Lactobacillus plantarum zFML004 with antibacterial activity and intestinal microecological regulation function and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a lactobacillus plantarum ZFML004 with antibacterial activity and intestinal microecological regulation function and application thereof. The present application provides a lactobacillus plantarum ZFML004 with a preservation number CCTCC NO: M2025330. The present application also simultaneously provides application of the above-mentioned lactobacillus plantarum ZFML004 in preparation of antibacterial and intestinal microecological regulation drugs; the drugs can relieve pulmonary fibrosis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a lactobacillus plantarum ZFML004 with antibacterial activity and intestinal microecological regulation function and application thereof. BACKGROUND

[0002] The lactobacillus plantarum has the probiotic properties of antibacterial, antioxidant, regulation of intestinal homeostasis, improvement of intestinal inflammation and immune regulation, and has a good promoting effect on human health; it can obtain energy from different sugars, and has high adaptability to various ecological niches such as dairy products, vegetables, wine and gastrointestinal tract of human and animals.

[0003] Lactic acid bacteria can antagonize the growth of pathogenic bacteria and degrade their toxins through the production of antibacterial substances, inhibition of pathogenic bacteria quorum sensing and regulation of immunity, and has inhibitory effect on various foodborne pathogenic bacteria such as salmonella, listeria monocytogenes and escherichia coli. Studies have found that the novel antibacterial peptide KMY15 produced by lactobacillus plantarum FB-2 has good antibacterial effect on gram-positive bacteria (staphylococcus aureus ATCC6538, listeria monocytogenes ATCC19115) and gram-negative bacteria (escherichia coli DH5α, salmonella typhimurium CICI10437). Studies have also shown that lactobacillus plantarum BXM2 exhibits antibacterial activity against oral pathogens (streptococcus mutans, prevotella intermedia) and foodborne pathogens (escherichia coli, staphylococcus aureus, shigella sonnei, salmonella typhimurium, proteus mirabilis), with an average antibacterial zone diameter of 11.00-21.67 mm.

[0004] Intestinal microbiota plays an important role in intestinal homeostasis, body development and resistance to pathogens, and can affect the nutrition, immunity and physiological function of the host, thereby having a significant impact on the overall health of the host. Supplementing certain probiotics in the body can not only promote the health of the body, but also improve the regulation of intestinal homeostasis in a direct or indirect manner. In addition, studies have found that intestinal flora imbalance can induce systemic inflammatory response, and further trigger the fibrosis process in the lungs. Certain flora (such as lactobacillus and bifidobacterium) can improve intestinal barrier function and reduce inflammatory response, thereby inhibiting the progression of pulmonary fibrosis. Therefore, it is of great value to develop functional bacterial agents with antibacterial function and intestinal flora regulation function.

[0005] CN11686454A invention “Lactobacillus plantarum with antioxidant and antibacterial function and application thereof” discloses that the lactobacillus plantarum WW has strong antibacterial effect on salmonella, enterobacter sakazakii and shigella, with an antibacterial circle diameter of more than 18 mm.

[0006] Invention CN117917475A, entitled "A plant lactobacillus p16 for regulating intestinal flora and its application, products and methods," discloses that: Plant lactobacillus ( Lactobacillus plantarum P16 can significantly inhibit the proliferation of pathogenic bacteria, can smoothly pass through the gastrointestinal tract to exert probiotic effects and improve health. Furthermore, this strain is sensitive to common antibiotics and has high safety, showing great potential in products that regulate intestinal flora balance. However, it was not disclosed that it has the function of alleviating pulmonary fibrosis.

[0007] Invention CN115500515A, entitled "Application of Lactobacillus plantarum in regulating intestinal flora," discloses that Lactobacillus plantarum (… Lactobacillus plantarum 9010 can promote the growth of beneficial microorganisms such as lactic acid bacteria, inhibit pathogenic bacteria, and regulate intestinal flora, thus possessing excellent application prospects, especially given the increasing importance and growing recognition of the impact of intestinal flora on human health. However, it was not stated that it has the function of alleviating pulmonary fibrosis.

[0008] The invention CN119320723A, entitled "A strain of fermenting lactobacillus and its compound preparations and applications," states that the most preferred *Lactobacillus plantarum* is *Lactobacillus plantarum*. Lactobacillus plantarum HXJS4-1 can be used to prepare IL-11 inhibitors, which can be used to prevent or treat conditions such as cardiovascular fibrosis and pulmonary fibrosis. However, it is not disclosed that it has gut microbiota regulation function.

[0009] Invention CN119736209A, entitled "An Edible Lactobacillus for Alleviating Pulmonary Fibrosis and Its Application," discloses that: *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ZYHL3 can significantly reduce lung inflammation and pulmonary fibrosis in mice and improve disease prognosis by regulating gut microbiota diversity and metabolic profile, thus providing a new approach for the prevention and treatment of pulmonary fibrosis and other idiopathic pulmonary fibrosis.

