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

Abstract

This application relates to the application of a strain of *Lactobacillus fermentum* MSJK0025 derived from breast milk in improving breast health (particularly improving mastitis). Specifically, it relates to the application of a strain of *Lactobacillus fermentum* MSJK0025 derived from breast milk in the preparation of a pharmaceutical composition for improving breast health (particularly improving mastitis), the pharmaceutical composition containing live *Lactobacillus fermentum* MSJK0025. It also relates to the application of a strain of *Lactobacillus fermentum* MSJK0025 derived from breast milk in the preparation of a probiotic agent for improving breast health (particularly improving mastitis), the probiotic agent containing live *Lactobacillus fermentum* MSJK0025. This application provides a novel application of a strain of *Lactobacillus fermentum* MSJK0025 derived from breast milk in improving breast health (particularly improving mastitis), utilizing *Lactobacillus fermentum* MSJK0025 to effectively alleviate breast-related inflammation and pathological changes, thereby improving breast health.

Description

Technical Field

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

[0002] Mastitis is a common inflammatory disease of the mammary glands in female mammals, including humans, and can be divided into lactating mastitis and non-lactating mastitis. In humans, lactating mastitis is most common in postpartum lactating women, usually caused by pathogenic bacterial infection and milk stasis; non-lactating mastitis can occur at any age. The main symptoms of mastitis are breast pain, redness, swelling, and fever, and it can further develop into a breast abscess.

[0003] 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. Its bacterial sources are of two types: 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" (e.g., ...). Figure 1 (As shown). In general, mastitis is a pathological process characterized by dysbiosis of the breast microecology. When this balance is disrupted, localized or diffuse infection can occur, leading to mastitis during lactation. Most cases of mastitis are caused by Staphylococcus infections, primarily Staphylococcus aureus and Staphylococcus epidermidis.

[0004] 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.

[0005] 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. Also in 2022, *Lactobacillus fermentum* CECT5716 was included in the *Postpartum Recovery Guidelines* for probiotic treatment of lactating mastitis, becoming the only probiotic strain included in the guidelines for lactating mastitis treatment. Research by Donnet Hughes et al. showed that bacteria can migrate from the mesenteric lymph nodes to the mammary glands as early as one day after delivery. Through breastfeeding, these bacteria enter the infant's body via breast milk, promoting the growth and colonization of beneficial gut bacteria, improving infant immunity, and reducing the incidence of gastrointestinal and upper respiratory tract infections. This further validates the "gut-mammary gland transfer pathway" of bacteria and provides a safe and effective treatment option for mastitis (especially lactating mastitis).

[0006] Currently, globally, only one probiotic strain, *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 domestic environment, significantly impacting the production continuity and cost of the final product. Therefore, there is an urgent need to develop a "Chinese-native strain" with the effect of improving mastitis and a reasonable price to meet market demand.

[0007] The *Lactobacillus fermentum* MSJK0025 involved in this application was also isolated from the breast milk of healthy mothers and belongs to the same species as *Lactobacillus fermentum* CECT5716, making them highly comparable. Studies show that *Lactobacillus fermentum* MSJK0025 excels in resistance to gastric acid, bile salts, intestinal colonization ability, and growth rate, and has completed safety evaluation tests and commercial batch production. Research data indicates that *Lactobacillus fermentum* MSJK0025 possesses advantages such as strong tolerance, good safety, and low production cost, demonstrating promising prospects for industrialization. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this application provides the application of a breast milk-derived Lactobacillus fermentum MSJK0025 in improving breast health (especially mastitis). Lactobacillus fermentum MSJK0025 can effectively alleviate breast inflammation and pathological changes, thereby improving breast health in female mammals, including humans.

[0009] Therefore, the first aspect of this application provides the use of a breast milk-derived Lactobacillus fermentum MSJK0025 in the preparation of a pharmaceutical composition for improving breast health; preferably, the pharmaceutical composition is used to improve mastitis.

[0010] The *Lactobacillus fermentum* MSJK0025 used in this application is the 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 inflammation and pathological changes in the breast, and therefore can be used in pharmaceutical compositions for improving breast health (especially mastitis).

