Use of breast milk-derived limosilactobacillus fermentum MSJK0025 in improvement of breast health

Abstract

The present application relates to the use of a strain of breast milk-derived Limosilactobacillus fermentum MSJK0025 in the improvement of breast health (improvement in mastitis, and prevention and / or treatment of breast cancer) in the technical field of microorganisms. Specifically provided are a pharmaceutical composition containing breast milk-derived Limosilactobacillus fermentum MSJK0025 for improving breast health, and a probiotic microbial agent containing breast milk-derived Limosilactobacillus fermentum MSJK0025 for improving breast health. Provided in the present application is the new use of a strain of breast milk-derived Limosilactobacillus fermentum MSJK0025 in the improvement of breast health. Limosilactobacillus fermentum MSJK0025 can be used to effectively improve breast health.

Description

Application of Lactobacillus mucinus fermentum MSJK0025 derived from breast milk in improving breast health Technical Field

[0001] This application relates to the field of microbial technology, and in particular to the application of a breast milk-derived fermenting Lactobacillus mucinus MSJK0025 in improving breast health. Background Technology

[0002] Mastitis is a common inflammatory disease of the mammary glands in female mammals, including humans, and can be divided into lactating and non-lactating mastitis. The physiological structure of the breast shows that the mammary gland is an ecosystem connecting the external and internal environments, with a large number of bacteria colonizing it, forming a balanced microecology under normal circumstances. There are two sources of bacteria: endogenous and exogenous. (1) Exogenous bacteria mainly migrate from the nipple skin and infant oral cavity to the mammary gland through the mammary ducts. (2) Endogenous bacteria migrate from the gastrointestinal tract to the mammary gland through the "gut-mammary gland transfer pathway" (as shown in Figure 1). In general, mastitis is a pathological process characterized by mammary gland microecological dysbiosis. Most mastitis is caused by Staphylococcus infection, mainly Staphylococcus aureus and Staphylococcus epidermidis.

[0003] Currently, the main treatment for mastitis is systemic antibiotic therapy. While effective, this approach has several drawbacks, such as the development of drug resistance, disruption of gut flora balance, and side effects. For breastfeeding mothers with mastitis, antibiotic use can also interfere with breastfeeding and adversely affect the infant, posing safety risks. Therefore, it is necessary to find alternative antibiotics for the improvement or treatment of mastitis.

[0004] In 2022, *Lactobacillus fermentum* LC40 (CECT5716) was included as a recommended treatment in the American Academy of Breastfeeding Medicine (ABM) Clinical Guideline #36: Mastitis Spectrum, 2022 Revision. However, globally, only *Lactobacillus fermentum* CECT5716 explicitly claims to have the effect of "improving mastitis." This strain is a star strain of the Spanish company Biosearch Life, isolated from the breast milk of healthy women. The raw material powder prepared from this strain is currently produced in Spain, with a high price, and the procurement of the powder is easily affected by the national environment, which has a significant impact on the production continuity and cost of the end product.

[0005] Breast cancer is one of the most common malignant tumors affecting women's breast health, typically occurring in the glandular epithelial tissue of the breast. Statistics show that its incidence rate accounts for 7%-10% of all malignant tumors, making it one of the most common malignant tumors that seriously impacts women's physical and mental health and even threatens their lives. Currently, the main treatments for breast cancer are surgery and Western medicine. However, surgery carries certain risks and is not suitable for older or frail patients; Western medicine often only treats the symptoms and not the root cause, with limited effectiveness and high cost. Therefore, there is a need to find new methods for improving or treating breast cancer.

[0006] The *Lactobacillus fermentum* MSJK0025 involved in this application was isolated from the breast milk of healthy postpartum women and belongs to the same species as *Lactobacillus fermentum* CECT5716, making them highly comparable. Studies have shown that *Lactobacillus fermentum* MSJK0025 excels in terms of resistance to gastric acid, bile salts, intestinal colonization ability, and growth rate. Furthermore, it has completed safety evaluation tests and commercial batch production, demonstrating promising prospects for industrialization. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this application provides the application of a breast milk-derived Lactobacillus fermentum MSJK0025 in improving breast health (improving mastitis, preventing and / or treating breast cancer). Lactobacillus fermentum MSJK0025 can effectively alleviate breast inflammation and pathological changes, as well as slow down the early growth of breast tumors, thereby effectively improving breast health.

[0008] Therefore, in a first aspect, this application provides a pharmaceutical composition comprising Limosilactobacillus fermentum MSJK0025, a breast milk-derived lactobacillus, for improving breast health.

[0009] In some implementations, the improvement of breast health includes improving mastitis and preventing and / or treating breast cancer.