[0010] The invention CN120037267A, "Application of Lactobacillus paracasei L9 in the Preparation of Agents to Improve Pulmonary Fibrosis in Aging Mice," discloses that Lactobacillus paracasei (… Lactobacillus paracasei The application of L9 in alleviating age-related pulmonary fibrosis and further elucidating the important role of gut microbiota and its metabolites in the disease.

[0011] The invention CN108495643A, entitled "Composition and Method of Use of Novel Lactobacillus Fermentation Strains," discloses that: Lactobacillus fermentation (… Lactobacillus fermentum Examples of inflammatory symptoms treated with NRRL B-67059 include lung inflammation (such as asthma, adult respiratory distress syndrome, bronchitis, lung inflammation, pulmonary fibrosis, and cystic fibrosis); however, it is not stated that it has gut microbiota regulation function. Summary of the Invention

[0012] The problem to be solved by this invention is to provide a *Lactobacillus plantarum* with antibacterial activity and intestinal microecological regulation function. Lactobacillus plantarum ZFML004 and its applications.

[0013] To address the above problems, this invention provides a *Lactobacillus plantarum* ZFML004, the taxonomic name of which is... Lactobacillus plantarum The accession number is CCTCC NO: M2025330.

[0014] The present invention also provides the application of the above-mentioned Lactobacillus plantarum ZFML004 in the preparation of antibacterial and intestinal microecological regulation (regulation of intestinal microecology) drugs.

[0015] As an improvement to the application of the present invention: the drug can alleviate pulmonary fibrosis.

[0016] As a further improvement to the application of the present invention, the antibacterial activity is against the following pathogenic bacteria: Escherichia coli DH5α, Salmonella enteritidis ATCC 14028, Salmonella typhimurium CMCC 50015, Staphylococcus aureus ATCC 25923, and Staphylococcus warwickii.

[0017] As a further improvement to the application of the present invention, the relief of pulmonary fibrosis is to improve lung inflammation in animals / humans with pulmonary fibrosis.

[0018] As a further improvement to the application of the present invention, the intestinal microecological regulation includes increasing the abundance of Bacillota and Thermodesulfobacteriota.

[0019] As a further improvement to the application of the present invention, the intestinal microecological regulation includes increasing the number of Trichophyton spp. ( Lachnospiraceae _NK4A136_group), Androgen spp. ( Adlercreutzia The relative abundance of ).

[0020] As a further improvement to the application of the present invention, Lactobacillus plantarum ZFML004 has adhesive properties and antioxidant capacity.

[0021] The present invention has the following beneficial effects:

[0022] 1) Pioneering the Androgen synergistic mechanism of the gut-lung axis

[0023] Lactobacillus plantarum ZFML004 uniquely induces Androgen spp. in pulmonary fibrosis intervention. Adlercreutzia The ability to accumulate Nrf2 in lung tissue directly inhibits the fibrosis process by activating the Nrf2 pathway.

[0024] It is well known in the industry that the metabolite of Andrade's bacteria (Enterococcus avium) inhibits ACE activity to reduce blood pressure and inhibit vascular smooth muscle proliferation and prevent atherosclerosis; therefore, it can be reasonably deduced that Lactobacillus plantarum ZFML004 can be used to treat the above-mentioned cardiovascular diseases. Adlercreutzia

[0025] 2) Bacteriostatic activity leap

[0026] The diameter of the bacteriostatic circle of Salmonella typhimurium CMCC 50015 is 26.13±1.00 mm, which is more than 45% higher than that of the known bacteriostatic strain Lactobacillus plantarum WW (CN11686454A, bacteriostatic circle >18 mm) (calculated based on the minimum threshold of 18 mm).

[0027] 3) Pathogenic antagonistic efficiency breakthrough

[0028] The co-aggregation rate of Salmonella enteritidis ATCC 14028 is as high as 64%, which is an absolute increase of 24.76 percentage points compared to the prior art (Lactobacillus plantarum 19-4 in CN118792209B has a co-aggregation rate of 39.24%), and a relative increase of more than 63%; at the same time, it also achieves strong co-aggregation of a wide range of pathogens such as Escherichia coli DH5α, Staphylococcus aureus ATCC 25923, etc., establishing a colonization-defense synergistic effect (self-aggregation / hydrophobicity≥80%). BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The colony map of Lactobacillus plantarum ZFML004.

[0030] Figure 2 The adhesion result map of Lactobacillus plantarum ZFML004.

[0031] Figure 2 In the middle:

[0032] A is the self-aggregation rate;

[0033] B~D are the hydrophobic rates of Lactobacillus plantarum ZFML004 to chloroform, xylene, and ethyl acetate, respectively.