[0011] In some embodiments, the pharmaceutical composition contains live Lactobacillus fermentum MSJK0025.

[0012] The inventors of this application discovered through research that the efficacy of fermented Lactobacillus mucinus MSJK0025 in improving mastitis is mainly manifested in live bacteria, rather than in the fermentation supernatant.

[0013] In addition to including live *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.

[0014] In some embodiments, the pharmaceutical composition is administered to female mammals, including humans, preferably female mammals suffering from mastitis.

[0015] The *Lactobacillus fermentum* MSJK0025 in the pharmaceutical composition of this application can effectively improve breast health (especially mastitis) symptoms. Tissue observation revealed that the inflammation and pathological changes of female mice with mastitis in the experimental group 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 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 of female mice with mastitis in the experimental group treated with *Lactobacillus fermentum* MSJK0025 was significantly reduced. Therefore, the pharmaceutical composition of this application can effectively improve breast health, especially in female mammals with mastitis.

[0016] The mammals mentioned in this application may be, for example, humans, rats, pigs, cattle, sheep, dogs, cats, rabbits, etc.

[0017] In some embodiments, the mastitis is non-lactational mastitis and lactational mastitis.

[0018] In this application, Lactobacillus fermentum MSJK0025 can be used to improve non-lactating mastitis as well as lactating mastitis, especially lactating (prolactinitis) mastitis.

[0019] The second aspect of this application provides the use of a breast milk-derived Lactobacillus fermentum MSJK0025 in the preparation of a probiotic agent for improving breast health; preferably, the probiotic agent is used to improve mastitis.

[0020] The *Lactobacillus fermentum* MSJK0025 used in this application is the 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 possesses excellent probiotic properties, effectively alleviating inflammation and pathological changes in the breast, and thus can be used in probiotic preparations for improving breast health (especially mastitis).

[0021] In some embodiments, the probiotic agent contains live Lactobacillus fermentum MSJK0025.

[0022] The inventors of this application have 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 fermentation supernatant, etc.

[0023] In addition to live bacteria of Lactobacillus fermentum MSJK0025, the probiotic agent in this application may also include other probiotics, and this application does not limit it.

[0024] In some embodiments, the probiotic agent is administered to female mammals, including humans, preferably female mammals suffering from mastitis.

[0025] The *Lactobacillus fermentum* MSJK0025 in the probiotic agent of this application can effectively improve breast health (especially mastitis) symptoms. Tissue observation showed that the inflammation and pathological changes of female mice with mastitis in the experimental group 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 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 of female mice with mastitis in the experimental group treated with *Lactobacillus fermentum* MSJK0025 was significantly reduced. Therefore, the probiotic agent of this application can effectively improve breast health, especially in female mammals with mastitis.

[0026] The mammals mentioned in this application may be, for example, humans, rats, pigs, cattle, sheep, dogs, cats, rabbits, etc.

[0027] In some embodiments, the mastitis is non-lactational mastitis and lactational mastitis.

[0028] In this application, Lactobacillus fermentum MSJK0025 can be used to improve non-lactating mastitis as well as lactating mastitis, especially lactating (prolactinitis) mastitis.

[0029] A third aspect of this application provides a pharmaceutical composition for improving breast health, the pharmaceutical composition containing live Lactobacillus fermentum MSJK0025; preferably, the pharmaceutical composition is used to improve mastitis.

[0030] In addition to including live *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.

[0031] The fourth aspect of this application provides a probiotic preparation for improving breast health, the probiotic preparation containing live Lactobacillus fermentum MSJK0025; preferably, the probiotic preparation is used to improve mastitis.

[0032] In addition to live bacteria of Lactobacillus fermentum MSJK0025, the probiotic agent in this application may also include other probiotics, and this application does not limit it.

[0033] 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 (especially mastitis). 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, indicating a significant reduction in inflammation. 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. Therefore, *Lactobacillus fermentum* MSJK0025 can be effectively applied to improve breast health (especially mastitis) and can be used as an antibiotic alternative for improving breast health (especially mastitis). Attached Figure Description

[0034] Figure 1 This is a diagram of the bacterial gut-mammary gland transfer pathway.