[0010] The *Lactobacillus fermentum* MSJK0025 used in the pharmaceutical composition described in this application is a strain isolated and screened in patent application number 202311070808.2, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26426. Through research on this strain, the inventors of this application have discovered that it can effectively alleviate breast inflammation and pathological changes, as well as slow the early growth of breast tumors, and therefore can be used in pharmaceutical compositions for improving breast health (improving mastitis, preventing and / or treating breast cancer).

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

[0012] In this application, Lactobacillus fermentum MSJK0025 can be used to improve non-lactating mastitis as well as lactating mastitis, especially lactating (prolactinitis) mastitis.

[0013] In this application, early breast cancer (or early-stage breast cancer) refers to a patient whose tumor is less than 3cm during surgery; no metastatic lymph nodes are palpable in the axilla, and there is no distant metastasis, or the ipsilateral axillary lymph nodes have no metastasis or only micrometastasis, and no distant metastasis; including non-invasive cancer.

[0014] The *Lactobacillus fermentum* MSJK0025 in this application can slow down the early growth of breast tumors (the tumor volume of female mice with breast cancer in the experimental group treated with *Lactobacillus fermentum* MSJK0025 was significantly smaller at week 13, and the tumor weight was significantly reduced at both weeks 13 and 17), but it does not improve the malignant development of breast tumors. Therefore, the breast cancer treated by the pharmaceutical composition described in this application is preferably early-stage breast cancer.

[0015] In some embodiments, the pharmaceutical composition contains live Lactobacillus fermentum MSJK0025.

[0016] The inventors of this application discovered through research that the efficacy of fermented Lactobacillus mucinus MSJK0025 in improving breast health, especially mastitis, is mainly manifested in live bacteria, rather than in the fermentation supernatant.

[0017] In addition to *Lactobacillus fermentum* MSJK0025, the pharmaceutical composition of this application may also include a pharmaceutically acceptable carrier. In this application, the pharmaceutically acceptable carrier includes, but is not limited to, any pharmaceutically acceptable excipient, surfactant, desiccant, diluent, carrier, adjuvant, or additive. Suitable pharmaceutically acceptable carriers may include, for example, magnesium carbonate, lactose, pectin, dextrin, starch, astragalus gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, and cocoa butter. In this application, the pharmaceutically acceptable carrier may be solid, semi-solid, or liquid.

[0018] In some embodiments, the pharmaceutical composition is administered to female mammals, including humans.

[0019] The *Lactobacillus fermentum* MSJK0025 in the pharmaceutical composition of this application can effectively improve mastitis symptoms and slow the early growth of breast tumors. Histological observation revealed that the inflammation and pathological changes in female mice with mastitis treated with *Lactobacillus fermentum* MSJK0025 were effectively alleviated. HE staining results showed that only a small number of inflammatory cells were present in the mammary alveoli of female mice with mastitis treated with *Lactobacillus fermentum* MSJK0025, indicating a significant reduction in inflammation. Serum sample analysis showed that the TNF-α secretion level in female mice with mastitis treated with *Lactobacillus fermentum* MSJK0025 was significantly reduced. Furthermore, the tumor volume in female mice with breast cancer treated with *Lactobacillus fermentum* MSJK0025 was significantly reduced at week 13, and the tumor weight was significantly reduced at both weeks 13 and 17. Therefore, the pharmaceutical composition of this application can effectively improve breast health (improve mastitis, prevent and / or treat breast cancer), and is thus effectively applicable to female mammals, including humans, especially to female mammals with breast health problems (such as mastitis, early breast cancer).

[0020] The mammals mentioned in this application may be, for example, humans, rats, pigs, cattle, sheep, dogs, cats, rabbits, etc.

[0021] The second aspect of this application provides a probiotic preparation containing Limosilactobacillus fermentum MSJK0025, derived from breast milk, for improving breast health.

[0022] In some implementations, the improvement of breast health includes improving mastitis and preventing and / or treating breast cancer.

[0023] The probiotic preparation described in this application contains *Lactobacillus fermentum* MSJK0025. Through research on this strain, the inventors of this application have found that this strain can effectively alleviate breast inflammation and pathological changes, as well as slow the early growth of breast tumors. Moreover, this strain is outstanding in terms of resistance to gastric acid, bile salts, intestinal colonization ability, and growth rate. It has also completed safety evaluation tests and commercial batch production, and can therefore be used in probiotic preparations for improving breast health (improving mastitis, preventing and / or treating breast cancer).

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

[0025] In some embodiments, the probiotic agent contains live Lactobacillus fermentum MSJK0025.

[0026] In addition to Lactobacillus fermentum MSJK0025, the probiotic agent in this application may also include other probiotics, and this application does not limit them.