[0034] E-H are the co-aggregation rates of Lactobacillus plantarum ZFML004 with Salmonella enteritidis ATCC 14028, Staphylococcus warneri, Escherichia coli DH5α, and Staphylococcus aureus ATCC 25923, respectively.

[0035] Figure 3 The antioxidant capacity result map of Lactobacillus plantarum ZFML004.

[0036] Figure 3 In the middle:

[0037] ​A is the result graph of the DPPH free radical scavenging rate of Lactobacillus plantarum ZFML004;

[0038] B is the result graph of the hydroxyl radical scavenging rate of Lactobacillus plantarum ZFML004;

[0039] C is the result graph of the ABTS free radical scavenging rate of Lactobacillus plantarum ZFML004.

[0040] Figure 4 is the result graph of HE staining of lung tissue of a pulmonary fibrosis mouse.

[0041] Figure 5 is the result graph of Masson staining of lung tissue of a pulmonary fibrosis mouse.

[0042] Figure 6 is the result of PCoA analysis.

[0043] Figure 7 is the result of analysis of flora at the level of door.

[0044] Figure 8 is the result of analysis of flora at the level of genus.

[0045] Figure 9 is the LEfSe evolutionary branch graph in the result of Lefse analysis.

[0046] Figure 10 is the LDA discriminant column chart in the result of Lefse analysis. DETAILED DESCRIPTION

[0047] The application will be further described below in conjunction with specific examples, but it should be noted that the protection scope of the application is not limited to this: in the following examples, if not specifically stated, the experimental operations adopted are conventional methods in the art; the equipment used is conventional equipment in the art; the reagents and materials involved, if not specified, are conventional products on the market or obtained by standard methods.

[0048] The MRS solid culture medium used in the application is a conventional MRS solid culture medium, and the components include 10 g of proteose peptone, 5 g of yeast extract powder, 20 g of glucose, 5 g of sodium acetate, 2 g of citric acid diammonium, 2 g of potassium phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate, 10 g of beef extract, 1 mL of Tween 80, 2% of agar, and pH 6.2-6.4.

[0049] The MRS liquid culture medium does not add 2% of agar.

[0050] Example 1, strain isolation and identification:

[0051] Strain isolation: 0.1 g of pickled cucumber (from a farm in Hangzhou, Zhejiang Province) was crushed and placed in a 10 mL centrifuge tube, 1 mL of normal saline was added to prepare a mixed sample liquid and recorded as the initial concentration, and the mixed sample liquid was placed on an ultraclean workbench and subjected to 10-fold serial gradient dilution. 100 μL of the mixed liquid at different gradients was taken and plated on MRS solid medium containing 1% calcium carbonate, and incubated in a 37℃ incubator for 48 h.

[0052] Strain identification: single colonies with typical lactic acid bacteria colony morphology were selected for further streaking and purification, and after microscopic examination and identification, strain ZFML004 was obtained, which was identified as Lactobacillus plantarum Lactobacillus plantarum ); can be inoculated in liquid medium to obtain pure culture and prepare preservation tubes (stored at -80℃).

[0053] The colony map of Lactobacillus plantarum Lactobacillus plantarum ) ZFML004 is shown in Figure 1 .

[0054] Its biological properties are as follows:

[0055] (1) Morphological characteristics: The growth morphology on MRS solid medium is a milky white colony, opaque, round, smooth surface, neat edge, central convex. The bacterial body shows uniform blue-violet after Gram staining, and belongs to Gram-positive bacteria.

[0056] (2) Culture characteristics: The optimum growth temperature is 37℃, and it is facultative anaerobic.

[0057] (3) Biological identification: identification is carried out by 16S rDNA PCR amplification, and the obtained results are subjected to homology comparison analysis in the GenBank database of NCBI, and the results show that the strain is Lactobacillus plantarum Lactobacillus plantarum ). The gene sequence is shown as SEQ ID No:1.

[0058] The preservation information of Lactobacillus plantarum Lactobacillus plantarum ) ZFML004 of the application is as follows:

[0059] Preservation name: Lactobacillus plantarum ZFML004 Lactobacillus plantarum ZFML004, preservation unit: China Center for Type Culture Collection, preservation address: Wuhan University, Wuhan, China, preservation number: CCTCC NO: M2025330, preservation time: February 28, 2025.

[0060] Example 2, antibacterial properties of Lactobacillus plantarum ZFML004

[0061] Preparation of indicator bacteria suspension: Escherichia coli DH5α, Salmonella enteritidis ATCC 14028, Salmonella typhimurium CMCC 50015, Staphylococcus warneri and Staphylococcus aureus ATCC 25923 were inoculated into LB liquid medium, respectively, and activated for 2 generations, and then stored at 4℃ for standby.

