Breast milk-derived limosilactobacillus fermentum MSJK0025 capable of improving intestinal flora condition and use thereof

Abstract

A breast milk-derived Limosilactobacillus fermentum MSJK0025 capable of improving the intestinal flora condition and the use thereof. The breast milk-derived Limosilactobacillus fermentum MSJK0025 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 26426. The breast milk-derived Limosilactobacillus fermentum is a new strain isolated from breast milk of healthy puerperae, and is referred to as Limosilactobacillus fermentum MSJK0025. The strain has a good antibiotic sensitivity and stable inheritance of traits, has a good tolerance to acid, bile salts and artificial gastric / intestinal fluid, and has a fast growth rate and good safety. Furthermore, the strain has a strong intestinal colonization capability and can effectively inhibit intestinal pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans, but has no inhibitory effects on intestinal probiotics such as Lactobacillus and Bifidobacterium.

Description

Breast milk-derived fermented Lactobacillus mucilaginosus MSJK0025 capable of regulating intestinal flora and its application Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a strain of human milk-derived fermentative Lactobacillus mucilaginosus MSJK0025 capable of regulating intestinal flora and its application. Background Art

[0002] A growing body of research indicates that commensal microbiota are inextricably linked to host health and disease. The intestinal microbiota can be considered the body's "microbial organ," regulating the host's metabolic, immune, endocrine, and neurological processes, both local and systemic, through mechanisms such as microbial composition, metabolites, and secondary metabolites, as well as commensal translocation. This influences the risk of obesity, type 2 diabetes, fatty liver disease, cardiovascular disease, autoimmune and inflammatory disorders, psychiatric and neurological diseases, and cancer.

[0003] Breast milk, an essential source of nutrition for infants in their early years, plays a vital role in their growth and development. Breast milk has been shown to contain live bacteria. These probiotic microorganisms form the foundation for infants to initially build their immune system and defend against disease. Through competitive exclusion, they displace pathogens that occupy the infant's intestines and establish colonization, improving the infant's intestinal environment through their probiotic properties.

[0004] Lactobacillus fermentum is a lactic acid-producing microorganism derived from breast milk and widely used in food, medicine, and agriculture. For example, the well-known strain Lactobacillus fermentum CECT5716, produced by the Spanish company BiosearchLife, is a probiotic isolated from breast milk. It can prevent and treat lactational mastitis, relieve symptoms such as breast swelling and pain, and enhance the immune and intestinal health of both mother and baby. This strain is included in the EU Qualification for Safety (QPS) recommended list of biological agents and on the International Dairy Federation's (IDF) "List of Microorganisms with a History of Safe Use in Food." In 2011, Lactobacillus fermentum was added to my country's "List of Bacteria Acceptable for Food Use." Furthermore, this strain has received GRAS (Generally Recognized as Safe) designation from the US Food and Drug Administration and can be used in infant formula. Infant formula containing Lactobacillus fermentum CECT5716 is available in many countries and regions, including Europe and Asia.

[0005] However, the health benefits of probiotics vary depending on the strain, and different strains of the same species may have different effects on the human body. Studies have also confirmed that certain strains of Lactobacillus muciniphila fermentans may have negative effects on the human body, such as reducing the integrity of the intestinal cell barrier. Furthermore, probiotics are generally taken orally, and to exert their beneficial properties, they need to pass the gastrointestinal barrier (particularly gastric acid and bile). Bile is secreted by the liver and enters the duodenum (part of the small intestine) through the gallbladder, where it has a strong killing effect on probiotics. The concentration of bile salts in the duodenum fluctuates between 0.03% and 0.3%, and food remains in the duodenum for approximately 4-6 hours. Under normal circumstances, the pH of gastric juice in fasting humans is between 0.9 and 1.5, and after a meal, it is above 3.5, and gastric emptying time is approximately 3 hours. Existing probiotic strains, including Lactobacillus muciniphila fermentans, often have varying stress resistance, which directly affects the efficacy of probiotics. At the same time, resistance to antibiotics is an important indicator for evaluating the safety of probiotics. Existing probiotic strains often have resistance genes in their genomes due to different isolation sources, making them resistant to antibiotics and posing a safety hazard. There are even some virulence factors in the genomes of existing strains. Although oral acute toxicology tests show safety, there is still the possibility of "gene migration", which poses a potential risk. Finally, probiotics need to colonize in the intestines to exert their efficacy, and the most important factor affecting the intestinal colonization of strains is their tolerance to gastric juice and bile salts. The tolerance of some existing probiotic strains varies, which often leads to their poor intestinal colonization ability. The above shortcomings in existing probiotics have greatly affected and restricted the probiotic effects of probiotics in practical applications.