[0027] In some embodiments, the probiotic agent is administered to female mammals, including humans.

[0028] The beneficial technical effects of this application are as follows: This application provides a novel application of *Lactobacillus fermentum* MSJK0025, derived from breast milk, in improving breast health (improving mastitis, preventing and / or treating breast cancer). In the experimental group treated with *Lactobacillus fermentum* MSJK0025, the inflammation and pathological changes in female mice with mastitis were effectively alleviated; HE staining results showed that only a small number of inflammatory cells were present in the mammary alveoli of female mice with mastitis in the experimental group treated with *Lactobacillus fermentum* MSJK0025, and the degree of inflammation was significantly reduced; serum sample detection results showed that the TNF-α secretion level in female mice with mastitis in the experimental group treated with *Lactobacillus fermentum* MSJK0025 was significantly reduced. Furthermore, in the experimental group treated with *Lactobacillus fermentum* MSJK0025, the tumor volume of female mice with breast cancer significantly decreased at week 13, and the tumor weight significantly decreased at both weeks 13 and 17. Therefore, fermented Lactobacillus mucinus MSJK0025 can be well applied in improving breast health (improving mastitis, preventing and / or treating breast cancer). Attached Figure Description

[0029] Figure 1 shows the gut-mammary gland transfer pathway of bacteria.

[0030] Figure 2 shows photographs of the mammary gland tissue of mother mice in each group in Example 1, "Establishment of a Mouse Model of Mammary Infection by Staphylococcus aureus". A. Blank control group; B. Staphylococcus aureus 10... 3 CFU / mL group; C. Staphylococcus aureus 10 4 CFU / mL group; D. Staphylococcus aureus 10 5 CFU / mL group; arrows indicate mammary glands.

[0031] Figure 3 shows HE-stained (200x) images of mammary gland tissue from each group of mother mice in Example 1, "Establishment of a Mouse Model of Mammary Infection by Staphylococcus aureus". A. Blank control group; B. Staphylococcus aureus 10... 3 CFU / mL group; C. Staphylococcus aureus 10 4 CFU / mL group; D. Staphylococcus aureus 10 5 CFU / mL group; arrows indicate inflammatory cells.

[0032] Figure 4 shows photographs of the appearance of mammary tissue in mice in each group during Example 2, "Efficacy Evaluation of Lactobacillus fumariformis MSJK0025 in Improving Mastitis". A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. 10 live Lactobacillus fumariformis MSJK0025 bacteria. 7 CFU / mL group; E. live Lactobacillus fermentum MSJK0025 10 8 CFU / mL group; F. Live Lactobacillus fermentum MSJK0025 10 9 CFU / mL group; arrows indicate mammary glands.

[0033] Figure 5 shows HE-stained (200x) images of mouse mammary tissue from each group in Example 2, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Improving Mastitis". A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. 10 live Lactobacillus fermentum MSJK0025 bacteria. 7 CFU / mL group; E. live Lactobacillus fermentum MSJK0025 10 8 CFU / mL group; F. Live Lactobacillus fermentum MSJK0025 10 9 CFU / mL group; arrows indicate inflammatory cells.

[0034] Figure 6 shows photographs of the appearance of mammary tissue in mice in each group in Example 3, "Evaluation of the Efficacy of Lactobacillus flavus MSJK0025 in Improving Mastitis". A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. Experimental group 1 (live Lactobacillus flavus MSJK0025); E. Experimental group 2 (live Lactobacillus flavus CECT5716); F. Experimental group 3 (fermentation supernatant of Lactobacillus flavus MSJK0025); G. Experimental group 4 (fermentation supernatant of Lactobacillus flavus CECT5716).

[0035] Figure 7 shows HE staining (200x) images of mouse mammary tissue from each group in Example 3, "Evaluation of the Efficacy of Lactobacillus fermentation MSJK0025 in Improving Mastitis". The groups are: A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. Experimental group 1 (live Lactobacillus fermentation MSJK0025); E. Experimental group 2 (live Lactobacillus fermentation CECT5716); F. Experimental group 3 (fermentation supernatant of Lactobacillus fermentation MSJK0025); G. Experimental group 4 (fermentation supernatant of Lactobacillus fermentation CECT5716).

[0036] Figure 8 shows photographs of the appearance of mammary tissue in mice in each group in Example 4, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Preventing Mastitis". Among them: A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. Experimental group 1 (live Lactobacillus fermentum MSJK0025); E. Experimental group 2 (live Lactobacillus fermentum CECT5716).