[0062] Preparation of Lactobacillus plantarum ZFML004 supernatant:

[0063] The bacterial liquid in the preservation tube obtained in Example 1 was dipped with an inoculation loop, streaked on MRS solid medium, and cultured at 37℃ for 24 h to form single colonies. The single colonies were picked into 5 mL of MRS liquid medium and cultured at 37℃ in a shaking bed for 12 h, which was the first generation; in the following, the inoculation was performed in MRS liquid medium at a inoculation amount of 1% (v / v) and cultured at 37℃ for 12 h, which was the second generation. The bacterial liquid activated for 2 times was centrifuged at 6000 r / min for 15 min at 4℃, filtered through a 0.22 μm filter membrane, and the Lactobacillus plantarum ZFML004 supernatant was obtained and stored at 4℃ for standby.

[0064] Bacteriostatic circle experiment: Escherichia coli DH5α, Salmonella enteritidis ATCC 14028, Salmonella typhimurium CMCC 50015, Staphylococcus warneri and Staphylococcus aureus ATCC 25923 were used as indicator bacteria, and the Oxford cup double-layer plate method was adopted, 100 μL of Lactobacillus plantarum ZFML004 supernatant was added to each hole, diffused at room temperature for 4 h, cultured in an incubator at 37℃ for 24 h, and the diameter of the bacteriostatic circle was measured to determine the bacteriostatic activity.

[0065] The results are shown in Table 1. The supernatant of the strain Lactobacillus plantarum ZFML004 of the application has bacteriostatic effect on both gram-positive bacteria and gram-negative bacteria, and obvious bacteriostatic circles can be observed, among which the bacteriostatic effect on Salmonella typhimurium CMCC 50015 is the most significant, and the diameter of the bacteriostatic circle reaches 26.13±1.00 mm. It is shown that the metabolites secreted during the culture of Lactobacillus plantarum ZFML004 have bacteriostatic effect on pathogenic bacteria, and the bacteriostatic activity has certain broad spectrum.

[0066] Table 1 Bacteriostatic properties of Lactobacillus plantarum ZFML004

[0067]

[0068] Example 3, adhesion of Lactobacillus plantarum ZFML004

[0069] Self-aggregation ability: Lactobacillus plantarum ZFML004 activated for 2 generations (the activation method is referred to Example 2) was centrifuged at 8000 ×g for 5 min at 4℃, the supernatant was discarded, and the bacteria were washed twice with PBS buffer (pH 7.4) and resuspended, and the absorbance (OD600 ) 0.5, and then the adjusted bacterial liquid was placed in a 37°C incubator for 24 h, and the upper bacterial liquid was collected to determine the absorbance, denoted as A t , with PBS as a blank control, denoted as A0, wherein LGG was used as a control strain. The calculation formula is as follows: self-aggregation rate (%) = (1-A t / A0) x 100.

[0070] Co-aggregation capacity: Lactobacillus plantarum ZFML004 and pathogenic strains (Staphylococcus aureus ATCC 25923, Escherichia coli DH5a, Salmonella enteritidis ATCC 14028, Staphylococcus warneri) were prepared into bacterial liquid with an absorbance (OD 600 ) of 0.5 (the bacterial suspension preparation method can refer to the above "self-aggregation capacity"), denoted as A1 and A2 (i.e., A1 and A2 are both 0.5), and then the pathogenic strains and the target strain were mixed in a 37°C incubator at a volume ratio of 1:1 for 24 h, and the upper bacterial liquid was collected to determine the absorbance, denoted as A 混 , wherein LGG was used as a control strain. The calculation formula is as follows: co-aggregation rate (%) = [(A1+A2) / 2-2A 混 / (A1+A2)]x100.

[0071] Surface hydrophobicity: Lactobacillus plantarum ZFML004 was prepared into a bacterial liquid with an absorbance (OD 600 ) of 0.5 (the bacterial suspension preparation method can refer to the above "self-aggregation capacity"), denoted as A0. 1 mL of xylene, chloroform, and ethyl acetate was added to 3 mL of bacterial suspension, respectively, and vortexed for 2 min to mix the two-phase system. Incubation at 37°C for 30 min, and the absorbance of the aqueous phase at 600 nm was determined, denoted as A1, wherein LGG was used as a control strain. The calculation formula is as follows: hydrophobicity rate (%) = (1-A1 / A0) x 100.

[0072] The results are as follows:

[0073] The self-aggregation results of the strains are shown in A of Figure 2 , and the self-aggregation rate of Lactobacillus plantarum ZFML004 is 78%, which is increased by 77.27% compared with the control strain LGG, so Lactobacillus plantarum ZFML004 shows high self-aggregation capacity.

[0074] The hydrophobicity results show (B-D of Figure 2 , the hydrophobicity rate of Lactobacillus plantarum ZFML004 to xylene, chloroform, and ethyl acetate is significantly higher than that of the control strain LGG, reaching more than 80%.