[0006] Summary of the Invention

[0007] In order to address the shortcomings of the existing technology, the present application provides a breast milk-derived fermented Lactobacillus mucus MSJK0025 that can regulate intestinal flora. This strain was isolated from the breast milk of healthy mothers in Linping District, Hangzhou City, Zhejiang Province in September 2021. It has good tolerance to acid, bile salts, and artificial gastric / intestinal fluid, and has strong intestinal colonization ability. It can effectively inhibit various intestinal pathogens such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans, but has no inhibitory effect on intestinal probiotics such as Lactobacillus and Bifidobacterium. It has broad application prospects in regulating intestinal flora.

[0008] To this end, the first aspect of the present application provides a strain of breast milk-derived fermentative Lactobacillus MSJK0025 that can regulate intestinal flora. The breast milk-derived fermentative Lactobacillus (Limosilactobacillus fermentum) MSJK0025 is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No.26426.

[0009] The inventors of this application obtained a strain that is non-hemolytic and has good acid resistance, resistance to artificial gastric juice, resistance to bile salts and resistance to artificial intestinal fluid by isolating and screening in healthy maternal breast milk. According to the analysis of experimental data such as cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence (sequence number OP861710.1 in GenBank), and with reference to relevant research papers such as Bergey's Manual of Systematic Bacteriology and International Journal of Systematic and Evolutionary Microbiology, it was identified as "fermentative mucus Lactobacillus" and officially named "fermentative mucus Lactobacillus MSJK0025". The strain was deposited in the General Microbiology Center of the China Culture Collection Administration on January 9, 2023, with the deposit number CGMCC No.26426.

[0010] In some embodiments, the nucleotide sequence of 16S rRNA of the human milk-derived Lactobacillus muciniphila MSJK0025 is shown in SEQ ID NO: 1, and the accession number in GeneBank is OP861710.1.

[0011] In the present application, the nucleotide sequence of the 16S rRNA is the gene sequence of the 16S rRNA of the breast milk-derived fermentative Lactobacillus mucilaginosus MSJK0025.

[0012] In some embodiments, the accession number of the complete genome sequence of the human milk-derived Lactobacillus mucofermentans MSJK0025 in GeneBank is CP121468.1.

[0013] In this application, through the whole genome data analysis of the breast milk-derived fermentative Lactobacillus mucinous MSJK0025, the results showed that: no virulence factors were detected, and it was judged that the strain had no pathogenic hazard; no antibiotic resistance genes were detected, it had good antibiotic sensitivity, and the traits could be stably inherited; and the strain could produce extracellular polysaccharides that were beneficial to intestinal colonization, and had a strong intestinal colonization ability, which is the basis for its prebiotic properties. The discovery of this strain is conducive to breaking the monopoly of European and American countries on the Chinese probiotics market.

[0014] The second aspect of the present application provides a use of the breast milk-derived fermented Lactobacillus mucilaginosus MSJK0025 as described in the first aspect of the present application in the preparation of a probiotic composition for inhibiting intestinal pathogens.

[0015] In the present application, the breast milk-derived fermented Lactobacillus mucilaginosus MSJK0025 has a strong colonization ability in the intestine and has a significant inhibitory effect on pathogenic bacteria in the intestine. Therefore, it can be well used in the preparation of a probiotic composition for regulating the balance of intestinal flora.

[0016] In some embodiments, the intestinal pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Candida albicans.

[0017] The inventors of the present application found through inhibition zone experiments that the breast milk-derived fermented Lactobacillus mucinous bacteria MSJK0025 described in the present application has inhibitory effects on intestinal pathogens: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans. The inhibitory effect is ranked as follows: Escherichia coli > Staphylococcus aureus > Salmonella typhimurium > Candida albicans; however, it has no inhibitory effect on probiotics in the intestine such as Lactobacillus and Bifidobacterium.

[0018] In some embodiments, the probiotic composition is an oral formulation.