[0037] Figure 9 shows HE staining (200x) images of mouse mammary tissue from each group in Example 4, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Preventing Mastitis". The groups are: A. Blank control group; B. Model control group; C. Positive drug (levofloxacin) group; D. Experimental group 1 (live Lactobacillus fermentum MSJK0025); E. Experimental group 2 (live Lactobacillus fermentum CECT5716).

[0038] Figure 10 shows a comparison of tumor volume in mice in the model control group and the fermented Lactobacillus mucin group in Example 5.

[0039] Figure 11 shows a comparison of tumor weight in mice in the model control group and the fermented *Lactobacillus mucinus* group in Example 5.

[0040] Figure 12 shows the morphological images of whole-body tumors in mice in Example 5, specifically in the model control group and the fermented *Lactobacillus mucinus* group; where A represents the model control group and B represents the fermented *Lactobacillus mucinus* group.

[0041] Biological Preservation Information

[0042] Limosilactobacillus fermentum MSJK0025, with accession number CGMCCNo.26426, is deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​January 9, 2023. Detailed Implementation

[0043] To make this application easier to understand, the following will describe this application in further detail with reference to embodiments.

[0044] The experimental animals used in Examples 1-4 below were provided by Spiford (Suzhou) Biotechnology Co., Ltd. (China), Certificate of Conformity: SCXK(Su)2022-0006. The experimental animal used in Example 5 below was a female MMTV-PyMT transgenic mouse, 6 weeks old, provided by Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd., Certificate of Conformity: SCXK(Su)2020-0009.

[0045] Example 1: Establishment of a mouse model of mastitis caused by Staphylococcus aureus infection

[0046] 1. Take a Staphylococcus aureus strain (China General Microbiological Culture Collection Center ATCC-29213) and inoculate it onto a nutrient agar slant, then incubate at 37°C.

[0047] 2. After 24 hours of culture, inoculate with 100 mL of nutrient broth liquid culture medium and continue to culture at 37°C.

[0048] 3. After culturing for another 16 hours, take 10 mL of bacterial suspension, centrifuge at 3000 rpm for 5 min, discard the supernatant, resuspend in an equal volume of physiological saline, centrifuge again, discard the supernatant, add 10 mL of sterile physiological saline and mix well to obtain the initial Staphylococcus aureus suspension.

[0049] 4. The bacterial suspension concentration is determined by viable cell count. Then, it is adjusted to the required concentration with sterile physiological saline and set aside for later use.

[0050] 5. Two hours before inoculation, separate the pups that have been receiving breast milk for 10 days from their mothers. Anesthetize the mother mouse, tie her up in a supine position, fully expose the fourth pair of mammary glands, disinfect the mammary glands with 75% alcohol, and use microscissors to cut off about 1 mm of the nipple tip.

[0051] 6. Using a passivated microsyringe (less than 30G), slowly insert it about 2mm into the lactiferous duct and inject 50μL of Staphylococcus aureus suspension. The bacterial concentration was determined by experimenting with three different dosages (10...). 3 CFU / mL, 10 4 CFU / mL and 10 5 (CFU / mL), see Table 1 for details.

[0052] 7. Three hours after inoculation, the pups were returned to their mothers. The mammary gland tissue of the mother mice was dissected and observed 24 hours after infection; the results are shown in Figures 2 and 3.

[0053] Table 1: Concentration grouping for Staphylococcus aureus infection model of mastitis

[0054] As shown in Figure 2, compared with the blank control group, 10 people infected with Staphylococcus aureus... 4 CFU / mL and 10 5 The mammary tissue of mice in the CFU / mL group showed slight darkening and mild bleeding. As shown in Figure 3, compared to the blank control group, mice infected with Staphylococcus aureus for 10... 3 CFU / mL, 10 4 CFU / mL and 10 5 In mice in the CFU / mL group, significant inflammatory cells were observed in the acinar lumen, and their number increased with increasing bacterial concentration. Based on the model exploration results, it was determined that a concentration of 10... 5CFU / mL is used as the concentration of Staphylococcus aureus in the infecting bacteria for mastitis.

[0055] Example 2: Evaluation of the efficacy of fermented Lactobacillus mucin MSJK0025 in improving mastitis

[0056] 1. Inoculate MRS agar slant with *Lactobacillus fermentum* MSJK0025 (China General Microbiological Culture Collection Center, CGMCC No. 26426) and incubate at 37°C for 24 h. Then inoculate with 100 mL of MRS broth and continue incubation at 37°C for 16 h. Take 10 mL of the bacterial suspension, centrifuge at 3000 rpm for 5 min, discard the supernatant, resuspend in an equal volume of physiological saline, centrifuge again, discard the supernatant, and add 10 mL of sterile physiological saline to prepare the initial bacterial suspension. Determine the viable cell concentration of the initial suspension by viable cell count. Then, adjust the concentration to the desired level with sterile physiological saline for later use.