[0075] The co-aggregation capacity results of the strains show (C of Figure 2Lactobacillus plantarum ZFML004 had co-aggregation ability to Staphylococcus aureus ATCC 25923, Escherichia coli DH5a, Salmonella enteritidis ATCC 14028, Staphylococcus warneri, etc. pathogenic bacteria, and there were differences in the co-aggregation ability to each pathogenic bacteria, and the co-aggregation rate to Salmonella enteritidis ATCC 14028 was as high as 64%. In summary, Lactobacillus plantarum ZFML004 has strong self-aggregation ability and co-aggregation ability with pathogenic bacteria, which can better play the role of probiotics; and because it shows strong hydrophobicity, it is helpful for the strain to play a role in the formation of biofilm, intercellular aggregation and interaction with other substances.

[0076] Example 4, antioxidant capacity of Lactobacillus plantarum ZFML004

[0077] Preparation of Lactobacillus plantarum ZFML004 bacterial suspension: the activated two generations (activation method refers to Example 2) of Lactobacillus plantarum ZFML004 were centrifuged at 8000 x g for 2 min, the supernatant was discarded, the collected bacterial cells were washed three times with deionized water, and the bacterial liquid concentration was adjusted to 1.0 x 10 9 CFU / mL, 4°C for standby.

[0078] DPPH free radical scavenging rate determination: the above prepared Lactobacillus plantarum ZFML004 bacterial suspension (as sample) was mixed with an equal volume of 0.2 mmol / L DPPH-ethanol solution, and then uniformly placed at 37°C in the dark for 30 min. After centrifugation at 6000 x g for 10 min, the supernatant was measured at a wavelength of 517 nm, and the absorbance was recorded as A 样品 , the control group used an equal volume of deionized water instead of the sample, and the absorbance was recorded as A 对照 . The blank group used an equal volume of anhydrous ethanol instead of the DPPH free radical solution (i.e., 0.2 mmol / L DPPH-ethanol solution), and the absorbance was recorded as A 空白 . The 0.5 mg / mL VC solution was used as a positive control. The calculation formula was as follows: DPPH free radical scavenging rate (%) = [1- (A 样品 -A 空白 ) / A 对照 ] x 100.

[0079] Hydroxyl radical scavenging rate determination: 1 mL of salicylic acid (5 mM) and 1 mL of FeSO4 (5 mM) were mixed, and then mixed with 1 mL of Lactobacillus plantarum ZFML004 bacterial suspension (as sample), 6 mL of deionized water was added, and then 1 mL of H2O2 (3 mM) was added to a volume of 10 mL. Incubate at 37°C for 30 min, take 1 mL from each tube, centrifuge at 6000 x g for 10 min, and measure the supernatant at 510 nm, and record the absorbance as A 样品The control group was replaced with an equal volume of deionized water, denoted as A. 对照 A 0.5 mg / mL vitamin C solution was used as a positive control. The calculation formula is as follows: Hydroxyl radical scavenging rate (%) = [(A 对照 -A 样品 ) / A 对照 ]×100.

[0080] ABTS free radical scavenging rate determination: Equal volumes of 7.4 mmol / L ABTS and 2.6 mmol / L K₂S₂O₈ were mixed and incubated at room temperature in the dark for 12 h. The mixture was then diluted with phosphate buffer (pH 7.4) to an OD₂ concentration of 100%. 734 =0.7±0.02, which is ABTS + Working solution. Take 0.8 mL of ABTS. + Mix the working solution with 0.2 mL of 95% ethanol, let stand for 6 min, and record the absorbance at 734 nm as A0. Take 0.8 mL of ABTS + The working solution and 0.2 mL of *Lactobacillus plantarum* ZFML004 suspension were mixed thoroughly and allowed to stand for 6 min. The absorbance measured at 734 nm was recorded as A. The calculation formula is as follows: ABTS free radical scavenging rate (%) = [(A0-A) / A0] × 100

[0081] The results are as follows Figure 3 As shown, Lactobacillus plantarum ZFML004 has certain antioxidant capacity, with DPPH free radical scavenging rate, hydroxyl free radical scavenging rate and ABTS free radical scavenging rate of 22.62%, 23.93% and 30.84% ​​respectively.

[0082] Example 5: Regulatory effect of Lactobacillus plantarum ZFML004 on bleomycin-induced pulmonary fibrosis in mice.

[0083] Animal experimental and feeding conditions: Thirty male C57BL / 6J mice, approximately 6-8 weeks old and weighing 18-20 g, were provided by the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences (ethics code AP2024-12-0395). Standard conditions (temperature 23±2℃, humidity 55±5%, and a 12-hour light / dark cycle) were maintained before the experiment, and the animals were allowed free access to food and water for 7 days to allow for acclimatization.

[0084] Preparation of experimental reagents:

[0085] 0.9% physiological saline: Accurately weigh 4.5 g NaCl and measure 500 mL of distilled water to prepare 500 mL of 0.9% physiological saline.