[0019] In the present application, the breast milk-derived fermented Lactobacillus mucin MSJK0025 has strong acid resistance, artificial gastric juice resistance, artificial intestinal juice resistance and bile salt resistance. Therefore, the probiotic composition including the breast milk-derived fermented Lactobacillus mucin MSJK0025 can be an oral preparation, and the breast milk-derived fermented Lactobacillus mucin MSJK0025 described in the present application can exert its probiotic effect through oral use.

[0020] The third aspect of the present application provides a probiotic composition, which includes the breast milk-derived fermented Lactobacillus mucilaginosus MSJK0025 as described in the first aspect of the present application.

[0021] In the present application, described breast milk source fermentation mucin lactobacillus MSJK0025 can produce the exopolysaccharide that is conducive to intestinal colonization, has stronger intestinal colonization ability, and this is the basis that it plays the effect of regulating intestinal flora.In addition, probiotics will play beneficial life function, must arrive human small intestine and large intestine with survival state, respond to the multiple coercion in digestive tract, and fermentation mucin lactobacillus MSJK0025 in the present application has stronger acid resistance and bile tolerance, can effectively respond to stomach acid environment and bile.That is, breast milk source fermentation mucin lactobacillus MSJK0025 described in the present application can be colonized in intestinal tract by adhesion, thus keeps interacting with host, plays its beneficial life effect, therefore can be used as a kind of probiotic composition with good effect.

[0022] The fourth aspect of the present application provides a pharmaceutical composition for regulating the balance of intestinal flora, wherein the pharmaceutical composition comprises the breast milk-derived fermented Lactobacillus mucosus MSJK0025 as described in the first aspect of the present application.

[0023] In the present application, the breast milk-derived fermented Lactobacillus mucin MSJK0025 can effectively colonize in the intestine, and has a significant inhibitory effect on pathogenic bacteria in the intestine (such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans), but has no inhibitory effect on beneficial bacteria in the intestine (such as Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus acidophilus). Therefore, the pharmaceutical composition including the breast milk-derived fermented Lactobacillus mucin MSJK0025 has a good effect on regulating the balance of gastrointestinal flora.

[0024] In the present application, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. In the present 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 be, for example, magnesium carbonate, lactose, pectin, dextrin, starch, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and cocoa butter. In the present application, the pharmaceutically acceptable carrier may be a solid, semisolid or liquid.

[0025] In a fifth aspect, the present application provides a functional food for regulating the balance of intestinal flora, wherein the functional food comprises the breast milk-derived fermented Lactobacillus mucinous MSJK0025 as described in the first aspect of the present application.

[0026] As mentioned above, the breast milk-derived fermented Lactobacillus mucin MSJK0025 described in the present application can effectively colonize in the intestine, and has a significant inhibitory effect on pathogenic bacteria in the intestine, but has no inhibitory effect on beneficial bacteria in the intestine. Therefore, the breast milk-derived fermented Lactobacillus mucin MSJK0025 described in the present application can also be used in functional foods that have the function of regulating the balance of intestinal flora.

[0027] In the present application, the functional food may be, for example, fermented milk, fermented fruits and vegetables, or enzymes.

[0028] The beneficial technical effect of the present application is: the breast milk source fermentation mucin lactobacillus provided by the present application is a new bacterial strain separated from healthy parturient breast milk, and named as fermentation mucin lactobacillus MSJK0025, it has good antibiotic sensitivity, and character can be stably inherited; better tolerance to acid, bile salts, artificial stomach / intestinal fluid; growth rate is fast, and safety is better (no hemolysis, no virulence factors detected, no antibiotic resistance genes detected). Simultaneously, this bacterial strain can produce the extracellular polysaccharide that is conducive to intestinal colonization, has stronger intestinal colonization ability, and can effectively suppress multiple intestinal pathogens, has the function of excellent regulation intestinal flora balance, and has broad application prospects. In addition, fermentation mucin lactobacillus MSJK0025 described in the present application has excellent probiotic performance, strengthens the development of localized probiotic bacterial strains, and is conducive to breaking the monopoly of countries such as Europe and the United States on Chinese probiotics market. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a plate colony diagram of breast milk samples of various dilution gradients after culture in Example 1.

[0030] FIG2 is a photograph of the streak separation of the MSJK0025 strain on a plate in Example 1.

[0031] FIG3 is a photograph of the colonies of the MSJK0025 strain on a plate in Example 1.

[0032] FIG4 is a Gram staining microscopic photograph of the MSJK0025 strain in Example 1.