[0057] 2. After determining the concentration of infectious bacteria in the mastitis model based on the experimental results of Example 1, another 48 lactating mice were randomly divided into six groups: 1 blank control group, 1 model control group, 1 positive drug group, and 3 experimental groups (three doses), with 8 mice in each group.

[0058] 3. Starting 3 days before the mastitis model was established, the positive drug group was given levofloxacin 55 mg / kg by gavage, and the three experimental groups were given 0.5 mL of live Lactobacillus fermentum MSJK0025 by gavage, at doses of 10 mg / kg and 10 mg / kg respectively. 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, the model control group and the blank control group were given 0.5 mL of physiological saline once a day.

[0059] 4. Except for the blank control group, all lactating mice were used to establish a Staphylococcus aureus-infected mastitis model according to the steps in Example 1, with 10 5 CFU / mL is used as the concentration of Staphylococcus aureus in the infecting bacteria for mastitis.

[0060] 5. After the model was established, the positive drug group continued to receive daily levofloxacin gavage intervention, the experimental group continued to receive daily gavage intervention with live Lactobacillus fermentum MSJK0025, and the model control group and blank control group were given an equal volume of physiological saline once a day for a total of 10 interventions (including before model establishment).

[0061] 6. Twenty-four hours after the last intervention, blood was collected from each group of mice, and the mice were euthanized. The skin was disinfected with alcohol, the abdominal skin was opened, and the mammary gland tissue on both sides of the mice was exposed. The appearance of the mammary gland tissue (color, texture, and presence of bleeding) was observed, photographed, and recorded. The results are shown in Figure 4. Mammary gland tissue was then subjected to subsequent pathological HE staining examination. The results are shown in Figure 5.

[0062] 7. Blood was collected from the orbital cavity of mice 24 hours after the last intervention, and the serum TNF-α level of mice was detected by ELISA kit. The results are shown in Table 2.

[0063] Table 2: TNF-α levels in serum samples from mice in each group

[0064] ***, p<0.001; **, p<0.005

[0065] As shown in Figure 4, compared with the blank control group, the mammary tissue of mice in the model group was slightly darker and accompanied by slight bleeding. In contrast, the positive drug group and the live Lactobacillus fermentum MSJK0025 group showed significantly different results. 9 No obvious inflammation or pathological changes were observed in the mammary tissue of mice in the CFU / mL group; meanwhile, compared with the model group, the number of live Lactobacillus fermentum MSJK0025 bacteria was 10. 7 CFU / mL group, 10 live Lactobacillus fermentum MSJK0025 8 The inflammation and pathological changes in the mammary tissue of mice in the CFU / mL group were somewhat reduced.

[0066] As shown in Figure 5, HE staining results indicated that, compared with the blank control group, the number of inflammatory cells in the acinar lumen of mice in the model control group was significantly increased; compared with the model control group, no obvious inflammatory cells were found in the mammary acinar lumen of mice in the positive drug group; 10 live Lactobacillus mucinus MSJK0025 were detected. 8 10 9 The mice in the CFU / mL group had a small number of inflammatory cells in their mammary alveoli, and the degree of inflammation was significantly reduced.

[0067] Table 2 shows that, compared with the blank control group, the serum TNF-α secretion level in the model control group mice was significantly increased; except for 10 live Lactobacillus fermentum MSJK0025 bacteria. 7 In the CFU / mL group, compared with the model control group, the positive drug group mice had 10 live Lactobacillus fermentum MSJK0025 bacteria. 8 10 9 The serum TNF-α secretion level was significantly reduced in the CFU / mL group.

[0068] The above results indicate that live *Lactobacillus fermentum* MSJK0025 can effectively improve mastitis symptoms in mice, and that 10 live *Lactobacillus fermentum* MSJK0025... 9 The CFU / mL group showed the best improvement.

[0069] Example 3: Evaluation of the efficacy components of fermented Lactobacillus mucin MSJK0025 in improving mastitis

[0070] I. Sample Preparation

[0071] Preparation of bacterial suspension and fermentation supernatant: *Lactobacillus fermentans* strain (MSJK0025 or CECT5716) was inoculated onto MRS slant agar and cultured at 37°C for 24 h. Afterward, it was transferred to 100 mL of MRS broth and fermented at 37°C for another 16 h. 10 mL of the bacterial suspension was then centrifuged at 3000 rpm for 5 min, and the fermentation supernatant was collected. The bacterial cells were resuspended in an equal volume of sterile physiological saline, centrifuged again, and the supernatant was discarded. An equal volume of sterile physiological saline was then added and mixed thoroughly to form the initial bacterial suspension. The viable cell concentration was then adjusted to 10⁻⁶ cells / mL with sterile physiological saline. 9 CFU / mL available for use.