[0086] 2.5% tribromoethanol solution: accurately weigh 1 g of tribromoethanol, dissolve in 1 mL of t-amyl alcohol, mix uniformly, then dilute to 40 mL with sterile normal saline, filter with 0.22 μm filter membrane, and store at 4°C for standby use.

[0087] 3 U / kg bleomycin sulfate solution: 2.4 mg of bleomycin sulfate is added to 4 mL of sterile normal saline, mixed uniformly until completely dissolved, filtered with 0.22 μm filter membrane, and stored for standby use.

[0088] Establishment of mouse pulmonary fibrosis model:

[0089] The pulmonary fibrosis mouse model was established by tracheal instillation. First, the mouse was anesthetized by intraperitoneal injection of 2.5% tribromoethanol solution (about 0.28 mL), and after about 1 min, the mouse was completely anesthetized and had stable breathing. The mouse was hung on a small animal operating table to naturally open its mouth, then the mouse's tongue was gently pulled out with forceps until the trachea was naturally open, and a 3 U / kg bleomycin sulfate solution (about 0.025 mL) was injected into the respiratory tract of the mouse using a pipette. Then the mouse was hung and gently shaken to evenly distribute the bleomycin solution in the lungs. During the whole process, the trachea of the mouse was kept open, and after the mouse's coughing sound became regular and stable, it was placed back in the mouse cage to wake up. The mouse was placed in a posture to ensure unobstructed breathing, and the mouse's condition was observed at any time.

[0090] Experimental grouping design: After adaptive feeding, randomly number into 3 groups, 10 in each group: normal control group (Ctrl group), modeling group (BLM group), and lactobacillus plantarum group (ZFML004 group). The specific grouping and feeding methods are as follows:

[0091] Normal control group (Ctrl group): after modeling with 3 U / kg bleomycin sulfate, continuously gavage with normal saline for 3 weeks, the gavage amount is 0.2 mL / 20 g;

[0092] Modeling group (BLM group): after modeling with 3 U / kg bleomycin sulfate, continuously gavage with normal saline for 3 weeks, the gavage amount is 0.2 mL / 20 g;

[0093] Lactobacillus plantarum group (ZFML004 group): after modeling with 3 U / kg bleomycin sulfate, continuously gavage with lactobacillus plantarum ZFML004 bacterial suspension with a concentration of 1×10 9 CFU / mL for 3 weeks, the gavage amount is 0.2 mL / 20 g.

[0094] After 3 weeks of continuous gavage, the mice were fasted for 12 h, anesthetized with a 2.5% solution of tribromoethanol (0.14 mL / 10 g), and the mouse samples (mouse lung tissue, colon contents, etc.) were stored at -80°C for subsequent determination.

[0095] After the lung tissue samples were obtained from the mice, they were first fixed in a 4% paraformaldehyde solution for 24-36 h to ensure the stability of the tissue structure. After that, the samples were dried and immersed in wax, and embedded in paraffin to facilitate subsequent slice preparation. The prepared paraffin-embedded samples were then precisely cut into 4 μm thick slices for further histological staining analysis. The slices were subjected to HE and Masson trichrome staining to evaluate the microscopic structure and pathological changes of the lung tissue. After staining, detailed microscopic examination was performed using a microscope, and corresponding image analysis was performed to obtain histological information. Figure 4 and Figure 5 The lung tissue staining results all showed that the intervention of ZFML004 greatly improved the lung inflammation of the lung fibrosis mice and alleviated the lung fibrosis process.

[0096] The HE staining results showed that the lung tissue of the Ctrl group had no obvious alveolar wall thickening, a small amount of alveolar mild expansion (green arrow), and no obvious fibrosis and inflammatory infiltration. The lung tissue of the BLM group showed a large range of alveolar wall moderate thickening, fibrous tissue hyperplasia, and a large number of inflammatory cell infiltration (green arrow), alveolar cavity stenosis, and a large number of alveolar moderate expansion (yellow arrow) around the periphery, and a large number of mononuclear cells in the alveolar cavity (blue arrow). The lung tissue of the ZFML004 group showed local alveolar wall mild thickening, fibrous tissue hyperplasia, and a small amount of inflammatory cell infiltration (green arrow) at the edge of the tissue, a small amount of alveolar mild expansion (yellow arrow) around the periphery, and a small amount of mononuclear cell increase in the alveolar cavity (blue arrow). The HE staining results of the mouse lung tissue were scored using the Szapiel scoring standard and the Ashcroft scoring standard, as shown in Tables 2 and 3, respectively, and the results are shown in Table 4:

[0097] Table 2, Szapiel Scoring Standard

[0098]

[0099] Table 3, Ashcroft Scoring Standard Table

[0100]

[0101] Table 4

[0102]