[0033] FIG5 is a scanning electron micrograph of the MSJK0025 strain in Example 1.

[0034] FIG6 is a photograph of a plate showing the hemolytic activity of fermented Lactobacillus mucilaginosus MSJK0025 in Example 2.

[0035] FIG7 is a typical graph of fluorescence intensity of zebrafish intestine after treatment with the sample (fermented Lactobacillus mucilaginosus MSJK0025) in Example 6.

[0036] FIG8 shows the fluorescence intensity of zebrafish intestine after being treated with the sample (fermented Lactobacillus mucilaginosus MSJK0025) for different time periods in Example 6.

[0037] FIG9 is a graph showing the antibacterial effects of fermented Lactobacillus mucilaginosus MSJK0025 on Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Candida albicans, Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus acidophilus in Example 7.

[0038] Biological deposit information

[0039] Limosilactobacillus fermentum MSJK0025, the preservation number is CGMCC No. 26426, the preservation unit is the General Microbiology Center of China Culture Collection Administration (abbreviated as: CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), and the preservation time is January 9, 2023. DETAILED DESCRIPTION

[0040] To make this application easier to understand, the following examples will be used to further illustrate this application. These examples 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 through commercial channels or conventional methods.

[0041] Example 1: Isolation, screening and identification of fermentative Lactobacillus mucilaginosus MSJK0025

[0042] 1. Separation and purification

[0043] Since most of the current probiotics are lactic acid bacteria, lactic acid bacteria are the main target strain for isolation and screening. During the growth and metabolism process, lactic acid bacteria will produce organic acids such as lactic acid. If calcium carbonate is added to the MRS medium, a transparent circle - "calcium-soluble circle" - will form around the colony during plate culture, which can be used as an indicator for the initial screening of lactic acid bacteria. The larger the calcium-soluble circle, the stronger the acid production ability. Therefore, colonies with larger calcium-soluble circles are preferentially selected for further streaking and separation. The specific separation and purification methods are as follows:

[0044] Fresh breast milk samples were diluted with sterile saline to 10 -1 , 10 -2 and 10 -3 , were spread on MRS medium containing 0.3% CaCO3, with two replicates for each gradient. The spread plates were then incubated in a 36°C incubator for 48 hours. The colonies on the plates of each dilution gradient after incubation are shown in Figure 1. Single colonies with a distinct transparent zone - the "calcium dissolution zone" that were white or milky white and growing well were selected for Gram staining and microscopic examination. Strains with a rod-shaped morphology and Gram-positive staining were selected and streaked more than 5 times to obtain pure strains. The streak separation photos and colony photos of the MSJK0025 strain are shown in Figures 2 and 3 respectively; the Gram staining microscopy photos and scanning electron micrographs of the MSJK0025 strain are shown in Figures 4 and 5 respectively. As can be seen from Figures 4 and 5, the Gram staining microscopy photos show that the MSJK0025 strain is a Gram-positive bacterium with a rod shape; at the same time, the scanning electron micrographs show that the bacteria are rod-shaped, with a smooth surface, no cilia or flagella, and no spores. Finally, the isolated pure strain was enriched and cultured in a 36°C incubator, and the bacterial solution was mixed with a 50% sterile glycerol aqueous solution in a ratio of 1:1 and stored in an ultra-low temperature refrigerator at -86°C.

[0045] 2. 16S rRNA sequencing screening

[0046] 16S rRNA sequencing was performed on all isolated strains. Comparison revealed that the 16S rRNA gene sequence of strain MSJK0025 had the highest similarity to that of Lactobacillus fermentum (Limosilactobacillus fermentum (CIP 102980)), reaching 100.00%. Therefore, it was preliminarily identified as Lactobacillus fermentum and designated MSJK0025. The 16S rRNA gene sequence of strain MSJK0025 is shown below:

[0047] 3. Identification and preservation of bacterial strains

[0048] To further clarify the taxonomic status of the MSJK0025 strain, identification was conducted from the perspectives of cell morphology, physiological and biochemical characteristics, and molecular biology (commissioned by the Institute of Microbiology, Chinese Academy of Sciences). The results of the cell morphology and physiological and biochemical characteristics of the MSJK0025 strain are shown in Table 1.

[0049] Table 1: Cell morphology and physiological and biochemical characteristics of MSJK0025 strain Note: F1 in the table represents the first generation of MSJK0025 strain, and F5 represents the fifth generation of MSJK0025 strain.