[0072] Drug solution preparation: Weigh levofloxacin and dissolve it in sterile water to prepare a 2.2 mg / mL solution for later use.

[0073] II. Evaluation Process

[0074] 1. Before the experiment, the participating female mice (with pups) underwent a 7-day acclimatization period. Then, healthy female mice 3-5 days after giving birth 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 administered 0.5 mL of the tested *Lactobacillus mucinus* MSJK0025 live bacteria (10 mL / day) via gavage daily. 9 CFU / mL dose); Group 2 was given 0.5 mL of live Lactobacillus fermentum CECT5716 via gavage daily (10 9 (CFU / mL dose); Experimental group 3 was given 0.5 mL of fermentation supernatant of *Lactobacillus mucinus* MSJK0025 by gavage daily; Experimental group 4 was given 0.5 mL of fermentation supernatant of *Lactobacillus mucinus* CECT5716 by gavage daily; Positive drug group was given levofloxacin 55 mg / kg by gavage daily; Model control group and blank control group were given 0.5 mL of physiological saline. Once daily for 3 consecutive days as pre-treatment.

[0075] 2. After three consecutive days of pre-administration, all female mice except the blank control group were inoculated with Staphylococcus aureus to establish the model (the modeling method was the same as in Example 2).

[0076] 3. After vaccination, each group continued to receive medication intervention as required in step 1, once daily for a total of 10 times (including pre-administration).

[0077] 4. Twenty-four hours after the last intervention, mice in each group were sacrificed, dissected, and subjected to histopathological observation (interstitial edema and inflammatory infiltration in the mammary epithelium and acini, etc.). Photos were taken and recorded; the results are shown in Figure 6. Mammary tissue was then subjected to subsequent histopathological HE staining; the results are shown in Figure 7.

[0078] 5. Blood was collected from the orbital cavity of mice 24 hours after the last intervention, and the serum TNF-α level of mice was detected by ELISA kit. The results are shown in Table 3.

[0079] Table 3: TNF-α levels in serum samples from mice in each group

[0080] Note: Compared with the model control group, **p<0.01

[0081] As shown in Figure 6, compared with the blank control group, the mammary tissue of mice in the model control group, experimental group 2, experimental group 3, and experimental group 4 showed a certain degree of redness and swelling and hardening of texture; while compared with the model control group, the positive drug group and experimental group 1 (live Lactobacillus fermentum MSJK0025 10) showed a certain degree of redness and swelling and hardening of texture of mammary tissue. 9 No obvious inflammation or pathological changes were observed in the mammary tissue of mice with CFU / mL.

[0082] As shown in Figure 7, HE staining results revealed that the mammary alveoli of the control group mice were intact, with no obvious inflammatory cell infiltration within the alveoli. Compared to the control group, the mammary tissue structure of the model control group mice was unclear, with a significant increase in inflammatory cells within the alveoli, and scattered hemorrhages were observed. Compared to the model control group, the positive drug group and experimental group 1 (live Lactobacillus fermentum MSJK0025 10) showed significantly different mammary gland structures. 9 In group 1, the mammary gland structure of mice with CFU / mL was clear, and the number of inflammatory cells in the acinar was significantly reduced; in group 2, the mammary gland structure of mice was unclear, and a large number of inflammatory cells were present in the acinar cavity; in group 3, a large number of inflammatory cells were present in the acinar cavity of the mammary gland of mice; in group 4, epithelial cells were shed from the acinar wall of the mammary gland tissue of mice, and a large number of inflammatory cells were present in the acinar cavity.

[0083] Table 3 shows that the serum results detected by ELISA in each group indicated that, compared with the blank control group, the TNF-α level in the model control group mice was significantly increased. Compared with the model control group, the positive drug group and experimental group 1 (live Lactobacillus fermentum MSJK0025 10) showed significantly higher levels of TNF-α. 9The serum TNF-α level in mice was significantly reduced (CFU / mL), p<0.01; the serum TNF-α level in mice in group 3 was slightly reduced but not significantly different; no reduction was found in the serum TNF-α level in groups 2 and 4.

[0084] The above results indicate that live *Lactobacillus fermentum* MSJK0025 can improve symptoms such as breast redness and hardening caused by *Staphylococcus aureus* infection and significantly reduce serum inflammatory factor levels (p<0.01), demonstrating its efficacy in improving mastitis caused by *Staphylococcus aureus* infection during lactation. This improvement is primarily observed in the live bacteria; the supernatant of *Lactobacillus fermentum* MSJK0025 does not show any effect in improving mastitis. However, neither live *Lactobacillus fermentum* CECT5716 nor its fermentation supernatant can improve the occurrence of *Staphylococcus aureus*-induced mastitis in lactating women.