[0103] Masson staining results showed that in the lung tissue of the Ctrl group, collagen fibers were blue arrows, and a small amount of intrinsic connective tissue (green arrow) was visible around the bronchus and blood vessels, and no obvious fibrosis was observed. In the lung tissue of the BLM group, collagen fibers were blue, and a large range of fibrosis was observed under the microscope, and the alveolar wall was moderately thickened (green arrow). In the lung tissue of the ZFML004 group, collagen fibers were blue, and partial fibrosis was observed at the edge of the tissue under the microscope, and the alveolar wall was slightly thickened (green arrow). The Masson quantitative results are shown in Table 5:

[0104] Table 5

[0105]

[0106] Therefore, in terms of ZFML004 improving lung inflammation in mice with pulmonary fibrosis, the BLM group scored 2 in the alveolitis score in the HE tissue score, while the score was 1 after ZFML004 intervention; in the fibrosis degree score, the BLM group scored 5 in the alveolitis score, while the score was 1 after ZFML004 intervention. The Masson quantitative results showed that the positive area accounted for 29.1376%, while the positive area accounted for 7.7686% after ZFML004 intervention, which decreased by 73.38%. The above results show that ZFML004 plays a significant role in alleviating the progression of pulmonary fibrosis in mice.

[0107] Example 6, Regulation of Lactobacillus plantarum ZFML004 on the intestinal microecology of mice, 16S rDNA analysis was performed on the colon contents of the mice obtained in Example 5:

[0108] Sample DNA extraction: According to the E.Z.N.A.® soil DNA kit instructions, the total genomic DNA of the microbial community was extracted, and the quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis. The DNA concentration and purity were determined using NanoDrop2000.

[0109] PCR amplification and sequencing library construction: Using the extracted DNA as a template, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR using an upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3', SEQ ID NO: 2) and a downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO: 3) carrying a Barcode sequence. The PCR reaction system was as follows: 5x TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, upstream primer (5 uM) 0.8 μL, downstream primer (5 uM) 0.8 μL, TransStart FastPfu DNA polymerase 0.4 μL, template DNA 10 ng, and the rest was supplemented to 20 μL. The amplification program was as follows: 95 °C pre-denaturation for 3 min, 27 cycles of 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s, then 72 °C stable extension for 10 min, and finally 4 °C storage. The PCR product was recovered using a 2% agarose gel, purified using a DNA gel recovery and purification kit, and detected and quantified using Qubit 4.0 (Thermo Fisher Scientific, USA). The purified PCR product was used to construct a library using a NEXTFLEX Rapid DNA-Seq Kit, and sequencing was performed using an Illumina Nextseq2000 platform (Shanghai Meiji Biopharmaceutical Technology Co., Ltd.).

[0110] High-throughput sequencing data analysis: The double-end raw sequencing sequences were quality controlled using fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software, and FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software was used for splicing. Using UPARSE v7.1 software (http: / / drive5.com / uparse / ), the quality-controlled spliced sequences were classified and operated according to 97% similarity OTU (Operational taxonomic unit) clustering and removing chimeras. RDP classifier (http: / / rdp.cme.msu.edu / , version 2.11) was used to align the Silva 16S rRNA gene database (v138) for OTU taxonomic annotation, with a confidence threshold of 70%, and the community composition of each sample was calculated at different species classification levels. PICRUSt2 (version 2.2.0) software was used for 16S functional prediction analysis.

[0111] To further analyze the regulatory effect of Lactobacillus plantarum ZFML004 on the intestinal microecology of mice, the Beta diversity results were analyzed, and it was found that Figure 6 The microbial community structure of each group was significantly different, and the BLM group sample was close to the right side of the PC1 axis, far from the Ctrl group, indicating that the intestinal flora of the mice induced by BLM changed significantly, and the microbial community structure showed high heterogeneity. The ZFML004 group (Lactobacillus plantarum ZFML004) was between the Ctrl group and the BLM group, and was closer to the Ctrl group, indicating that the intake of Lactobacillus plantarum ZFML004 had a restoring effect on the intestinal microbial flora of mice induced by BLM. Lactobacillus plantarum Figure 7 At the phylum level, it was found that (Bacteroidota) and (Bacillota) were the main phyla detected, and their relative abundance accounted for more than 60% of the total microbial community. Compared with the BLM group, the relative abundance of (Bacillota), (Thermodesulfobacteriota), etc. increased after the intervention of Lactobacillus plantarum ZFML004. Figure 8 At the genus level, it was found that (Lactobacillus) and (Bifidobacterium) were the main genera detected, and their relative abundance accounted for more than 60% of the total microbial community. Compared with the BLM group, the relative abundance of (Lactobacillus) and (Bifidobacterium) increased after the intervention of Lactobacillus plantarum ZFML004. Lachnospiraceae ​The relative abundance of *Lactobacillus plantarum* (NK4A136 group) was significantly increased, and it has been reported to have potential anti-colitis activity as a potentially beneficial bacterium. Compared with the BLM group, *Lactobacillus plantarum* ZFML004 intervention significantly increased the abundance of *Andekella spp.* (NK4A136 group). Adlercreutzia The abundance was significantly upregulated. It should be noted that this genus has been shown to improve the intestinal microecological environment in mice by metabolizing isoflavones to exert anti-inflammatory effects. Furthermore, studies have found that *Androgen Androgen* has a direct protective effect on the cardiovascular system; its metabolite equol can inhibit angiotensin-converting enzyme (ACE) activity, reduce vascular resistance, and thus lower blood pressure. In addition, studies have found that *Androgen Androgen* can reduce plaque lipid cores by inhibiting vascular smooth muscle proliferation and foam cell formation, thereby combating atherosclerosis.