[0035] Figure 2 Images of the mammary gland tissue from each group of mother mice in Example 1, "Establishment of a Mouse Model of Mammary Infection by Staphylococcus aureus", are shown. 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.

[0036] Figure 3 Images of HE-stained (200x) mammary tissue from mother mice in each group of Example 1, "Establishment of a Mouse Model of Mammary Infection by Staphylococcus aureus," are shown. 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.

[0037] Figure 4 The images shown are photographs of the mammary tissue of mice in each group in Example 2, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Improving Mastitis". The groups are: 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 8CFU / mL group; F. Live Lactobacillus fermentum MSJK0025 10 9 CFU / mL group; arrows indicate mammary glands.

[0038] Figure 5 Images of HE staining (200x) of mouse mammary tissue from each group in Example 2, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Improving Mastitis," are shown. 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.

[0039] Figure 6 The images shown are photographs of the appearance of mammary tissue in mice in Example 3, "Evaluation of the Efficacy of Lactobacillus flavus 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 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).

[0040] Figure 7 Images of HE staining (200x) of mouse mammary tissue from each group in Example 3, "Evaluation of the Efficacy of Lactobacillus fermentation MSJK0025 in Improving Mastitis", are shown. 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).

[0041] Figure 8 The images shown are photographs of the appearance of mammary tissue in each group of mice 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).

[0042] Figure 9 Images of HE staining (200x) of mouse mammary tissue from each group in Example 4, "Efficacy Evaluation of Lactobacillus fermentum MSJK0025 in Preventing Mastitis", are shown. 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).

[0043] Biological Preservation Information

[0044] 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

[0045] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0046] The experimental animals used in the following examples were provided by Spiford (Suzhou) Biotechnology Co., Ltd. (China), certificate of conformity: SCXK(Su)2022-0006.

[0047] Example 1: Establishment of a mouse model of mastitis caused by Staphylococcus aureus infection

[0048] 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.

[0049] 2. After 24 hours of culture, inoculate with 100 mL of nutrient broth liquid culture medium and continue to culture at 37°C.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 7. Three hours after inoculation, the pups were returned to their mothers. Twenty-four hours after infection, the mammary gland tissue of the mother mice was dissected and examined; the results were as follows... Figure 2 and 3 As shown.

[0055] Table 1: Concentration grouping for Staphylococcus aureus infection model of mastitis

[0056]

[0057]

[0058] from Figure 2 It can be seen that, compared with the blank control group, infection with Staphylococcus aureus 10 4 CFU / mL and 10 5 The mammary tissue of mice in the CFU / mL group showed slight darkening and mild bleeding. From Figure 3 It can be seen that, compared with the blank control group, infection with Staphylococcus aureus 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... 5 CFU / mL is used as the concentration of Staphylococcus aureus in the infecting bacteria for mastitis.

[0059] Example 2: Evaluation of the efficacy of fermented Lactobacillus mucin MSJK0025 in improving mastitis

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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).

[0065] 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 as follows: Figure 4 As shown. Breast tissue was taken for subsequent pathological HE staining examination, and the results are as follows. Figure 5 As shown.

[0066] 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.

[0067] Table 2: TNF-α levels in serum samples from mice in each group

[0068] Group TNF-α content (pg / mL) Blank control group 9.01±1.81 Model control group 54.27±11.30 Positive drug group: Levofloxacin (55 mg / kg) 11.97±2.10*** <![CDATA[Low-dose group: viable Lactobacillus mucosae fermentum 10 7 CFU / mL group]]> 42.22±7.44 <![CDATA[Medium-dose group: viable Lactobacillus mucosae fermentum 10 8 CFU / mL group]]> 11.77±6.38** <![CDATA[High-dose group: 10 9 CFU / mL of live Lactobacillus mucosae fermentum]]> 14.40±3.81***

[0069] ***, p<0.001; **, p<0.005

[0070] from Figure 4 It was observed that, compared to the blank control group, the mammary tissue of the mice in the model group was slightly darker and accompanied by mild bleeding. In contrast, the positive control 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.

[0071] from Figure 5 HE staining results showed 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, there were no obvious inflammatory cells in the mammary acinar lumen of mice in the positive drug group; 10 live Lactobacillus mucinus MSJK0025 fermentation bacteria 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.