[0050] The cell morphology, physiological and biochemical characteristics of strain MSJK0025 (Table 1), as well as the 16S rRNA gene sequence (GenBank sequence number OP861710.1) of strain MSJK0025, were analyzed. With reference to relevant research papers such as the Bergey's Manual of Systematic Bacteriology and the International Journal of Systematic and Evolutionary Microbiology, the strain was identified as Lactobacillus mucilaginosus and officially named "Lactobacillus mucilaginosus MSJK0025." This strain was deposited on January 9, 2023, at the General Microbiology Center of the China Culture Collection Administration (CMC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 26426.

[0051] Example 2: Hemolytic test of fermented Lactobacillus mucilaginosus MSJK0025

[0052] After activation, fermentation Lactobacillus mucilaginosus MSJK0025 was inoculated on a blood plate, with hemolytic Staphylococcus aureus as a positive control, and anaerobically cultured at 36°C for 48 hours to observe its hemolytic activity. The plate photograph after culture is shown in FIG6 .

[0053] As shown in Figure 6, the positive control (hemolytic Staphylococcus) had colonies growing and hemolysis, while the fermented Lactobacillus mucilaginosus MSJK0025 had colonies growing and no hemolysis. This indicates that the fermented Lactobacillus mucilaginosus MSJK0025 is non-hemolytic and will not cause bacterial hemolytic disease, and is therefore safe.

[0054] Example 3: Antibiotic sensitivity test of fermented Lactobacillus mucilaginosus MSJK0025

[0055] The KB method was used to determine the sensitivity of Lactobacillus fermentum MSJK0025 to 30 common antibiotics. The antibiotic susceptibilities of F1 and F5 generation strains were tested. The results are shown in Table 2.

[0056] Table 2: Antibiotic susceptibility test results of Lactobacillus mucilaginosus MSJK0025 Note: F1 in the table represents the first generation of MSJK0025 strain, and F5 represents the fifth generation of MSJK0025 strain.

[0057] As shown in Table 2, fermentative mucus Lactobacillus MSJK0025 has good antibiotic sensitivity, is not resistant to most antibiotics, and its traits can be stably inherited.

[0058] Example 4: Whole genome sequencing and analysis of fermentative Lactobacillus mucilaginosus MSJK0025

[0059] The whole genome of Lactobacillus fermentum MSJK0025 was sequenced and submitted to Genebank, obtaining the sequence number CP121468.1. The ANI value (average nucleotide identity, ANI) between the whole genome sequence of this strain and the genome of the standard strain of Lactobacillus fermentum was 97.6284%.

[0060] The results of the whole genome data analysis of fermentative Lactobacillus mucilaginosus MSJK0025 are shown in Tables 3-5 respectively.

[0061] Table 3: Genes related to regulating intestinal flora in Lactobacillus mucilaginosus MSJK0025

[0062] Table 4: Intestinal colonization-related genes in the whole genome of Lactobacillus mucilaginosus MSJK0025

[0063] Table 5: Genome structure of Lactobacillus mucilaginosus MSJK0025

[0064] By analyzing the whole genome data of Lactobacillus mucilaginosus MSJK0025 in Table 3-5, the following conclusions were obtained:

[0065] (1) No virulence factors were detected, indicating that the strain had no pathogenicity;

[0066] (2) no antibiotic resistance genes were detected;

[0067] (3) The strain contains exopolysaccharides that are beneficial for intestinal colonization;

[0068] (4) The bacteria have strong acid resistance, adhesion, bile tolerance, competitiveness, and persistence, which are conducive to colonization in the intestine.

[0069] Example 5: In vitro tolerance test of fermented Lactobacillus mucilaginosus MSJK0025

[0070] 1. Experimental plan

[0071] The control groups in the following experiments were all: the well-known commercial strain fermented Lactobacillus mucilaginosus CECT5716; the experimental groups were all: fermented Lactobacillus mucilaginosus MSJK0025.

[0072] Acid resistance test: MRS broth was used as the basal medium. At a 1% inoculum, the strain was inoculated into a control medium (basal medium, pH 6.0), basal MRS medium at pH 2.0, pH 3.0, pH 4.0, and pH 5.0. The culture was incubated at 37°C for 17 hours. A 10-fold serial dilution was performed with sterile saline, and then the colonies were counted. The survival rate was determined using the formula: P = lgP1 / lgP0

[0073] Wherein, P is the logarithmic ratio of viable bacteria of the test strain, %; P0 is the number of viable bacteria in the control group, CFU / mL; P1 is the number of viable bacteria in the experimental group, CFU / mL.