[0085] Example 4: Evaluation of the efficacy of fermented Lactobacillus mucinus MSJK0025 in preventing mastitis

[0086] I. Sample Preparation

[0087] Same as Example 3.

[0088] II. Evaluation Process

[0089] 1. Before the experiment, the participating female mice (with pups) underwent a 7-day acclimatization period. Then, healthy female mice 3-5 days after giving birth 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 administered 0.5 mL of the tested *Lactobacillus mucinus* MSJK0025 live bacteria (10 mL / day) via gavage daily. 9 CFU / mL dose); Group 2 was given 0.5 mL of live Lactobacillus fermentum CECT5716 via gavage daily (10 9 (CFU / mL dose); the positive control group received levofloxacin 55 mg / kg by gavage daily; the model control group and the blank control group received 0.5 mL of normal saline. Once daily for 10 consecutive days as pre-treatment.

[0090] 2. After the intervention was completed on the 10th day, all female mice except the blank control group were inoculated with Staphylococcus aureus to establish the model (the modeling method was the same as in Example 2).

[0091] 3. Twenty-four hours after inoculation, mice in each group were sacrificed, dissected, and subjected to histopathological observation (interstitial edema and inflammatory infiltration within the mammary epithelium and acini, etc.). Photos were taken and recorded; the results are shown in Figure 8. Mammary tissue was then subjected to subsequent histopathological HE staining; the results are shown in Figure 9.

[0092] 4. Blood was collected from the orbital cavity of mice 24 hours after inoculation, and the serum TNF-α level of mice was detected by ELISA kit. The results are shown in Table 4.

[0093] Table 4: TNF-α levels in serum samples from mice in each group

[0094] Note: Compared with the model control group, *p<0.05

[0095] As shown in Figure 8, compared with the blank control group, the mammary tissue of mice in the positive drug group was dark red. The mammary tissue of mice in the other groups showed a certain degree of redness and swelling compared with the blank control group. There was no visible difference between the groups.

[0096] As shown in Figure 9, HE staining results indicated that the mammary alveolar cavities of mice in the blank control group were intact, with no obvious inflammatory cell infiltration within the cavities. Compared with the blank control group, the mammary tissue structures of mice in the model control group, experimental group 1, and experimental group 2 were unclear, and the number of inflammatory cells within the alveolar cavities was significantly increased, with scattered hemorrhages visible. There were no visible differences among the three groups. Compared with the model control group, the number of inflammatory cells within the mammary alveoli of mice in the positive drug group was significantly reduced.

[0097] Table 4 shows that the serum TNF-α levels in the model control group mice were significantly increased compared with the blank control group. Compared with the model control group mice, the serum TNF-α levels in the positive drug group mice were significantly decreased (p<0.05), while the serum TNF-α levels in experimental groups 1 and 2 were slightly decreased, but there was no significant difference, and there was no significant difference between the two experimental groups.

[0098] The above results indicate that, under the experimental conditions, live *Lactobacillus mucinus* MSJK0025 fermentation bacteria have the effect of improving mastitis caused by *Staphylococcus aureus* infection during lactation, but cannot prevent the occurrence of mastitis caused by *Staphylococcus aureus* infection during lactation, and this effect is mainly manifested in the live bacteria rather than the fermentation supernatant. Under the experimental conditions, neither live *Lactobacillus mucinus* CECT5716 fermentation bacteria nor the fermentation supernatant had the effect of improving or preventing mastitis caused by *Staphylococcus aureus* infection during lactation.

[0099] Example 5: Evaluation of the efficacy of fermented Lactobacillus mucinus MSJK0025 in the prevention and treatment of breast cancer

[0100] 1. Animal grouping and administration

[0101] Female MMTV-PyMT transgenic mice (6 weeks old) were used. After acclimatization, the mice were randomly divided into two groups: a model control group and a fermented Lactobacillus mucin group, with 32 mice in each group and a total of 64 mice.

[0102] Starting at 6 weeks of age, the model control group and the *Lactobacillus fermentum* group were administered the drug, respectively. The *Lactobacillus fermentum* group received the drug three times a week, with each mouse receiving 0.5 mL of sample orally via gavage. The model control group received the drug three times a week, with each mouse receiving 0.5 mL of water orally via gavage. The sample preparation method for the *Lactobacillus fermentum* group was as follows: 20 mg of *Lactobacillus fermentum* MSJK0025 dry powder was weighed and added to 20 mL of water to prepare a bacterial powder solution for later use.