[0072] 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.

[0073] 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.

[0074] Example 3: Evaluation of the efficacy components of fermented Lactobacillus mucin MSJK0025 in improving mastitis

[0075] I. Sample Preparation

[0076] 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.

[0077] Drug solution preparation: Weigh levofloxacin and dissolve it in sterile water to prepare a 2.2 mg / mL solution for later use.

[0078] II. Evaluation Process

[0079] 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.

[0080] 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).

[0081] 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).

[0082] 4. Twenty-four hours after the last intervention, mice in each group were sacrificed, dissected, and subjected to histopathological observation (interstitial edema of mammary epithelium and acini, inflammatory infiltration, etc.). Photos were taken and recorded. Results are as follows: Figure 6 As shown. Breast tissue was taken for subsequent pathological HE staining examination, and the results are as follows. Figure 7 As shown.

[0083] 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.

[0084] Table 3: TNF-α levels in serum samples from mice in each group

[0085]

[0086] Note: Compared with the model control group, **p<0.01

[0087] from Figure 6 It can be seen that, 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 bacteria of 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.

[0088] from Figure 7 HE staining results showed that the mammary alveolar cavities of mice in the blank control group were intact, with no obvious inflammatory cell infiltration. Compared with the blank control group, the mammary tissue structure of mice in the model control group was unclear, and the number of inflammatory cells in the alveolar cavities was significantly increased, with scattered hemorrhages visible. Compared with the model control group, the positive drug group and experimental group 1 (live Lactobacillus fermentum MSJK0025 10) showed significantly higher levels of inflammatory cells. 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.

[0089] 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. 9 The 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.

[0090] 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.

[0091] Example 4: Evaluation of the efficacy of fermented Lactobacillus mucinus MSJK0025 in preventing mastitis

[0092] I. Sample Preparation

[0093] Same as Example 3.

[0094] II. Evaluation Process

[0095] 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.

[0096] 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).

[0097] 3. Twenty-four hours after inoculation, mice in each group were sacrificed, dissected, and subjected to histopathological observation (interstitial edema of mammary epithelium and acini, inflammatory infiltration, etc.). Photos were taken and recorded. Results are as follows: Figure 8 As shown. Breast tissue was taken for subsequent pathological HE staining examination, and the results are as follows. Figure 9 As shown.

[0098] 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.

[0099] Table 4: TNF-α levels in serum samples from mice in each group

[0100]

[0101] Note: Compared with the model control group, *p<0.05

[0102] from Figure 8 It can be seen that, compared with the blank control group, the mammary tissue of mice in the positive drug group was dark red, and 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.

[0103] from Figure 9 HE staining results showed that the mammary alveolar cavities of mice in the blank control group were intact, and no obvious inflammatory cell infiltration was observed in 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 in 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 in the mammary alveoli of mice in the positive drug group was significantly reduced.

[0104] 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.

[0105] 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.

[0106] 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. The use of Limosilactobacillus fermentum MSJK0025, derived from breast milk, in the preparation of a pharmaceutical composition for improving breast health; said pharmaceutical composition is used to improve mastitis.

2. The application according to claim 1, characterized in that, The pharmaceutical composition contains live Lactobacillus fermentum MSJK0025.

3. The application according to claim 1 or 2, characterized in that, The pharmaceutical composition is administered to female mammals, including humans, specifically female mammals suffering from mastitis.

4. The application according to claim 1 or 2, characterized in that, The mastitis referred to includes non-lactating mastitis and lactating mastitis.

5. The use of Lactobacillus fermentum MSJK0025 derived from breast milk in the preparation of a probiotic agent for improving breast health; said probiotic agent is used to improve mastitis.

6. The application according to claim 5, characterized in that, The probiotic agent contains live Lactobacillus fermentum MSJK0025.

7. The application according to claim 5 or 6, characterized in that, The probiotic preparation is intended for use by female mammals, including humans, specifically female mammals suffering from mastitis.

8. The application according to claim 5 or 6, characterized in that, The mastitis referred to includes non-lactating mastitis and lactating mastitis.

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