[0112] In addition, studies have found that the genus *Alternaria* (…) Alistipes ) has been shown to be closely related to the activation of the PI3K / Akt pathway, Prevotella NK3B31 group ( Prevotellaceae_NK3B31_group The IL-17R signaling pathway is associated with an increased pathogenesis of fibrosis. In summary, the intervention of Lactobacillus plantarum ZFML004 can regulate gut microbiota-related flora and thus improve pulmonary fibrosis in mice through the gut-lung axis.

[0113] To more intuitively reflect the specific differences in gut microbiota among the groups, LefSe analysis was performed. Figure 9 , Figure 10), linear discriminant analysis (LDA) threshold set as LDA≥4, the results showed that there were great differences in intestinal flora among the three groups of mice. In the BLM group, Erysipelotrichales, Erysipelotrichaceae and Dubosiella were dominant. Bacteria in Erysipelotrichales and Erysipelotrichaceae were involved in various biochemical processes through their metabolic activities, including the production of short-chain fatty acids. In addition, they also played an important role in the stability and diversity of intestinal microbial community, which was of great significance to the health of the host. In the Ctrl group, Clostridia, Bacillota, Lachnospiraceae, Lachnospirales, Faecalibaculum and Lachnospiraceae NK4A136 group were dominant. Clostridia could promote intestinal barrier repair and immune regulation. Bacillota was the dominant group of Firmicutes, which contained a large number of short-chain fatty acid (SCFA)-producing bacteria, maintained the acidic environment of the intestine, and inhibited the colonization of pathogens. Lachnospiraceae dominated butyrate production and anti-inflammation, and enhanced barrier function. In the BLM group, Erysipelotrichales, Erysipelotrichaceae and Dubosiella were dominant. Studies have found that Erysipelotrichales contains various opportunistic pathogens (such as Erysipelotrichales), promotes inflammation, and is associated with metabolic disorders. In the Lactobacillus plantarum ZFML004 group, Bacteroidia, Bacteroidota and Bacteroidales were dominant. Bacteroides has been reported to play an important role in maintaining intestinal health, promoting digestion and nutrient absorption, and regulating the immune system. Therefore, it further illustrates that Lactobacillus plantarum ZFML004 plays an important role in improving intestinal inflammation and intestinal homeostasis.

[0114] In addition:

[0115] Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum ) P16, Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarumThe 9010 samples were tested according to the method described in Example 5 above. In the bleomycin-induced mouse pulmonary fibrosis model, the sample did not show any function in alleviating the pathological state of pulmonary fibrosis (according to the Ashcroft scoring system, the degree of fibrosis was not significantly different from that of the BLM group), indicating that it does not have the ability to alleviate the progression of pulmonary fibrosis.

[0116] Lactobacillus fermentum ( Lactobacillus fermentum NRRL B-67059, Lactobacillus plantarum ( Lactobacillus plantarum HXJS4-1, Lactobacillus plantarum ( Lactiplantibacillus plantarum ZYHL3, Lactobacillus paracasei ( Lactobacillus paracasei L9 does not possess the gut-lung axis synergistic mechanism with Enterogermina. When tested according to the method in Example 5 above, the Masson quantitative results showed that the positive area accounted for approximately 15-22%.

[0117] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It is worth noting that those skilled in the art can discover many variations and modifications to the present invention, and any modifications, equivalent substitutions, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Lactobacillus plantarum ZFML004, characterized by: The taxonomic name of the Lactobacillus plantarum is Lactobacillus plantarum , and the accession number is CCTCC NO: M2025330.

2. The use of Lactobacillus plantarum ZFML004 in the preparation of an antibacterial drug according to claim 1, characterized in that: the antibacterial is against the following pathogenic bacteria: Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Staphylococcus warneri. The drug can alleviate pulmonary fibrosis.

3. Use according to claim 2, characterized in that: The alleviation of pulmonary fibrosis is to improve the lung inflammation of pulmonary fibrosis animals / humans.

4. Use according to claim 3, characterized in that: Lactobacillus plantarum ZFML004 has adhesion and antioxidant capacity.

5. Use according to claim 4, characterized in that: ​

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