[0103] 2. Efficacy testing

[0104] When the animals reached 13, 15, 17, and 19 weeks of age, 8 mice from each group were selected for the following tests:

[0105] (1) Weigh yourself;

[0106] (2) Use calipers to measure the volume of the tumor in the breast (if it is not measured, record it as 0). Since there are multiple diffuse tumors in different locations on the ventral side of the mouse, only the largest tumor near the breast is selected for measurement and recording.

[0107] (3) After euthanizing the mice, remove the tumors from the whole body, weigh and record the tumor weight, and take pictures;

[0108] (4) Blood collection and serum separation: The levels of CA153, VEGF and CEA in serum were detected by ELISA kit.

[0109] 3. Experimental Results

[0110] (1) Expression levels of CA153, VEGF and CEA in mouse serum

[0111] The expression levels of CA153, VEGF, and CEA in mouse serum were detected using the mouse carcinoembryonic antigen (CEA; CD66) ELISA research kit (MM-0633M1, Jiangsu Enzyme Immunosorbent Assay Co., Ltd., China), the mouse vascular endothelial growth factor (VEGF) ELISA research kit (MM-0128M1, Jiangsu Enzyme Immunosorbent Assay Co., Ltd., China), and the mouse carbohydrate antigen 15-3 (CA15-3) ELISA research kit (MM-47737M1, Jiangsu Enzyme Immunosorbent Assay Co., Ltd., China). The results are shown in Table 5.

[0112] Table 5: Expression levels of CA153, VEGF, and CEA in mouse serum (n=8)

[0113] Note: Compared with the model control group, *p<0.05, **p<0.01

[0114] As shown in Table 5, compared with the model control group, the expression of VEGF and CEA in the serum of mice in the fermented Lactobacillus group decreased slightly at week 13, with the decrease in CEA expression being more significant. However, it increased significantly at week 19, and the differences were not significant at other times.

[0115] (2) Mouse tumor volume

[0116] The tumor volumes of mice in the model control group and the fermented *Lactobacillus mucinus* group are shown in Table 6 and Figure 10.

[0117] Table 6: Results of tumor volume detection in mice (n=8)

[0118] Note: Compared with the model control group, *p<0.05, **p<0.01

[0119] As shown in Table 6 and Figure 10, compared with the model control group, the tumor volume of mice in the *Lactobacillus fermentum* group was significantly smaller at week 13, with no significant difference at other times. This indicates that administration of *Lactobacillus fermentum* MSJK0025 can slow the growth of early-stage breast tumors.

[0120] (3) Mouse tumor weight

[0121] The tumor weights of mice in the model control group and the fermented Lactobacillus mucin group are shown in Table 7 and Figure 11, and whole-body tumor images of mice are shown in Figure 12.

[0122] Table 7: Results of tumor weight detection in mice (n=8)

[0123] Note: Compared with the model control group, *p<0.05

[0124] As shown in Table 7 and Figure 11, compared with the model control group, the tumor weight of mice in the *Lactobacillus fermentum* group was significantly reduced at weeks 13 and 17, with no significant difference at other times. Similarly, as shown in Figure 12, compared with the model control group, the tumor weight and tumor size of mice in the *Lactobacillus fermentum* group were significantly reduced at weeks 13 and 17, with no significant difference at other times. This indicates that administration of *Lactobacillus fermentum* MSJK0025 can slow the growth of early-stage breast tumors.

[0125] The above results indicate that *Lactobacillus fermentum* MSJK0025 has the effect of slowing down the early growth of breast tumors, but it does not improve the malignant development of breast tumors. Therefore, *Lactobacillus fermentum* MSJK0025 can be used in the prevention and treatment of breast cancer (especially the treatment of early-stage breast cancer).

[0126] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. A pharmaceutical composition comprising Limosilactobacillus fermentum MSJK0025, derived from breast milk, for improving breast health.

2. The pharmaceutical composition according to claim 1, characterized in that, The improvement of breast health includes improving mastitis and preventing and / or treating breast cancer.

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

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

5. The pharmaceutical composition according to any one of claims 1-3, characterized in that, The pharmaceutical composition is to be administered to female mammals, including humans.

6. A probiotic preparation containing *Limosilactobacillus fermentum* MSJK0025 derived from breast milk for improving breast health.

7. The probiotic agent according to claim 6, characterized in that, The improvement of breast health includes improving mastitis and preventing and / or treating breast cancer.

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

9. The probiotic agent according to any one of claims 6-8, characterized in that, The probiotic agent contains live Lactobacillus fermentum MSJK0025.

10. The probiotic preparation according to any one of claims 6-8, characterized in that, The probiotic preparation is intended for use by female mammals, including humans.

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