacillus fermentum group. The rimosiractobacillus fermentum group received 0.5 mL of the sample orally per mouse three times a week, while the model control group received 0.5 mL of water orally per mouse three times a week. For the rimosiractobacillus fermentum group, 20 mg of rimosiractobacillus fermentum MSJK0025 dried powder was weighed, 20 ml of water was added, and the sample was prepared as a bacterial powder solution and stored until use.

[0088] 2. Measurement of effectiveness

[0089] At 13, 15, 17, and 19 weeks of age, eight mice were selected from each group, and the following measurements were performed.

[0090] (1) Weighing body weight

[0091] (2) Since diffuse tumors were present in multiple locations on the ventral side of the mice, the tumor volume in the mammary gland area was measured using calipers (recorded as 0 if measurement was not possible), and only the largest tumor in the vicinity of the mammary gland was measured and recorded.

[0092] (3) After the mice were sacrificed, tumors were removed from all parts of the body, their weight was recorded, and photographs were taken.

[0093] (4) Blood samples were collected, serum was separated, and the concentrations of CA153, VEGF, and CEA in the serum were measured using an ELISA kit.

[0094] 3. Experimental Results

[0095] (1) Expression levels of CA153, VEGF, and CEA in mouse serum

[0096] The expression levels of CA153, VEGF, and CEA in mouse serum were measured using the following ELISA research kits: mouse carcinoembryonic antigen (CEA, CD66) ELISA research kit (MM-0633M1, Jiangsu Enzyme Immunology Co., Ltd., China), mouse vascular endothelial growth factor (VEGF) ELISA research kit (MM-0128M1, Jiangsu Enzyme Immunology Co., Ltd., China), and mouse glycan antigen 15-3 (CA15-3) ELISA research kit (MM-47737M1, Jiangsu Enzyme Immunology Co., Ltd., China). The results are shown in Table 5.

[0097] Table 5: Expression levels of CA153, VEGF, and CEA in mouse serum (n=8) [Table 5] Note: Comparison with the model control group, *p±<±0.05, **p±<±0.01

[0098] Table 5 shows that, compared to the model control group, the expression of VEGF and CEA in the serum of mice in the rimosiractobacillus fermentum group was slightly lower at week 13, with a significant decrease in CEA expression at this time. However, it significantly increased at week 19, and the difference was not significant at other times.

[0099] (2) Mouse tumor volume

[0100] The tumor volumes of the mouse control group and the rimosiractobacillus fermentum group are shown in Table 6 and Figure 10.

[0101] Table 6: Measurement results of mouse tumor volume (n=8) [Table 6] Note: Comparison with the model control group, *p<0.05, **p<0.01

[0102] Table 6 and Figure 10 show that, compared to the model control group, the tumor volume of mice in the rimosiractobacillus fermentum group was significantly reduced at week 13, but the difference was not significant at other times. This indicates that administration of rimosiractobacillus fermentum MSJK0025 can slow the growth of early mammary gland tumors.

[0103] (3) Mouse tumor weight

[0104] The tumor weights of the mouse control group and the rimosiractobacillus fermentum group are shown in Table 7 and Figure 11, and images of the whole-body tumors in mice are shown in Figure 12.

[0105] Table 7: Measurement results of mouse tumor weight (n=8) [Table 7] Note: Comparison with the model control group, *p<0.05

[0106] Table 7 and Figure 11 show that, compared to the model control group, the tumor weight of mice in the rimosiractobacillus fermentum group was significantly reduced at both weeks 13 and 17, but the difference was not significant at other times. Simultaneously, Figure 12 shows that, compared to the model control group, the tumor size of mice in the rimosiractobacillus fermentum group was significantly reduced at both weeks 13 and 17, but the difference was not significant at other times. This indicates that administration of rimosiractobacillus fermentum MSJK0025 can slow the growth of early mammary gland tumors. [Industrial applicability]

[0107] Based on the above results, Rimosiractobacillus fermentum MSJK0025 has the effect of slowing the early growth of mammary gland tumors, but does not show any improvement in the malignant progression of mammary gland tumors. Therefore, Rimosiractobacillus fermentum MSJK0025 can be used for the prevention and treatment of breast cancer (especially the treatment of early-stage breast cancer). It should be understood that the embodiments described above are for illustrative purposes only and do not limit the present application. While this application is described based on representative embodiments, it should be understood that the terminology used herein is descriptive, not restrictive. This application may be modified within the scope of the claims and may be amended without departing from the scope and spirit of this application. While this application relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein. Rather, this application can be extended to all other methods and applications having similar functions.

Claims

1. A pharmaceutical composition for improving breast health containing breast milk-derived limosilactobacillus fermentum MSJK0025.

2. The pharmaceutical composition according to claim 1, characterized in that the improvement of breast health includes the improvement of mastitis and the prevention and / or treatment of breast cancer.

3. The pharmaceutical composition according to claim 2, characterized in that the mastitis is selected from at least one of non-lactation mastitis and lactation mastitis, and the breast cancer is early-stage breast cancer.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the pharmaceutical composition contains live rimosilactobacillus fermentum MSJK0025 bacteria.

5. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the target of administration of the pharmaceutical composition is female mammals, including humans.

6. A probiotic preparation for improving breast health containing breast milk-derived limosilactobacillus fermentum MSJK0025.

7. The probiotic formulation according to claim 6, characterized in that the improvement of breast health includes the improvement of mastitis and the prevention and / or treatment of breast cancer.

8. The probiotic preparation according to claim 7, characterized in that the mastitis is selected from at least one of non-lactation mastitis and lactation mastitis, and the breast cancer is early-stage breast cancer.

9. The probiotic preparation according to any one of claims 6 to 8, characterized in that the probiotic preparation contains live rimosilactobacillus fermentum MSJK0025 bacteria.

10. The probiotic preparation according to any one of claims 6 to 8, characterized in that the target recipient of the probiotic preparation is female mammals, including humans.

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