[0074] Bile salt tolerance test: Culture the bacteria in MRS broth for approximately 17 hours; aspirate 1 mL of the bacterial solution and inoculate it into four sterile centrifuge tubes containing 10 mL of MRS broth; prepare a high-concentration bile salt stock solution, filter-sterilize it, and add it to the centrifuge tubes to adjust the bile salt content to 0%, 0.1%, 0.2%, and 0.3%, respectively. Then, incubate at 36°C and determine the number of viable cells at 0, 2, 4, and 6 hours. The survival rate is determined by the formula: P = lgP1 / lgP0

[0075] Where P is the survival rate of the test strain, %; P0 is the number of viable bacteria in the control group, CFU / mL; P1 is the number of viable bacteria in the experimental group, CFU / mL.

[0076] Artificial gastric / intestinal fluid resistance test: Lactobacillus mucosa fermentation MSJK0025 and Lactobacillus mucosa fermentation CECT5716 were cultured in MRS broth medium for about 17 hours. Shake the bacterial solution, take 10 mL and centrifuge at 4000 rpm for 4 minutes, wash the bacterial slurry three times with PBS buffer, and then resuspend in 10 mL of artificial gastric fluid / artificial intestinal fluid, and digest at 37°C. Samples were taken at 0, 1, 2, and 3 hours to determine the number of viable bacteria in the artificial gastric fluid group; samples were taken at 0, 2, 4, and 6 hours to determine the number of viable bacteria in the artificial intestinal fluid group. The survival rate determination formula is: P = lgP1 / lgP0

[0077] Where P is the logarithmic ratio of viable bacteria of the test strain, %; P0 is the number of viable bacteria at 0 hours of digestion, CFU / mL; P1 is the number of viable bacteria at n hours of digestion, CFU / mL.

[0078] 2. Results and Analysis

[0079] (1) Acid tolerance

[0080] Lactic acid bacteria produce large amounts of organic acids during metabolism, resulting in a certain degree of acid resistance, a key factor in their ability to pass the gastric barrier as probiotics. Furthermore, acid resistance is a crucial factor influencing bacterial yield during fermentation. Table 6 shows the survival rates of Lactobacillus mucilaginosus MSJK0025 and CECT5716 cultured at different pH values.

[0081] Table 6: Acid tolerance results of fermentative Lactobacillus mucilaginosus MSJK0025 and CECT5716

[0082] As shown in Table 6, with the increase of pH value, the logarithmic ratio of the number of viable bacteria of strains MSJK0025 and CECT5716 showed an increasing trend, and the increase of the two was relatively close.

[0083] (2) Bile salt tolerance

[0084] Bile is secreted by the liver and enters the duodenum (a part of the small intestine) through the gallbladder, where it has a strong killing effect on probiotics. Bile salt concentrations in the duodenum fluctuate between 0.03% and 0.3%, and food resides in the duodenum for approximately 4-6 hours. The survival rates of Lactobacillus muciflorus fermentans MSJK0025 and CECT5716 at different bile salt concentrations are shown in Table 7.

[0085] Table 7: Bile salt tolerance results of Lactobacillus mucilaginosus MSJK0025 and CECT5716

[0086] As shown in Table 7, the bile salt tolerance of strain MSJK0025 was slightly lower than that of strain CECT5716, but the difference was not significant.

[0087] (3) Tolerance to artificial gastric juice

[0088] Under normal circumstances, the pH of gastric juice in humans is between 0.9 and 1.5 when fasting, and above 3.5 after a meal. Gastric emptying takes approximately 3 hours. The survival rates of Lactobacillus muciflorus MSJK0025 and CECT5716 in artificial gastric juice at different pH values ​​are shown in Table 8.

[0089] Table 8: Tolerance results of fermentative Lactobacillus mucilaginosus MSJK0025 and CECT5716 to artificial gastric juice

[0090] As shown in Table 8, strain MSJK0025 maintained a 21.20% survival rate after 1 hour in artificial gastric fluid at pH 1.5, while strain CECT5716 showed no detectable viable cells. As pH increased, the tolerance of both strains increased and remained relatively consistent. Overall, strain MSJK0025 exhibited slightly better gastric acid resistance than strain CECT5716.

[0091] (4) Tolerance to artificial intestinal fluid

[0092] The intestine is the main site for probiotic colonization in the digestive tract. The survival rates of Lactobacillus muciflorus MSJK0025 and CECT5716 treated in artificial intestinal fluid for different time periods are shown in Table 9.

[0093] Table 9: Tolerance results of fermentative Lactobacillus mucilaginosus MSJK0025 and CECT5716 to artificial intestinal fluid

[0094] As shown in Table 9, the logarithmic ratios of viable bacteria in both strains MSJK0025 and CECT5716 decreased slightly with prolonged digestion time, but the decrease was minimal and the differences were not significant. After 6 h of digestion, the logarithmic ratios of viable bacteria were both around 99%. This data suggests that strain MSJK0025 has a strong ability to survive in artificial intestinal fluid and is similar to that of strain CECT5716.

[0095] 3. Conclusion

[0096] Lactobacillus fermentans CECT5716 (from breast milk) from Biosearch Life, Spain, is a well-known commercial strain. Test data showed that strain MSJK0025's resistance to acid, bile salts, gastric juice, and intestinal fluid was similar to that of CECT5716.

[0097] Example 6: Colonization detection of fermented Lactobacillus mucilaginosus MSJK0025 in the zebrafish intestine

[0098] 1. Determination of intestinal colonization MTC (maximum detectable concentration)

[0099] Wild-type AB zebrafish, 5 days post-fertilization (DPF), were randomly selected and plated in 6-well plates. Thirty zebrafish were treated in each well (experimental group). Samples were administered in water (concentrations shown in Table 10). A normal control group was also established, with a volume of 3 mL per well. After treatment at 28°C for 24 hours, the MTC of the samples on intestinal colonization in normal zebrafish was measured. The results are shown in Table 10.

[0100] Table 10: Experimental results of intestinal colonization concentration of fermented Lactobacillus mucilaginosus MSJK0025 (n=30)

[0101] As shown in Table 10, under the test conditions, the MTC of the intestinal colonization experiment of fermentative Lactobacillus mucilaginosus MSJK0025 was 2.50×10 9 CFU / mL.

[0102] 2. Determination of intestinal colonization time

[0103] Using DIO green fluorescence to label Lactobacillus fermentans MSJK0025 and Lactobacillus fermentans CECT5716, wild-type AB zebrafish 5 days after fertilization were randomly selected and placed in a 6-well plate. Each well (experimental group) was treated with 30 zebrafish. 2.50×10 9 CFU / mL and fermented Lactobacillus mucilaginosus CECT5716 2.50×10 9 CFU / mL, the volume of each well is 3mL. Four biological replicates were set up in parallel. After treating at 28℃ for 24h, the probiotics were removed, and the treatment was continued at 28℃ for 0, 6, 24 and 48h. Ten zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for observation, photography and preservation (as shown in Figure 7). NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, analyze the fluorescence intensity of the zebrafish intestine, and use the statistical analysis results of this indicator to evaluate the intestinal colonization of the samples. The results are shown in Table 11 and Figure 8. The statistical processing results are expressed as mean±SE. SPSS 26.0 software was used for statistical analysis, and p<0.05 indicated that the difference was statistically significant.

[0104] Table 11: Results of colonization experiment of Lactobacillus mucilaginosus MSJK025 in zebrafish intestine (n=10) Note: Compared with fermentation of Lactobacillus mucilaginosus MSJK0025 at 0 h, ***p<0.001; compared with fermentation of Lactobacillus mucilaginosus MSJK0025 at 48 h, # p<0.05; compared with Lactobacillus fermentans CECT5716 0h, $ p<0.05, $$$ p<0.001

[0105] As shown in Table 11 and Figure 8, under the test conditions, both Lactobacillus mucilaginosus MSJK0025 and Lactobacillus mucilaginosus CECT5716 were able to colonize the zebrafish intestine, with effective colonization times of 24 and 6 hours, respectively. There was no difference in fluorescence intensity between the two strains at 0, 6, and 24 hours, but a significant difference was observed at 48 hours.

[0106] Example 7: Antibacterial experiment of fermentation of Lactobacillus mucilaginosus MSJK0025

[0107] Fermented Lactobacillus mucilaginosus MSJK0025 was cultured in MRS liquid medium for approximately 17 hours. Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Candida albicans, Lactobacillus plantarum, Bifidobacterium longum, and Lactobacillus acidophilus were activated and prepared as indicator bacteria. The indicator bacteria suspension was evenly spread on nutrient agar plates. An Oxford cup was placed vertically on the surface of the plate and gently pressed to ensure close contact with the culture medium. 200 μL of the fermented Lactobacillus mucilaginosus MSJK0025 suspension was pipetted into the Oxford cup and incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured. The results are shown in Table 12 and Figure 9.

[0108] Table 12: Antibacterial test results of fermented Lactobacillus mucilaginosus MSJK0025

[0109] As can be seen from Table 12, fermented mucus lactobacillus MSJK0025 has inhibitory effects on pathogenic bacteria in the intestine: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans, and the inhibitory effect is ranked as follows: Escherichia coli > Staphylococcus aureus > Salmonella typhimurium > Candida albicans; while it has no inhibitory effect on probiotics in the intestine: Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus acidophilus.

[0110] Example 8: Regulatory Effect of Fermented Lactobacillus mucilaginosus MSJK0025 on Intestinal Flora

[0111] Thirty healthy mice were randomly divided into two groups. One group was the experimental group (15 mice). They were gavaged with 1.5 mL of culture medium of fermented Lactobacillus mucilaginosus MSJK0025 every day. The number of viable bacteria of fermented Lactobacillus mucilaginosus MSJK0025 in the culture medium was ≥1×10 9 CFU / mL for 5 consecutive days; the other group was the control group, which was fed with 1.5mL of blank MRS culture medium every day for 5 consecutive days. The feces of mice in each group were taken to detect the changes in the intestinal microorganisms of mice, and the test results were expressed as lg10 n The results are shown in Table 13. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 value indicated a statistically significant difference.

[0112] Table 13: Regulatory effects of fermented Lactobacillus mucilaginosus MSJK0025 on intestinal flora: Note: Compared with the control group, **P<0.05

[0113] As shown in Table 13, compared with the control group, gavage feeding of Lactobacillus mucilaginosus MSJK0025 significantly increased the levels of Lactobacillus and Bifidobacterium in the mouse intestines, while reducing the levels of Escherichia coli and Clostridium perfringens to a certain extent. This indicates that Lactobacillus mucilaginosus MSJK0025 has a regulatory effect on the intestinal flora.

[0114] It should be noted that the embodiments described above are only used to explain the present application and do not constitute any limitation to the present application. The present application has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present application may be modified as specified within the scope of the claims of the present application, and the invention may be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein. On the contrary, the present application can be extended to all other methods and applications with the same function.

Claims

1. A breast milk-derived fermented Lactobacillus mucosa MSJK0025 capable of regulating intestinal flora, characterized in that: The breast milk-derived fermented mucus lactobacillus (Limosilactobacillus fermentum) MSJK0025 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No.26426.

2. The breast milk-derived fermented Lactobacillus mucosa according to claim 1, characterized in that: The nucleotide sequence of 16S rRNA of the breast milk-derived fermentative Lactobacillus mucosa MSJK0025 is shown in SEQ ID NO: 1, and its accession number in GeneBank is OP861710.

1.

3. The breast milk-derived fermented Lactobacillus mucosa according to claim 1 or 2, characterized in that: The accession number of the complete genome sequence of the breast milk-derived fermentative Lactobacillus mucosa in GeneBank is CP121468.

1.

4. A use of the breast milk-derived fermented Lactobacillus mucilaginosus MSJK0025 as claimed in any one of claims 1 to 3 in the preparation of a probiotic composition for inhibiting intestinal pathogens.

5. The use according to claim 4, characterized in that: The intestinal pathogenic bacteria include one or more of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Candida albicans.

6. The use according to claim 4, characterized in that: The probiotic composition is an oral preparation.

7. A probiotic composition, characterized in that: The probiotic composition comprises the breast milk-derived fermented Lactobacillus mucosa according to any one of claims 1 to 3.

8. A drug for regulating the balance of intestinal flora, characterized in that: The medicine comprises the breast milk-derived fermented Lactobacillus mucosa as described in any one of claims 1 to 3.

9. A food for regulating the balance of intestinal flora, characterized in that: The food comprises the breast milk-derived fermented Lactobacillus mucosa as claimed in any one of claims 1 to 3.

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